text:
Andrew Fire
George D. Smith Professor of Molecular and Genetic Medicine and Professor of Pathology and of Genetics
Web page: http://genome-www.stanford.edu/group/fire/
Professor, Pathology
Professor, Genetics
Member, Bio-X
Member, Cardiovascular Institute
Member, Maternal & Child Health Research Institute (MCHRI)
Member, Stanford Cancer Institute
Member, Wu Tsai Neurosciences Institute
Administrative Appointments
Professor of Pathology and Genetics, Stanford University School of Medicine (2003 - Present)
Assistant Professor -> Professor of Biology (Adjunct), Johns Hopkins University (1989 - 2009)
Scientific Staff, Carnegie Institution of Washington (1989 - 2003)
Staff Associate, Carnegie Institution of Washington (1986 - 1989)
afire@stanford.edu
University - Faculty Department:&nbspPathology Position: Professor
Departments of Pathology & Genetics
Stanford Univ. School of Medicine
300 Pasteur Drive - L235
Stanford, California 94305-5324
Fire Lab
Current Research and Scholarly Interests
->What We Do:
Our lab studies the mechanisms by which cells and organisms respond to genetic change.
The genetic landscape faced by a living cell is constantly changing. Developmental transitions, environmental shifts, and pathogenic invasions lend a dynamic character to both the genome and its activity pattern.We study a variety of natural mechanisms that are utilized by cells adapting to genetic change. These include mechanisms activated during normal development and systems for detecting and responding to foreign or unwanted genetic activity. At the root of these studies are questions of how a cell can distinguish "self" versus "nonself" and "wanted" versus "unwanted" gene expression.
We primarily make use of the nematode C. elegans in our experimental studies. C. elegans is small, easily cultured, and can readily be made to accept foreign DNA or RNA. The results of such experiments have outlined a number of concerted responses that recognize (and in most cases work to silence) the foreign nucleic acid. One such mechanism ("RNAi") responds to double stranded character in RNA: either as introduced experimentally into the organism or as produced from foreign DNA that has not undergone selection to avoid a dsRNA response. Much of the current effort in the lab is directed toward a molecular understanding of the RNAi machinery and its roles in the cell. RNAi is not the only cellular defense against unwanted nucleic acid, and substantial current effort in the lab is also directed at identification of other triggers and mechanisms used in recognition and response to foreign information.
->Who we are:
PI: Andrew Fire, Professor of Pathology and Genetics, Stanford University School of Medicine
Dae-Eun Jeong (Ph.D. Pohang University of Science and Technology, Life Sciences, 2017)
Maya Kasowski (M.D./Ph.D. Yale University School of Medicine, 2016)
Matthew McCoy (Ph.D. Washington University, Molecular Genetics and Genomics 2018)
Massa Shoura (Ph.D. Univ. Texas at Dallas, Molecular-Cellular Biology 2013, Bioengineering 2014)
Lamia Wahba (Ph.D. Johns Hopkins University, Biology, 2013)
Nelson Hall (Stanford Bioengineering Ph.D. Program; B.S. MIT, 2016)
Nimit Jain (Stanford Bioengineering Ph.D. Program; B.S. Yale, 2011)
Alizeh Ahmad (Stanford Human Biology, 2019)
Visiting Scientist (Stanford Thinking-Matters Fellows Program):
Saumya Sankaran (Ph.D. Stanford, Biology, 2016)
Laboratory Manager:
Karen Artiles (Ph.D. UC Santa Cruz, 2008)
Laboratory Specialist:
Krisztina Perez
-> Joining The Fire Lab
We welcome new applicants to the lab.
Prospective postdoctoral applicants should send a resume and summary of research to Dr. Fire (afire <at> stanford <dot> edu), and arrange to have 3-4 letters of reference likewise sent to this address.
Prospective graduate students are encouraged to apply to the Stanford Genetics Ph.D. program (or to any of the biosciences Ph.D. programs): http://biosciences.stanford.edu/prospective/
Rotation Students: We welcome rotation students from any program at Stanford, with Spring being the preferred quarter. Email the PI.
We occasionally have positions for undergraduate researchers in the lab (especially summers, and particularly straightforward for current or incoming Stanford students). Email the PI at the above address.
2022-23 Courses
Advanced Genetics
GENE 205 (Win)
Computational Analysis of Biological Information: Introduction to Python for Biologists
GENE 218, MI 218, PATH 218 (Sum)
Genetics and Developmental Biology Training Camp
DBIO 200, GENE 200 (Aut)
Genetics of Viral Emergence and Emerging Viruses
Independent Studies (22)
Directed Investigation
BIOE 392 (Aut, Win, Spr, Sum)
Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
Directed Reading in Genetics
GENE 299 (Aut, Win, Spr, Sum)
Directed Reading in Immunology
IMMUNOL 299 (Win, Spr)
Directed Reading in Pathology
PATH 299 (Aut, Win, Spr, Sum)
Early Clinical Experience in Immunology
Early Clinical Experience in Pathology
Medical Scholars Research
Out-of-Department Advanced Research Laboratory in Bioengineering
BIOE 191X (Aut, Win, Spr)
Out-of-Department Undergraduate Research
BIO 199X (Aut, Win, Spr)
Supervised Study
Teaching in Cancer Biology
CBIO 260 (Spr)
Teaching in Immunology
Writing of Original Research for Engineers
ENGR 199W (Spr)
Prior Year Courses
GENE 218 (Spr)
C. Elegans Genetics
Stanford Advisees
Doctoral Dissertation Reader (AC)
Alex Adams, Ching Pin Cheng, Jennifer Doherty, Kelsey Fryer, Lynnette Jackson, Sedona Murphy, Rahul Nagvekar, Ben Ober-Reynolds, Lucero Rogel, Kei Yamaya, Alyssa Yoxsimer
Postdoctoral Faculty Sponsor
Orkan Ilbay, Dae-Eun Jeong, Matthew McCoy
Doctoral Dissertation Advisor (AC)
Usman Enam, Ivan Zheludev
Doctoral Dissertation Co-Advisor (AC)
Emily Greenwald
Graduate and Fellowship Programs
Cancer Biology (Phd Program)
Genetics (Phd Program)
Immunology (Phd Program)
Challenges and opportunities in interventions for chronic kidney disease of unknown origin (CKDu): report from the International Society of Nephrology Consortium of Collaborators on CKDu. Kidney international Smyth, B., Glaser, J., Butler-Dawson, J., Nanayakkara, N., Wegman, D. H., Anand, S., Levin, A., International Society of Nephrologys International Consortium of Collaborators on Chronic Kidney Disease of Unknown Etiology (i3C), Caplin, B., Correa Rotter, R., Eckardt, K., Fire, A., Friedman, D., Herath, C., Jha, V., Wijewickrama, E., Yang, C., Bajpai, D., Pippias, M., Ulasi, I., Nangaku, M. 2023; 103 (1): 6-12
View details for DOI 10.1016/j.kint.2022.10.013
View details for PubMedID 36603985
Restriction Endonuclease-Based Modification-Dependent Enrichment (REMoDE) of DNA for Metagenomic Sequencing. Applied and environmental microbiology Enam, S. U., Cherry, J. L., Leonard, S. R., Zheludev, I. N., Lipman, D. J., Fire, A. Z. 2022: e0167022
Metagenomic sequencing is a swift and powerful tool to ascertain the presence of an organism of interest in a sample. However, sequencing coverage of the organism of interest can be insufficient due to an inundation of reads from irrelevant organisms in the sample. Here, we report a nuclease-based approach to rapidly enrich for DNA from certain organisms, including enterobacteria, based on their differential endogenous modification patterns. We exploit the ability of taxon-specific methylated motifs to resist the action of cognate methylation-sensitive restriction endonucleases that thereby digest unwanted, unmethylated DNA. Subsequently, we use a distributive exonuclease or electrophoretic separation to deplete or exclude the digested fragments, thus enriching for undigested DNA from the organism of interest. As a proof of concept, we apply this method to enrich for the enterobacteria Escherichia coli and Salmonella enterica by 11- to 142-fold from mock metagenomic samples and validate this approach as a versatile means to enrich for genomes of interest in metagenomic samples. IMPORTANCE Pathogens that contaminate the food supply or spread through other means can cause outbreaks that bring devastating repercussions to the health of a populace. Investigations to trace the source of these outbreaks are initiated rapidly but can be drawn out due to the labored methods of pathogen isolation. Metagenomic sequencing can alleviate this hurdle but is often insufficiently sensitive. The approach and implementations detailed here provide a rapid means to enrich for many pathogens involved in foodborne outbreaks, thereby improving the utility of metagenomic sequencing as a tool in outbreak investigations. Additionally, this approach provides a means to broadly enrich for otherwise minute levels of modified DNA, which may escape unnoticed in metagenomic samples.
View details for DOI 10.1128/aem.01670-22
Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection. Cell reports methods Hall, R. N., Weill, U., Drees, L., Leal-Ortiz, S., Li, H., Khariton, M., Chai, C., Xue, Y., Rosental, B., Quake, S. R., Sanchez Alvarado, A., Melosh, N. A., Fire, A. Z., Rink, J. C., Wang, B. 2022; 2 (10): 100298
Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating the measurement of expression kinetics in both isolated cells and whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a foundation for the development and expansion of transgenic techniques in this unique model system.
View details for DOI 10.1016/j.crmeth.2022.100298
Context-dependent DNA polymerization effects can masquerade as DNA modification signals. BMC genomics Takahashi, Y., Shoura, M., Fire, A., Morishita, S. 2022; 23 (1): 249
BACKGROUND: Single molecule measurements of DNA polymerization kinetics provide a sensitive means to detect both secondary structures in DNA and deviations from primary chemical structure as a result of modified bases. In one approach to such analysis, deviations can be inferred by monitoring the behavior of DNA polymerase using single-molecule, real-time sequencing with zero-mode waveguide. This approach uses a Single Molecule Real Time (SMRT)-sequencing measurement of time between fluorescence pulse signals from consecutive nucleosides incorporated during DNA replication, called the interpulse duration (IPD).RESULTS: In this paper we present an analysis of loci with high IPDs in two genomes, a bacterial genome (E. coli) and a eukaryotic genome (C. elegans). To distinguish the potential effects of DNA modification on DNA polymerization speed, we paired an analysis of native genomic DNA with whole-genome amplified (WGA) material in which DNA modifications were effectively removed. Adenine modification sites for E. coli are known and we observed the expected IPD shifts at these sites in the native but not WGA samples. For C. elegans, such differences were not observed. Instead, we found a number of novel sequence contexts where IPDs were raised relative to the average IPDs for each of the four nucleotides, but for which the raised IPD was present in both native and WGA samples.CONCLUSION: The latter results argue strongly against DNA modification as the underlying driver for high IPD segments for C. elegans, and provide a framework for separating effects of DNA modification from context-dependent DNA polymerase kinetic patterns inherent in underlying DNA sequence for a complex eukaryotic genome.
View details for DOI 10.1186/s12864-022-08471-2
Lymphoid blast transformation in an MPN with BCR-JAK2 treated with ruxolitinib: putative mechanisms of resistance. Blood advances Chen, J. A., Hou, Y., Roskin, K. M., Arber, D. A., Bangs, C. D., Baughn, L. B., Cherry, A. M., Ewalt, M. D., Fire, A. Z., Fresard, L., Kearney, H. M., Montgomery, S. B., Ohgami, R. S., Pearce, K. E., Pitel, B. A., Merker, J. D., Gotlib, J. 2021; 5 (17): 3492-3496
The basis for acquired resistance to JAK inhibition in patients with JAK2-driven hematologic malignancies is not well understood. We report a patient with a myeloproliferative neoplasm (MPN) with a BCR activator of RhoGEF and GTPase (BCR)-JAK2 fusion with initial hematologic response to ruxolitinib who rapidly developed B-lymphoid blast transformation. We analyzed pre-ruxolitinib and blast transformation samples using genome sequencing, DNA mate-pair sequencing (MPseq), RNA sequencing (RNA-seq), and chromosomal microarray to characterize possible mechanisms of resistance. No resistance mutations in the BCR-JAK2 fusion gene or transcript were identified, and fusion transcript expression levels remained stable. However, at the time of blast transformation, MPseq detected a new IKZF1 copy-number loss, which is predicted to result in loss of normal IKZF1 protein translation. RNA-seq revealed significant upregulation of genes negatively regulated by IKZF1, including IL7R and CRLF2. Disease progression was also characterized by adaptation to an activated B-cell receptor (BCR)-like signaling phenotype, with marked upregulation of genes such as CD79A, CD79B, IGLL1, VPREB1, BLNK, ZAP70, RAG1, and RAG2. In summary, IKZF1 deletion and a switch from cytokine dependence to activated BCR-like signaling phenotype represent putative mechanisms of ruxolitinib resistance in this case, recapitulating preclinical data on resistance to JAK inhibition in CRLF2-rearranged Philadelphia chromosome-like acute lymphoblastic leukemia.
View details for DOI 10.1182/bloodadvances.2020004174
An essential role for the piRNA pathway in regulating the ribosomal RNA pool in C.elegans. Developmental cell Wahba, L., Hansen, L., Fire, A. Z. 2021
Piwi-interacting RNAs (piRNAs) are RNA effectors with key roles in maintaining genome integrity and promoting fertility in metazoans. In Caenorhabditis elegans loss of piRNAs leads to a transgenerational sterility phenotype. The plethora of piRNAs and their ability to silence transcripts with imperfect complementarity have raised several (non-exclusive) models for the underlying drivers of sterility. Here, we report the extranuclear and transferable nature of the sterility driver, its suppression via mutations disrupting the endogenous RNAi and poly-uridylation machinery, and copy-number amplification at the ribosomal DNA locus. In piRNA-deficient animals, several small interfering RNA (siRNA) populations become increasingly overabundant in the generations preceding loss of germline function, including ribosomal siRNAs (risiRNAs). A concomitant increase in uridylated sense rRNA fragments suggests that poly-uridylation may potentiate RNAi-mediated gene silencing of rRNAs. We conclude that loss of the piRNA machinery allows for unchecked amplification of siRNA populations, originating from abundant highly structured RNAs, to deleterious levels.
View details for DOI 10.1016/j.devcel.2021.07.014
PLP-1 is essential for germ cell development and germline gene silencing in C. elegans. Development (Cambridge, England) Vishnupriya, R., Thomas, L., Wahba, L., Fire, A., Subramaniam, K. 2020
The germ line genome is guarded against invading foreign genetic elements by small RNA-dependent gene-silencing pathways. Components of these pathways localize to, or form distinct aggregates in the vicinity of, germ granules. These components and their dynamics in and out of granules are currently being intensively studied. Here, we report the identification of PLP-1, a C. elegans protein related to the human single-stranded nucleic acid-binding protein called Pur-alpha, as a component of germ granules in C. elegans We show that PLP-1 is essential for silencing different types of transgenes in the germ line, and for suppressing the expression of several endogenous genes controlled by the germline gene-silencing pathways. Our results reveal that PLP-1 functions downstream of small RNA biogenesis during initiation of gene silencing. Based on these results and the earlier findings that Pur-alpha proteins interact with both RNA and protein, we propose PLP-1 couples certain RNAs with their protein partners in the silencing complex. Its orthologs localized on RNA granules may similarly contribute to germline gene silencing in other organisms.
View details for DOI 10.1242/dev.195578
Doubling of the known set of RNA viruses by metagenomic analysis of an aquatic virome. Nature microbiology Wolf, Y. I., Silas, S., Wang, Y., Wu, S., Bocek, M., Kazlauskas, D., Krupovic, M., Fire, A., Dolja, V. V., Koonin, E. V. 2020
RNA viruses in aquatic environments remain poorly studied. Here, we analysed the RNA virome from approximately 10l water from Yangshan Deep-Water Harbour near the Yangtze River estuary in China and identified more than 4,500 distinct RNA viruses, doubling the previously known set of viruses. Phylogenomic analysis identified several major lineages, roughly, at the taxonomic ranks of class, order and family. The 719-member-strong Yangshan virus assemblage is the sister clade to the expansive class Alsuviricetes and consists of viruses with simple genomes that typically encode only RNA-dependent RNA polymerase (RdRP), capping enzyme and capsid protein. Several clades within the Yangshan assemblage independently evolved domain permutation in the RdRP. Another previously unknown clade shares ancestry with Potyviridae, the largest known plant virus family. The 'Aquatic picorna-like viruses/Marnaviridae' clade was greatly expanded, with more than 800 added viruses. Several RdRP-linked protein domains not previously detected in any RNA viruses were identified, such as the small ubiquitin-like modifier (SUMO) domain, phospholipase A2 and PrsW-family protease domain. Multiple viruses utilize alternative genetic codes implying protist (especially ciliate) hosts. The results reveal a vast RNA virome that includes many previously unknown groups. However, phylogenetic analysis of the RdRPs supports the previously established five-branch structure of the RNA virus evolutionary tree, with no additional phyla.
View details for DOI 10.1038/s41564-020-0755-4
An Extensive Meta-Metagenomic Search Identifies SARS-CoV-2-Homologous Sequences in Pangolin Lung Viromes. mSphere Wahba, L., Jain, N., Fire, A. Z., Shoura, M. J., Artiles, K. L., McCoy, M. J., Jeong, D. 2020; 5 (3)
In numerous instances, tracking the biological significance of a nucleic acid sequence can be augmented through the identification of environmental niches in which the sequence of interest is present. Many metagenomic data sets are now available, with deep sequencing of samples from diverse biological niches. While any individual metagenomic data set can be readily queried using web-based tools, meta-searches through all such data sets are less accessible. In this brief communication, we demonstrate such a meta-metagenomic approach, examining close matches to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in all high-throughput sequencing data sets in the NCBI Sequence Read Archive accessible with the "virome" keyword. In addition to the homology to bat coronaviruses observed in descriptions of the SARS-CoV-2 sequence (F. Wu, S. Zhao, B. Yu, Y. M. Chen, et al., Nature 579:265-269, 2020, https://doi.org/10.1038/s41586-020-2008-3; P. Zhou, X. L. Yang, X. G. Wang, B. Hu, et al., Nature 579:270-273, 2020, https://doi.org/10.1038/s41586-020-2012-7), we note a strong homology to numerous sequence reads in metavirome data sets generated from the lungs of deceased pangolins reported by Liu et al. (P. Liu, W. Chen, and J. P. Chen, Viruses 11:979, 2019, https://doi.org/10.3390/v11110979). While analysis of these reads indicates the presence of a similar viral sequence in pangolin lung, the similarity is not sufficient to either confirm or rule out a role for pangolins as an intermediate host in the recent emergence of SARS-CoV-2. In addition to the implications for SARS-CoV-2 emergence, this study illustrates the utility and limitations of meta-metagenomic search tools in effective and rapid characterization of potentially significant nucleic acid sequences.IMPORTANCE Meta-metagenomic searches allow for high-speed, low-cost identification of potentially significant biological niches for sequences of interest.
View details for DOI 10.1128/mSphere.00160-20
Aberrant B cell repertoire selection associated with HIV neutralizing antibody breadth. Nature immunology Roskin, K. M., Jackson, K. J., Lee, J., Hoh, R. A., Joshi, S. A., Hwang, K., Bonsignori, M., Pedroza-Pacheco, I., Liao, H., Moody, M. A., Fire, A. Z., Borrow, P., Haynes, B. F., Boyd, S. D. 2020
A goal of HIV vaccine development is to elicit antibodies with neutralizing breadth. Broadly neutralizing antibodies (bNAbs) to HIV often have unusual sequences with long heavy-chain complementarity-determining region loops, high somatic mutation rates and polyreactivity. A subset of HIV-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. We sequenced antibody heavy-chain repertoires in a large cohort of HIV-infected individuals with bNAb responses or no neutralization breadth and uninfected controls, identifying consistent features of bNAb repertoires, encompassing thousands of B cell clones per individual, with correlated T cell phenotypes. These repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. Our data indicate that the development of numerous B cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of HIV neutralizing antibody breadth.
Transcription polymerase-catalyzed emergence of novel RNA replicons. Science (New York, N.Y.) Jain, N. n., Blauch, L. R., Szymanski, M. R., Das, R. n., Tang, S. K., Yin, Y. W., Fire, A. Z. 2020
Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as information carrier. Two classes of pathogenic RNAs (Hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting and interrupted rolling circle synthesis) and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.
View details for DOI 10.1126/science.aay0688
Intron and gene size expansion during nervous system evolution. BMC genomics McCoy, M. J., Fire, A. Z. 2020; 21 (1): 360
The evolutionary radiation of animals was accompanied by extensive expansion of gene and genome sizes, increased isoform diversity, and complexity of regulation.Here we show that the longest genes are enriched for expression in neuronal tissues of diverse vertebrates and of invertebrates. Additionally, we show that neuronal gene size expansion occurred predominantly through net gains in intron size, with a positional bias toward the 5' end of each gene.We find that intron and gene size expansion is a feature of many genes whose expression is enriched in nervous systems. We speculate that unique attributes of neurons may subject neuronal genes to evolutionary forces favoring net size expansion. This process could be associated with tissue-specific constraints on gene function and/or the evolution of increasingly complex gene regulation in nervous systems.
Deconvolution of nucleic-acid length distributions: a gel electrophoresis analysis tool and applications. Nucleic acids research Ziraldo, R., Shoura, M. J., Fire, A. Z., Levene, S. D. 2019
Next-generation DNA-sequencing (NGS) technologies, which are designed to streamline the acquisition of massive amounts of sequencing data, are nonetheless dependent on various preparative steps to generate DNA fragments of required concentration, purity and average size (molecular weight). Current automated electrophoresis systems for DNA- and RNA-sample quality control, such as Agilent's Bioanalyzer and TapeStation products, are costly to acquire and use; they also provide limited information for samples having broad size distributions. Here, we describe a software tool that helps determine the size distribution of DNA fragments in an NGS library, or other DNA sample, based on gel-electrophoretic line profiles. The software, developed as an ImageJ plug-in, allows for straightforward processing of gel images, including lane selection and fitting of univariate functions to intensity distributions. The user selects the option of fitting either discrete profiles in cases where discrete gel bands are visible or continuous profiles, having multiple bands buried under a single broad peak. The method requires only modest imaging capabilities and is a cost-effective, rigorous alternative characterization method to augment existing techniques for library quality control.
View details for DOI 10.1093/nar/gkz534
Ribosome clearance during RNA interference. RNA (New York, N.Y.) Pule, M. N., Glover, M. L., Fire, A. Z., Arribere, J. A. 2019
In the course of identifying and cleaving RNA, the RNAi machinery must encounter and contend with the megadalton-sized ribosomes that carry out translation. We investigated this interface by examining the fate of actively translated mRNAs subjected to RNAi in C. elegans. Quantifying RNA levels (RNA-seq) and ongoing translation (Ribo-seq), we found there is a greater fold repression of ongoing translation than expected from loss of RNA alone, observing stronger translation repression relative to RNA repression for multiple, independent double-stranded RNA triggers, and for multiple genes. In animals that lack the RNA helicase SKI complex and the ribosome rescue factor PELOTA, ribosomes stall on the 3' edges of mRNAs at and upstream of the RNAi trigger. One model to explain these observations is that ribosomes are actively cleared from mRNAs by SKI and PELO during or following mRNA cleavage. Our results expand prior studies that show a role for the SKI RNA helicase complex in removing RNA targets following RNAi in flies and plants, illuminating the widespread role of the nonstop translation surveillance in RNA silencing during RNAi. Our results are also consistent with proposals that RNAi can attack messages during active translation.
View details for DOI 10.1261/rna.070813.119
Target-dependent nickase activities of the CRISPR-Cas nucleases Cpf1 and Cas9. Nature microbiology Fu, B. X., Smith, J. D., Fuchs, R. T., Mabuchi, M., Curcuru, J., Robb, G. B., Fire, A. Z. 2019
Clustered regularly interspaced short palindromic repeats (CRISPR) machineries are prokaryotic immune systems that have been adapted as versatile gene editing and manipulation tools. We found that CRISPR nucleases from two families, Cpf1 (also known as Cas12a) and Cas9, exhibit differential guide RNA (gRNA) sequence requirements for cleavage of the two strands of target DNA in vitro. As a consequence of the differential gRNA requirements, both Cas9 and Cpf1 enzymes can exhibit potent nickase activities on an extensive class of mismatched double-stranded DNA (dsDNA) targets. These properties allow the production of efficient nickases for a chosen dsDNA target sequence, without modification of the nuclease protein, using gRNAs with a variety of patterns of mismatch to the intended DNA target. In parallel to the nicking activities observed with purified Cas9 in vitro, we observed sequence-dependent nicking for both perfectly matched and partially mismatched target sequences in a Saccharomyces cerevisiae system. Our findings have implications for CRISPR spacer acquisition, off-target potential of CRISPR gene editing/manipulation, and tool development using homology-directed nicking.
CLONALITY: POINT ESTIMATION ANNALS OF APPLIED STATISTICS Tian, L., Liu, Y., Fire, A. Z., Boyd, S. D., Olshen, R. A. 2019; 13 (1): 113–31
View details for DOI 10.1214/18-AOAS1197
Assessment and Maintenance of Unigametic Germline Inheritance for C.elegans. Developmental cell Artiles, K. L., Fire, A. Z., Frokjar-Jensen, C. 2019
The recent work of Besseling and Bringmann (2016) identified a molecular intervention for C.elegans inwhich premature segregation of maternal and paternal chromosomes in the fertilized oocyte can produce viable animals exhibiting a non-Mendelian inheritance pattern. Overexpression in embryos of a single protein regulating chromosome segregation (GPR-1) provides a germline derived clonally from a single parental gamete. We present a collection of strains and cytological assays to consistently generate and track non-Mendelian inheritance. These tools allow reproducible and high-frequency (>80%) production of non-Mendelian inheritance, the facile and simultaneous homozygosis for all nuclear chromosomes in a single generation, the precise exchange of nuclear and mitochondrial genomes between strains, and the assessments of non-canonical mitosis events. We show the utility of these strains by demonstrating a rapid assessment of cell lineage requirements (AB versus P1) for a set of genes (lin-2, lin-3, lin-12, and lin-31) with roles in C.elegans vulval development.
Maternal Ribosomes Are Sufficient for Tissue Diversification during Embryonic Development in C.elegans. Developmental cell Cenik, E. S., Meng, X., Tang, N. H., Hall, R. N., Arribere, J. A., Cenik, C., Jin, Y., Fire, A. 2019
Caenorhabditis elegans provides an amenable system to explore whether newly composed ribosomes are required to progress through development. Despite the complex pattern of tissues that are formed during embryonic development, we found that null homozygotes lacking any of the five different ribosomal proteins (RPs) can produce fully functional first-stage larvae, with similar developmental competence seen upon complete deletion of the multi-copy ribosomal RNA locus. These animals, relying on maternal but not zygotic contribution of ribosomal components, are capable of completing embryogenesis. In the absence of new ribosomal components, the resulting animals are arrested before progression from the first larval stage and fail in two assays for postembryonic plasticity of neuronal structure. Mosaic analyses of larvae that are a mixture of ribosome-competent and non-competent cells suggest a global regulatory mechanism in which ribosomal insufficiency in a subset of cells triggers organism-wide growth arrest.
Prospective Biopsy-Based Study of CKD of Unknown Etiology in Sri Lanka CLINICAL JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY Anand, S., Montez-Rath, M. E., Adasooriya, D., Ratnatunga, N., Kambham, N., Wazil, A., Wijetunge, S., Badurdeen, Z., Ratnayake, C., Karunasena, N., Schensul, S. L., Valhos, P., Haider, L., Bhalla, V., Levin, A., Wise, P. H., Chertow, G. M., Barry, M., Fire, A. Z., Nanayakkara, N. 2019; 14 (2): 224–32
View details for DOI 10.2215/CJN.07430618
Prospective Biopsy-Based Study of Chronic Kidney Disease of Unknown Etiology in Sri Lanka. Clinical journal of the American Society of Nephrology : CJASN Anand, S., Montez-Rath, M. E., Adasooriya, D., Ratnatunga, N., Kambham, N., Wazil, A., Wijetunge, S., Badurdeen, Z., Ratnayake, C., Karunasena, N., Schensul, S. L., Valhos, P., Haider, L., Bhalla, V., Levin, A., Wise, P. H., Chertow, G. M., Barry, M., Fire, A. Z., Nanayakkara, N. 2019
BACKGROUND AND OBJECTIVES: A kidney disease of unknown cause is common in Sri Lanka's lowland (dry) region. Detailed clinical characterizations of patients with biopsy-proven disease are limited, and there is no current consensus on criteria for a noninvasive diagnosis.DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We designed a prospective study in a major Sri Lankan hospital servicing endemic areas to ascertain pathologic and clinical characteristics of and assess risk factors for primary tubulointerstitial kidney disease. We used logistic regression to determine whether common clinical characteristics could be used to predict the presence of primary tubulointerstitial kidney disease on kidney biopsy.RESULTS: From 600 new patients presenting to a tertiary nephrology clinic over the course of 1 year, 87 underwent kidney biopsy, and 43 (49%) had a biopsy diagnosis of primary tubulointerstitial kidney disease. On detailed biopsy review, 13 (30%) had evidence of moderate to severe active kidney disease, and six (15%) had evidence of moderate to severe chronic tubulointerstitial kidney disease. Patients with tubulointerstitial kidney disease were exclusively born in endemic provinces; 91% spent a majority of their lifespan there. They were more likely men and farmers (risk ratio, 2.0; 95% confidence interval, 1.2 to 2.9), and they were more likely to have used tobacco (risk ratio, 1.7; 95% confidence interval, 1.0 to 2.3) and well water (risk ratio, 1.5; 95% confidence interval, 1.1 to 2.0). Three clinical characteristics-age, urine dipstick for protein, and serum albumin-could predict likelihood of tubulointerstitial kidney disease on biopsy (model sensitivity of 79% and specificity of 84%). Patients referred for kidney biopsy despite comorbid diabetes or hypertension did not experience lower odds of tubulointerstitial kidney disease.CONCLUSIONS: A primary tubulointerstitial kidney disease occurs commonly in specific regions of Sri Lanka with characteristic environmental and lifestyle exposures.PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2019_01_18_CJASNPodcast_19_02_.mp3.
Epidemiology, molecular, and genetic methodologies to evaluate causes of CKDu around the world: report of the Working Group from the ISN International Consortium of Collaborators on CKDu. Kidney international Anand, S. n., Caplin, B. n., Gonzalez-Quiroz, M. n., Schensul, S. L., Bhalla, V. n., Parada, X. n., Nanayakkara, N. n., Fire, A. n., Levin, A. n., Friedman, D. J. 2019; 96 (6): 1254–60
Recompleting the Caenorhabditis elegans genome. Genome research Yoshimura, J. n., Ichikawa, K. n., Shoura, M. J., Artiles, K. L., Gabdank, I. n., Wahba, L. n., Smith, C. L., Edgley, M. L., Rougvie, A. E., Fire, A. Z., Morishita, S. n., Schwarz, E. M. 2019
Caenorhabditis elegans was the first multicellular eukaryotic genome sequenced to apparent completion. Although this assembly employed a standard C. elegans strain (N2), it used sequence data from several laboratories, with DNA propagated in bacteria and yeast. Thus, the N2 assembly has many differences from any C. elegans available today. To provide a more accurate C. elegans genome, we performed long-read assembly of VC2010, a modern strain derived from N2. Our VC2010 assembly has 99.98% identity to N2 but with an additional 1.8 Mb including tandem repeat expansions and genome duplications. For 116 structural discrepancies between N2 and VC2010, 97 structures matching VC2010 (84%) were also found in two outgroup strains, implying deficiencies in N2. Over 98% of N2 genes encoded unchanged products in VC2010; moreover, we predicted ≥53 new genes in VC2010. The recompleted genome of C. elegans should be a valuable resource for genetics, genomics, and systems biology.
View details for DOI 10.1101/gr.244830.118
A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition MOLECULAR CELL Mohr, G., Silas, S., Stamos, J. L., Makarova, K. S., Markham, L. M., Yao, J., Lucas-Elio, P., Sanchez-Amat, A., Fire, A. Z., Koonin, E., Lambowitz, A. M. 2018; 72 (4): 700-+
View details for DOI 10.1016/j.molcel.2018.09.013
A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition. Molecular cell Mohr, G., Silas, S., Stamos, J. L., Makarova, K. S., Markham, L. M., Yao, J., Lucas-Elio, P., Sanchez-Amat, A., Fire, A. Z., Koonin, E. V., Lambowitz, A. M. 2018
Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion.
A Small RNA Isolation and Sequencing Protocol and Its Application to Assay CRISPR RNA Biogenesis in Bacteria BIO-PROTOCOL Silas, S., Jain, N., Stadler, M., Fu, B., Sanchez-Amat, A., Fire, A. Z., Arribere, J. 2018; 8 (4)
View details for DOI 10.21769/BioProtoc.2727
Nonsense mRNA suppression via nonstop decay ELIFE Arribere, J. A., Fire, A. Z. 2018; 7
Nonsense-mediated mRNA decay is the process by which mRNAs bearing premature stop codons are recognized and cleared from the cell. While considerable information has accumulated regarding recognition of the premature stop codon, less is known about the ensuing mRNA suppression. During the characterization of a second, distinct translational surveillance pathway (nonstop mRNA decay), we trapped intermediates in nonsense mRNA degradation. We present data in support of a model wherein nonsense-mediated decay funnels into the nonstop decay pathway in Caenorhabditis elegans. Specifically, our results point to SKI-exosome decay and pelota-based ribosome removal as key steps facilitating suppression and clearance of prematurely-terminated translation complexes. These results suggest a model in which premature stop codons elicit nucleolytic cleavage, with the nonstop pathway disengaging ribosomes and degrading the resultant RNA fragments to suppress ongoing expression.
Intricate and Cell Type-Specific Populations of Endogenous Circular DNA (eccDNA) in Caenorhabditis elegans and Homo sapiens. G3 (Bethesda, Md.) Shoura, M. J., Gabdank, I., Hansen, L., Merker, J., Gotlib, J., Levene, S. D., Fire, A. Z. 2017; 7 (10): 3295-3303
Investigations aimed at defining the 3D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. Here, we define eccDNA distributions in Caenorhabditis elegans and in three human cell types, utilizing a set of DNA topology-dependent approaches for enrichment and characterization. The use of parallel biophysical, enzymatic, and informatic approaches provides a comprehensive profiling of eccDNA robust to isolation and analysis methodology. Results in human and nematode systems provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture, in which endogenous genomic DNA circles are present in normal physiological states, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples.
View details for PubMedCentralID PMC5633380
A novel TRIP11-FLT3 fusion in a patient with a myeloid/lymphoid neoplasm with eosinophilia CANCER GENETICS Chung, A., Hou, Y., Ohgami, R. S., Von Gehr, A., Fisk, D. G., Roskin, K. M., Li, X., Gojenola, L., Bangs, C. D., Arber, D. A., Fire, A. Z., Cherry, A. M., Zehnder, J. L., Gotlib, J., Merker, J. D. 2017; 216: 10–15
FLT3 fusions are associated with myeloid and lymphoid neoplasms with eosinophilia. We describe a patient presenting with clinicopathologic features of both chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) and systemic mastocytosis (SM). The bone marrow demonstrated a myeloproliferative neoplasm with eosinophilia and aggregates of atypical mast cells. Cytogenetic analysis revealed a t(13;14)(q12;q32), which was subsequently molecularly characterized as a novel TRIP11-FLT3 rearrangement. A KIT D816V mutation was also identified. The patient rapidly transformed to T-lymphoblastic leukemia/lymphoma and expired shortly after diagnosis. This is the fifth FLT3 fusion gene described in the literature; the presence of both myeloid and lymphoid neoplasms implicates involvement of an early hematopoietic progenitor by rearranged FLT3. We suggest that leukemias and lymphomas with FLT3 fusion genes exhibit similar clinicopathologic features to, and should be included in, the WHO category of "Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1, or with PCM1-JAK2."
Type III CRISPR-Cas systems can provide redundancy to counteract viral escape from type I systems ELIFE Silas, S., Lucas-Elio, P., Jackson, S. A., Aroca-Crevillen, A., Hansen, L. L., Fineran, P. C., Fire, A. Z., Sanchez-Amat, A. 2017; 6
CRISPR-Cas-mediated defense utilizes information stored as spacers in CRISPR arrays to defend against genetic invaders. We define the mode of target interference and role in antiviral defense for two CRISPR-Cas systems in Marinomonas mediterranea. One system (type I-F) targets DNA. A second system (type III-B) is broadly capable of acquiring spacers in either orientation from RNA and DNA, and exhibits transcription-dependent DNA interference. Examining resistance to phages isolated from Mediterranean seagrass meadows, we found that the type III-B machinery co-opts type I-F CRISPR-RNAs. Sequencing and infectivity assessments of related bacterial and phage strains suggests an 'arms race' in which phage escape from the type I-F system can be overcome through use of type I-F spacers by a horizontally-acquired type III-B system. We propose that the phage-host arms race can drive selection for horizontal uptake and maintenance of promiscuous type III interference modules that supplement existing host type I CRISPR-Cas systems.
Sequence-Modified Antibiotic Resistance Genes Provide Sustained Plasmid-Mediated Transgene Expression in Mammals MOLECULAR THERAPY Lu, J., Zhang, F., Fire, A. Z., Kay, M. A. 2017; 25 (5): 1187-1198
Conventional plasmid vectors are incapable of achieving sustained levels of transgene expression in vivo even in quiescent mammalian tissues because the transgene expression cassette is silenced. Transcriptional silencing results from the presence of the bacterial plasmid backbone or virtually any DNA sequence of >1 kb in length placed outside of the expression cassette. Here, we show that transcriptional silencing can be substantially forestalled by increasing the An/Tn sequence composition in the plasmid bacterial backbone. Increasing numbers of An/Tn sequences increased sustained transcription of both backbone sequences and adjacent expression cassettes. In order to recapitulate these expression profiles in compact and portable plasmid DNA backbones, we engineered the standard kanamycin or ampicillin antibiotic resistance genes, optimizing the number of An/Tn sequence without altering the encoded amino acids. The resulting vector backbones yield sustained transgene expression from mouse liver, providing generic DNA vectors capable of sustained transgene expression without additional genes or mammalian regulatory elements.
View details for DOI 10.1016/j.ymthe.2017.03.003
On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires. mBio Silas, S. n., Makarova, K. S., Shmakov, S. n., Páez-Espino, D. n., Mohr, G. n., Liu, Y. n., Davison, M. n., Roux, S. n., Krishnamurthy, S. R., Fu, B. X., Hansen, L. L., Wang, D. n., Sullivan, M. B., Millard, A. n., Clokie, M. R., Bhaya, D. n., Lambowitz, A. M., Kyrpides, N. C., Koonin, E. V., Fire, A. Z. 2017; 8 (4)
Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium-Arthrospira platensis Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown.IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis, a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic.
Intricate and Cell Type-Specific Populations of Endogenous Circular DNA (eccDNA) in Caenorhabditis elegans and Homo sapiens G3: GENES, GENOMES, GENETICS Shoura, M., Gabdank, I., Merker, J., Gotlib, J., Levene, S., Fire, A. 2017; 7: 3295-3303
High-Throughput Characterization of Cascade type I-E CRISPR Guide Efficacy Reveals Unexpected PAM Diversity and Target Sequence Preferences. Genetics Fu, B. X., Wainberg, M. n., Kundaje, A. n., Fire, A. Z. 2017; 206 (4): 1727–38
Interactions between Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) RNAs and CRISPR-associated (Cas) proteins form an RNA-guided adaptive immune system in prokaryotes. The adaptive immune system utilizes segments of the genetic material of invasive foreign elements in the CRISPR locus. The loci are transcribed and processed to produce small CRISPR RNAs (crRNAs), with degradation of invading genetic material directed by a combination of complementarity between RNA and DNA and in some cases recognition of adjacent motifs called PAMs (Protospacer Adjacent Motifs). Here we describe a general, high-throughput procedure to test the efficacy of thousands of targets, applying this to the Escherichia coli type I-E Cascade (CRISPR-associated complex for antiviral defense) system. These studies were followed with reciprocal experiments in which the consequence of CRISPR activity was survival in the presence of a lytic phage. From the combined analysis of the Cascade system, we found that (i) type I-E Cascade PAM recognition is more expansive than previously reported, with at least 22 distinct PAMs, with many of the noncanonical PAMs having CRISPR-interference abilities similar to the canonical PAMs; (ii) PAM positioning appears precise, with no evidence for tolerance to PAM slippage in interference; and (iii) while increased guanine-cytosine (GC) content in the spacer is associated with higher CRISPR-interference efficiency, high GC content (>62.5%) decreases CRISPR-interference efficiency. Our findings provide a comprehensive functional profile of Cascade type I-E interference requirements and a method to assay spacer efficacy that can be applied to other CRISPR-Cas systems.
An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline CELL Frokjaer-Jensen, C., Jain, N., Hansen, L., Davis, M. W., Li, Y., Zhao, D., Rebora, K., Millet, J. R., Liu, X., Kim, S. K., Dupuy, D., Jorgensen, E. M., Fire, A. Z. 2016; 166 (2): 343-357
Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure.
View details for DOI 10.1016/j.cell.2016.05.072
Translation readthrough mitigation NATURE Arribere, J. A., Cenik, E. S., Jain, N., Hess, G. T., Lee, C. H., Bassik, M. C., Fire, A. Z. 2016; 534 (7609): 719-?
A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR–Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3′ untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3′ UTR sequences to reduce mature protein expression in tissue culture assays, including 3′ UTR sequences from the hypomorphic 'Constant Spring' haemoglobin stop codon variant. We suggest that 3′ UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.
View details for DOI 10.1038/nature18308
Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo NUCLEIC ACIDS RESEARCH Fu, B. X., Onge, R. P., Fire, A. Z., Smith, J. D. 2016; 44 (11): 5365-5377
Cas9, a CRISPR-associated RNA-guided nuclease, has been rapidly adopted as a tool for biochemical and genetic manipulation of DNA. Although complexes between Cas9 and guide RNAs (gRNAs) offer remarkable specificity and versatility for genome manipulation, mis-targeted events occur. To extend the understanding of gRNA::target homology requirements, we compared mutational tolerance for a set of Cas9::gRNA complexes in vitro and in vivo (in Saccharomyces cerevisiae). A variety of gRNAs were tested with variant libraries based on four different targets (with varying GC content and sequence features). In each case, we challenged a mixture of matched and mismatched targets, evaluating cleavage activity on a wide variety of potential target sequences in parallel through high-throughput sequencing of the products retained after cleavage. These experiments evidenced notable and consistent differences between in vitro and S. cerevisiae (in vivo) Cas9 cleavage specificity profiles including (i) a greater tolerance for mismatches in vitro and (ii) a greater specificity increase in vivo with truncation of the gRNA homology regions.
View details for DOI 10.1093/nar/gkw417
Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY Levin, M., King, J. J., Glanville, J., Jackson, K. J., Looney, T. J., Hoh, R. A., Mari, A., Andersson, M., Greiff, L., Fire, A. Z., Boyd, S. D., Ohlin, M. 2016; 137 (5): 1535-1544
Specific immunotherapy (SIT) is the only treatment with proved long-term curative potential in patients with allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B-cell repertoires are not well understood.We sought to characterize the IgE sequences expressed by allergen-specific B cells and track the fate of these B-cell clones during SIT.We used high-throughput antibody gene sequencing and identification of allergen-specific IgE with combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and the nasal mucosa from aeroallergen-sensitized subjects before and during the first year of subcutaneous SIT.Of 52 distinct allergen-specific IgE heavy chains from 8 allergic donors, 37 were also detected by using high-throughput antibody gene sequencing of blood samples, nasal mucosal samples, or both. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships.In the future, combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies might aid assessment of SIT mechanisms and efficacy.
View details for DOI 10.1016/j.jaci.2015.09.027
Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody. Cell Bonsignori, M., Zhou, T., Sheng, Z., Chen, L., Gao, F., Joyce, M. G., Ozorowski, G., Chuang, G., Schramm, C. A., Wiehe, K., Alam, S. M., Bradley, T., Gladden, M. A., Hwang, K., Iyengar, S., Kumar, A., Lu, X., Luo, K., Mangiapani, M. C., Parks, R. J., Song, H., Acharya, P., Bailer, R. T., Cao, A., Druz, A., Georgiev, I. S., Kwon, Y. D., Louder, M. K., Zhang, B., Zheng, A., Hill, B. J., Kong, R., Soto, C., Mullikin, J. C., Douek, D. C., Montefiori, D. C., Moody, M. A., Shaw, G. M., Hahn, B. H., Kelsoe, G., Hraber, P. T., Korber, B. T., Boyd, S. D., Fire, A. Z., Kepler, T. B., Shapiro, L., Ward, A. B., Mascola, J. R., Liao, H., Kwong, P. D., Haynes, B. F. 2016; 165 (2): 449-463
Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.
A streamlined tethered chromosome conformation capture protocol BMC GENOMICS Gabdank, I., Ramakrishnan, S., Villeneuve, A. M., Fire, A. Z. 2016; 17
Identification of locus-locus contacts at the chromatin level provides a valuable foundation for understanding of nuclear architecture and function and a valuable tool for inferring long-range linkage relationships. As one approach to this, chromatin conformation capture-based techniques allow creation of genome spatial organization maps. While such approaches have been available for some time, methodological advances will be of considerable use in minimizing both time and input material required for successful application.Here we report a modified tethered conformation capture protocol that utilizes a series of rapid and efficient molecular manipulations. We applied the method to Caenorhabditis elegans, obtaining chromatin interaction maps that provide a sequence-anchored delineation of salient aspects of Caenorhabditis elegans chromosome structure, demonstrating a high level of consistency in overall chromosome organization between biological samples collected under different conditions. In addition to the application of the method to defining nuclear architecture, we found the resulting chromatin interaction maps to be of sufficient resolution and sensitivity to enable detection of large-scale structural variants such as inversions or translocations.Our streamlined protocol provides an accelerated, robust, and broadly applicable means of generating chromatin spatial organization maps and detecting genome rearrangements without a need for cellular or chromatin fractionation.
Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein SCIENCE Silas, S., Mohr, G., Sidote, D. J., Markham, L. M., Sanchez-Amat, A., Bhaya, D., Lambowitz, A. M., Fire, A. Z. 2016; 351 (6276): 932-?
CRISPR systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in type I and II CRISPR systems by the acquisition of short segments of DNA (spacers) from invasive elements. In several type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA.
View details for DOI 10.1126/science.aad4234
Chikungunya Virus Sequences Across the First Epidemic in Nicaragua, 2014-2015. American journal of tropical medicine and hygiene Wang, C., Saborio, S., Gresh, L., Eswarappa, M., Wu, D., Fire, A., Parameswaran, P., Balmaseda, A., Harris, E. 2016; 94 (2): 400-403
Chikungunya is caused by the mosquito-borne arthrogenic alphavirus, chikungunya virus (CHIKV). Chikungunya was introduced into the Americas in late 2013 and Nicaragua in mid-2014. Here, we sequenced five imported and 30 autochthonous Nicaraguan CHIKV from cases identified in the first epidemic in the country between August 2014 and April 2015. One full-length and two partial genomic sequences were obtained by deep sequencing; Sanger methodology yielded 33 E1 sequences from five imported and 28 autochthonous cases. Phylogenetic analysis indicates that Nicaraguan CHIKV all belonged to the Asian genotype, Caribbean clade. Moreover, E1 gene sequences revealed accumulation of mutations in later months of the epidemic, including four silent mutations in 11 autochthonous cases and three non-synonymous mutations in three autochthonous cases. No mutations contributing to increased transmissibility by Aedes albopictus were identified in the E1 gene. This represents the most comprehensive set of CHIKV sequences available from the Americas to date.
View details for DOI 10.4269/ajtmh.15-0497
Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans. Genetics Bell, R. T., Fu, B. X., Fire, A. Z. 2016; 202 (2): 381-388
The proliferation of CRISPR/Cas9-based methods in Caenorhabditis elegans has enabled efficient genome editing and precise genomic tethering of Cas9 fusion proteins. Experimental designs using CRISPR/Cas9 are currently limited by the need for a protospacer adjacent motif (PAM) in the target with the sequence NGG. Here we report the characterization of two modified Cas9 proteins in C. elegans that recognize NGA and NGCG PAMs. We found that each variant could stimulate homologous recombination with a donor template at multiple loci and that PAM specificity was comparable to that of wild-type Cas9. To directly compare effectiveness, we used CRISPR/Cas9 genome editing to generate a set of assay strains with a common single-guide RNA (sgRNA) target sequence, but that differ in the juxtaposed PAM (NGG, NGA, or NGCG). In this controlled setting, we determined that the NGA PAM Cas9 variant can be as effective as wild-type Cas9. We similarly edited a genomic target to study the influence of the base following the NGA PAM. Using four strains with four NGAN PAMs differing only at the fourth position and adjacent to the same sgRNA target, we observed that efficient homologous replacement was attainable with any base in the fourth position, with an NGAG PAM being the most effective. In addition to demonstrating the utility of two Cas9 mutants in C. elegans and providing reagents that permit CRISPR/Cas9 experiments with fewer restrictions on potential targets, we established a means to benchmark the efficiency of different Cas9::PAM combinations that avoids variations owing to differences in the sgRNA sequence.
View details for DOI 10.1534/genetics.115.185041
Associations between nucleosome phasing, sequence asymmetry, and tissue-specific expression in a set of inbred Medaka species BMC GENOMICS Nakatani, Y., Mello, C. C., Hashimoto, S., Shimada, A., Nakamura, R., Tsukahara, T., Qu, W., Yoshimura, J., Suzuki, Y., Sugano, S., Takeda, H., Fire, A., Morishita, S. 2015; 16
Transcription start sites (TSSs) with pronounced and phased nucleosome arrays downstream and nucleosome-depleted regions upstream of TSSs are observed in various species.We have characterized sequence variation and expression properties of this set of TSSs (which we call "Nucleocyclic TSSs") using germline and somatic cells of three medaka (Oryzias latipes) inbred isolates from different locations. We found nucleocyclic TSSs in medaka to be associated with higher gene expression and characterized by a clear boundary in sequence composition with potentially-nucleosome-destabilizing A/T-enrichment upstream (p < 10(-60)) and nucleosome- accommodating C/G-enrichment downstream (p < 10(-40)) that was highly conserved from an ancestor. A substantial genetic distance between the strains facilitated the in-depth analysis of patterns of fixed mutations, revealing a localization-specific equilibrium between the rates of distinct mutation categories that would serve to maintain the conserved sequence anisotropy around TSSs. Downstream of nucleocyclic TSSs, C to T, T to C, and other mutation rates on the sense strand increased around first nucleosome dyads and decreased around first linkers, which contrasted with genomewide mutational patterns around nucleosomes (p < 5 %). C to T rates are higher than G to A rates around nucleosome associated with germline nucleocyclic TSS sites (p < 5 %), potentially due to the asymmetric effect of transcription-coupled repair.Our results demonstrate an atypical evolutionary process surrounding nucleocyclic TSSs.
Functional relevance of "seed" and "non-seed" sequences in microRNA-mediated promotion of C. elegans developmental progression RNA Zhang, H., Artiles, K. L., Fire, A. Z. 2015; 21 (11): 1980-1992
The founding heterochronic microRNAs, lin-4 and let-7, together with their validated targets and well-characterized phenotypes in C. elegans, offer an opportunity to test functionality of microRNAs in a developmental context. In this study, we defined sequence requirements at the microRNA level for these two microRNAs, evaluating lin-4 and let-7 mutant microRNAs for their ability to support temporal development under conditions where the wild-type lin-4 and let-7 gene products are absent. For lin-4, we found a strong requirement for seed sequences, with function drastically affected by several central mutations in the seed sequence, while rescue was retained by a set of mutations peripheral to the seed. let-7 rescuing activity was retained to a surprising degree by a variety of central seed mutations, while several non-seed mutant effects support potential noncanonical contributions to let-7 function. Taken together, this work illustrates both the functional partnership between seed and non-seed sequences in mediating C. elegans temporal development and a diversity among microRNA effectors in the contributions of seed and non-seed regions to activity.
Landscape of target:guide homology effects on Cas9-mediated cleavage. Nucleic acids research Fu, B. X., Hansen, L. L., Artiles, K. L., Nonet, M. L., Fire, A. Z. 2014; 42 (22): 13778-13787
To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9::guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in 'seed' sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide::target duplex (positions 13-18 relative to the PAM). Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage. In addition, the first-base Guanine constraint commonly imposed by gRNA expression systems has little effect on overall cleavage efficiency. Taken together, these studies provide an in vitro understanding of the complexities of Cas9-gRNA interaction and cleavage beyond the general paradigm of site determination based on the 'seed' sequence and PAM.
View details for DOI 10.1093/nar/gku1102
Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans GENETICS Arribere, J. A., Bell, R. T., Fu, B. X., Artiles, K. L., Hartman, P. S., Fire, A. Z. 2014; 198 (3): 837-U842
Facilitated by recent advances using CRISPR/Cas9, genome editing technologies now permit custom genetic modifications in a wide variety of organisms. Ideally, modified animals could be both efficiently made and easily identified with minimal initial screening and without introducing exogenous sequence at the locus of interest or marker mutations elsewhere. To this end, we describe a coconversion strategy, using CRISPR/Cas9 in which screening for a dominant phenotypic oligonucleotide-templated conversion event at one locus can be used to enrich for custom modifications at another unlinked locus. After the desired mutation is identified among the F1 progeny heterozygous for the dominant marker mutation, F2 animals that have lost the marker mutation are picked to obtain the desired mutation in an unmarked genetic background. We have developed such a coconversion strategy for Caenorhabditis elegans, using a number of dominant phenotypic markers. Examining the coconversion at a second (unselected) locus of interest in the marked F1 animals, we observed that 14-84% of screened animals showed homologous recombination. By reconstituting the unmarked background through segregation of the dominant marker mutation at each step, we show that custom modification events can be carried out recursively, enabling multiple mutant animals to be made. While our initial choice of a coconversion marker [rol-6(su1006)] was readily applicable in a single round of coconversion, the genetic properties of this locus were not optimal in that CRISPR-mediated deletion mutations at the unselected rol-6 locus can render a fraction of coconverted strains recalcitrant to further rounds of similar mutagenesis. An optimal marker in this sense would provide phenotypic distinctions between the desired mutant/+ class and alternative +/+, mutant/null, null/null, and null/+ genotypes. Reviewing dominant alleles from classical C. elegans genetics, we identified one mutation in dpy-10 and one mutation in sqt-1 that meet these criteria and demonstrate that these too can be used as effective conversion markers. Coconversion was observed using a variety of donor molecules at the second (unselected) locus, including oligonucleotides, PCR products, and plasmids. We note that the coconversion approach described here could be applied in any of the variety of systems where suitable coconversion markers can be identified from previous intensive genetic analyses of gain-of-function alleles.
Immunoglobulin Gene Insertions and Deletions in the Affinity Maturation of HIV-1 Broadly Reactive Neutralizing Antibodies CELL HOST & MICROBE Kepler, T. B., Liao, H., Alam, S. M., Bhaskarabhatla, R., Zhang, R., Yandava, C., Stewart, S., Anasti, K., Kelsoe, G., Parks, R., Lloyd, K. E., Stolarchuk, C., Pritchett, J., Solomon, E., Friberg, E., Morris, L., Karim, S. S., Cohen, M. S., Walter, E., Moody, M. A., Wu, X., Altae-Tran, H. R., Georgiev, I. S., Kwong, P. D., Boyd, S. D., Fire, A. Z., Mascola, J. R., Haynes, B. F. 2014; 16 (3): 304-313
Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events.
View details for DOI 10.1016/j.chom.2014.08.006
Human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements. Cell host & microbe Jackson, K. J., Liu, Y., Roskin, K. M., Glanville, J., Hoh, R. A., Seo, K., Marshall, E. L., Gurley, T. C., Moody, M. A., Haynes, B. F., Walter, E. B., Liao, H., Albrecht, R. A., García-Sastre, A., Chaparro-Riggers, J., Rajpal, A., Pons, J., Simen, B. B., Hanczaruk, B., Dekker, C. L., Laserson, J., Koller, D., Davis, M. M., Fire, A. Z., Boyd, S. D. 2014; 16 (1): 105-114
B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual's secreted antibody response to the vaccine, and the expanded clones are enriched with those expressing influenza-specific monoclonal antibodies. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.
Effects of Aging, Cytomegalovirus Infection, and EBV Infection on Human B Cell Repertoires JOURNAL OF IMMUNOLOGY Wang, C., Liu, Y., Xu, L. T., Jackson, K. J., Roskin, K. M., Pham, T. D., Laserson, J., Marshall, E. L., Seo, K., Lee, J., Furman, D., Koller, D., Dekker, C. L., Davis, M. M., Fire, A. Z., Boyd, S. D. 2014; 192 (2): 603-611
Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by CMV is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or EBV infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of IGH gene rearrangements to study the BCR repertoires over two successive years in 27 individuals ranging in age from 20 to 89 y. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG Ig genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, whereas CMV infection correlates with the proportion of highly mutated Ab genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.
View details for DOI 10.4049/jimmunol.1301384
Gamete-Type Dependent Crossover Interference Levels in a Defined Region of Caenorhabditis elegans Chromosome V. G3 (Bethesda, Md.) Gabdank, I., Fire, A. Z. 2014; 4 (1): 117-120
In certain organisms, numbers of crossover events for any single chromosome are limited ("crossover interference") so that double crossover events are obtained at much lower frequencies than would be expected from the simple product of independent single-crossover events. We present a number of observations during which we examined interference over a large region of Caenorhabditis elegans chromosome V. Examining this region for multiple crossover events in heteroallelic configurations with limited dimorphism, we observed high levels of crossover interference in oocytes with only partial interference in spermatocytes.
A requirement for ERK-dependent Dicer phosphorylation in coordinating oocyte-to-embryo transition in C. elegans. Developmental cell Drake, M. n., Furuta, T. n., Suen, K. M., Gonzalez, G. n., Liu, B. n., Kalia, A. n., Ladbury, J. E., Fire, A. Z., Skeath, J. B., Arur, S. n. 2014; 31 (5): 614–28
Signaling pathways and small RNAs direct diverse cellular events, but few examples are known of defined signaling pathways directly regulating small RNA biogenesis. We show that ERK phosphorylates Dicer on two conserved residues in its RNase IIIb and double-stranded RNA (dsRNA)-binding domains and that phosphorylation of these residues is necessary and sufficient to trigger Dicer's nuclear translocation in worms, mice, and human cells. Phosphorylation of Dicer on either site inhibits Dicer function in the female germline and dampens small RNA repertoire. Our data demonstrate that ERK phosphorylates and inhibits Dicer during meiosis I for oogenesis to proceed normally in Caenorhabditis elegans and that this inhibition is released before fertilization for embryogenesis to proceed normally. The conserved Dicer residues, their phosphorylation by ERK, and the consequences of the resulting modifications implicate an ERK-Dicer nexus as a fundamental component of the oocyte-to-embryo transition and an underlying mechanism coupling extracellular cues to small RNA production.
Comprehensive whole-genome sequencing of an early-stage primary myelofibrosis patient defines low mutational burden and non-recurrent candidate genes. Haematologica Merker, J. D., Roskin, K. M., Ng, D., Pan, C., Fisk, D. G., King, J. J., Hoh, R., Stadler, M., Okumoto, L. M., Abidi, P., Hewitt, R., Jones, C. D., Gojenola, L., Clark, M. J., Zhang, B., Cherry, A. M., George, T. I., Snyder, M., Boyd, S. D., Zehnder, J. L., Fire, A. Z., Gotlib, J. 2013; 98 (11): 1689-1696
In order to identify novel somatic mutations associated with classic BCR/ABL1-negative myeloproliferative neoplasms, we performed high-coverage genome sequencing of DNA from peripheral blood granulocytes and cultured skin fibroblasts from a patient with MPL W515K-positive primary myelofibrosis. The primary myelofibrosis genome had a low somatic mutation rate, consistent with that observed in similar hematopoietic tumor genomes. Interfacing of whole-genome DNA sequence data with RNA expression data identified three somatic mutations of potential functional significance: a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; a 19-base pair deletion involving a potential regulatory region in the 5'-untranslated region of BRD2, implicated in transcriptional regulation and cell cycle control; and a non-synonymous point mutation in KIAA0355, an uncharacterized protein. Additional mutations in three genes (CAP2, SOX30, and MFRP) were also evident, albeit with no support for expression at the RNA level. Re-sequencing of these six genes in 178 patients with polycythemia vera, essential thrombocythemia, and myelofibrosis did not identify recurrent somatic mutations in these genes. Finally, we describe methods for reducing false-positive variant calls in the analysis of hematologic malignancies with a low somatic mutation rate. This trial is registered with ClinicalTrials.gov (NCT01108159).
View details for DOI 10.3324/haematol.2013.092379
Unusual DNA packaging characteristics in endoreduplicated Caenorhabditis elegans oocytes defined by in vivo accessibility to an endogenous nuclease activity EPIGENETICS & CHROMATIN Gu, S. G., Goszczynski, B., McGhee, J. D., Fire, A. Z. 2013; 6
Germ cells in animals are highly specialized to preserve the genome. A distinct set of chromatin structures must be properly established in germ cells to maintain cell fate and genome integrity. We describe DNA-surface interactions in activated Caenorhabditis elegans oocytes that are revealed through the activity of an endogenous nuclease ('endocleavage').Our analysis began with an unexpected observation that a majority (>50%) of DNA from ovulated but unfertilized C. elegans oocytes can be recovered in fragments of approximately 500 base pairs or shorter, cleaved at regular intervals (10 to 11 nt) along the DNA helix. In some areas of the genome, DNA cleavage patterns in these endoreduplicated oocytes appear consistent from cell-to-cell, indicating coherent rotational positioning of the DNA in chromatin. Particularly striking in this analysis are arrays of sensitive sites with a periodicity of approximately 10 bp that persist for several hundred base pairs of genomic DNA, longer than a single nucleosome core. Genomic regions with a strong bias toward a 10-nt periodic occurrence of A(n)/T(n) (so-called PATC regions) appear to exhibit a high degree of rotational constraint in endocleavage phasing, with a strong tendency for the periodic A(n)/T(n) sites to remain on the face of the helix protected from nuclease digestion.The present analysis provides evidence for an unusual structure in C. elegans oocytes in which genomic DNA and associated protein structures are coherently linked.
View details for DOI 10.1186/1756-8935-6-37
Conserved translatome remodeling in nematode species executing a shared developmental transition. PLoS genetics Stadler, M., Fire, A. 2013; 9 (10)
Nematodes of the genus Caenorhabditis enter a developmental diapause state after hatching in the absence of food. To better understand the relative contributions of distinct regulatory modalities to gene expression changes associated with this developmental transition, we characterized genome-wide changes in mRNA abundance and translational efficiency associated with L1 diapause exit in four species using ribosome profiling and mRNA-seq. We found a strong tendency for translational regulation and mRNA abundance processes to act synergistically, together effecting a dramatic remodeling of the gene expression program. While gene-specific differences were observed between species, overall translational dynamics were broadly and functionally conserved. A striking, conserved feature of the response was strong translational suppression of ribosomal protein production during L1 diapause, followed by activation upon resumed development. On a global scale, ribosome footprint abundance changes showed greater similarity between species than changes in mRNA abundance, illustrating a substantial and genome-wide contribution of translational regulation to evolutionary maintenance of stable gene expression.
View details for DOI 10.1371/journal.pgen.1003739
The transcription start site landscape of C. elegans GENOME RESEARCH Saito, T. L., Hashimoto, S., Gu, S. G., Morton, J. J., Stadler, M., Blumenthal, T., Fire, A., Morishita, S. 2013; 23 (8): 1348-1361
More than half of C. elegans pre-mRNAs lose their original 5' ends in a process termed "trans-splicing" in which the RNA extending from the transcription start site (TSS) to the site of trans-splicing of the primary transcript, termed the "outron", is replaced with a 22nt spliced leader. This complicates the mapping of TSSs, leading to a lack of available TSS mapping data for these genes. We used growth at low temperature and nuclear isolation to enrich for transcripts still containing outrons, applying a modified SAGE capture procedure and high throughput sequencing to characterize 5' termini in this transcript population. We report from this data both a landscape of 5' end utilization for C. elegans and a representative collection of TSSs for 7,351 trans-spliced genes. TSS distributions for individual genes were often dispersed, with a greater average number of TSSs for trans-spliced genes, suggesting that trans-splicing may remove selective pressure for a single TSS. Upstream of newly defined TSSs, we observed well-known motifs (including TATAA box and SP1) as well as novel motifs. A number of these motifs showed association with tissue-specific expression and/or conservation among six worm species. Comparing TSS features between trans-spliced and non-trans-spliced genes, we found stronger signals among outron TSSs for preferentially-positioning of flanking nucleosomes and for downstream Pol II enrichment. Our data provides an enabling resource for both experimental and theoretical analysis of gene structure and function in C. elegans.
Convergent antibody signatures in human dengue. Cell host & microbe Parameswaran, P., Liu, Y., Roskin, K. M., Jackson, K. K., Dixit, V. P., Lee, J., Artiles, K. L., Zompi, S., Vargas, M. J., Simen, B. B., Hanczaruk, B., McGowan, K. R., Tariq, M. A., Pourmand, N., Koller, D., Balmaseda, A., Boyd, S. D., Harris, E., Fire, A. Z. 2013; 13 (6): 691-700
Dengue is the most prevalent mosquito-borne viral disease in humans, and the lack of early prognostics, vaccines, and therapeutics contributes to immense disease burden. To identify patterns that could be used for sequence-based monitoring of the antibody response to dengue, we examined antibody heavy-chain gene rearrangements in longitudinal peripheral blood samples from 60 dengue patients. Comparing signatures between acute dengue, postrecovery, and healthy samples, we found increased expansion of B cell clones in acute dengue patients, with higher overall clonality in secondary infection. Additionally, we observed consistent antibody sequence features in acute dengue in the highly variable major antigen-binding determinant, complementarity-determining region 3 (CDR3), with specific CDR3 sequences highly enriched in acute samples compared to postrecovery, healthy, or non-dengue samples. Dengue thus provides a striking example of a human viral infection where convergent immune signatures can be identified in multiple individuals. Such signatures could facilitate surveillance of immunological memory in communities.
Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature Liao, H., Lynch, R., Zhou, T., Gao, F., Alam, S. M., Boyd, S. D., Fire, A. Z., Roskin, K. M., Schramm, C. A., Zhang, Z., Zhu, J., Shapiro, L., Mullikin, J. C., Gnanakaran, S., Hraber, P., Wiehe, K., Kelsoe, G., Yang, G., Xia, S., Montefiori, D. C., Parks, R., Lloyd, K. E., Scearce, R. M., Soderberg, K. A., Cohen, M., Kamanga, G., Louder, M. K., Tran, L. M., Chen, Y., Cai, F., Chen, S., Moquin, S., Du, X., Joyce, M. G., Srivatsan, S., Zhang, B., Zheng, A., Shaw, G. M., Hahn, B. H., Kepler, T. B., Korber, B. T., Kwong, P. D., Mascola, J. R., Haynes, B. F. 2013; 496 (7446): 469-476
Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.
Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level. Molecular therapy : the journal of the American Society of Gene Therapy Gracey Maniar, L. E., Maniar, J. M., Chen, Z., Lu, J., Fire, A. Z., Kay, M. A. 2013; 21 (1): 131-138
Current efforts in nonviral gene therapy are plagued by a pervasive difficulty in sustaining therapeutic levels of delivered transgenes. Minicircles (plasmid derivatives with the same expression cassette but lacking a bacterial backbone) show sustained expression and hold promise for therapeutic use where persistent transgene expression is required. To characterize the widely-observed silencing process affecting expression of foreign DNA in mammals, we used a system in which mouse liver presented with either plasmid or minicircle consistently silences plasmid but not minicircle expression. We found that preferential silencing of plasmid DNA occurs at a nuclear stage that precedes transport of mRNA to the cytoplasm, evident from a consistent >25-fold minicircle/plasmid transcript difference observed in both nuclear and total RNA. Among possible mechanisms of nuclear silencing, our data favor chromatin-linked transcriptional blockage rather than targeted degradation, aberrant processing, or compromised mRNA transport. In particular, we observe dramatic enrichment of H3K27 trimethylation on plasmid sequences. Also, it appears that Pol II can engage the modified plasmid chromatin, potentially in a manner that is not productive in the synthesis of high levels of new transcript. We outline a scenario in which sustained differences at the chromatin level cooperate to determine the activity of foreign DNA.
View details for DOI 10.1038/mt.2012.244
Contributions of mRNA abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C-elegans heterochronic miRNA targets GENOME RESEARCH Stadler, M., Artiles, K., Pak, J., Fire, A. 2012; 22 (12): 2418-2426
miRNAs are post-transcriptional regulators of gene activity that reduce protein accumulation from target mRNAs. Elucidating precise molecular effects that animal miRNAs have on target transcripts has proven complex, with varied evidence indicating that miRNA regulation may produce different molecular outcomes in different species, systems, and/or physiological conditions. Here we use high-throughput ribosome profiling to analyze detailed translational parameters for five well-studied targets of miRNAs that regulate C. elegans developmental timing. For two targets of the miRNA lin-4 (lin-14 and lin-28), functional down-regulation was associated with decreases in both overall mRNA abundance and ribosome loading; however, these changes were of substantially smaller magnitude than corresponding changes observed in protein abundance. For three functional targets of the let-7 miRNA family for which down-regulation is critical in temporal progression of the animal (daf-12, hbl-1, and lin-41), we observed only modest changes in mRNA abundance and ribosome loading. lin-41 provides a striking example in that populations of ribosome-protected fragments from this gene remained essentially unchanged during the L3-L4 time interval when lin-41 activity is substantially down-regulated by let-7. Spectra of ribosomal positions were also examined for the five lin-4 and let-7 target mRNAs as a function of developmental time, with no indication of miRNA-induced ribosomal drop-off or significant pauses in translation. These data are consistent with models in which physiological regulation by this set of C. elegans miRNAs derives from combinatorial effects including suppressed recruitment/activation of translational machinery, compromised stability of target messages, and post- or peri-translational effects on lifetimes of polypeptide products.
Whole Genome Sequence Analysis of Primary Myelofibrosis. 54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Merker, J. D., Roskin, K., Ng, D., Pan, C., Fisk, D. G., Jones, C. D., Gojenola, L., Clark, M. J., Zhang, B., Cherry, M., Snyder, M., Boyd, S. D., Zehnder, J. L., Fire, A. Z., Gotlib, J. AMER SOC HEMATOLOGY. 2012
Protection from Feed-Forward Amplification in an Amplified RNAi Mechanism CELL Pak, J., Maniar, J. M., Mello, C. C., Fire, A. 2012; 151 (4): 885-899
The effectiveness of RNA interference (RNAi) in many organisms is potentiated through the signal-amplifying activity of a targeted RNA-directed RNA polymerase (RdRP) system that can convert a small population of exogenously-encountered dsRNA fragments into an abundant internal pool of small interfering RNA (siRNA). As for any biological amplification system, we expect an underlying architecture that will limit the ability of a randomly encountered trigger to produce an uncontrolled and self-escalating response. Investigating such limits in Caenorhabditis elegans, we find that feed-forward amplification is limited by biosynthetic and structural distinctions at the RNA level between (1) triggers that can produce amplification and (2) siRNA products of the amplification reaction. By assuring that initial (primary) siRNAs can act as triggers but not templates for activation, and that the resulting (secondary) siRNAs can enforce gene silencing on additional targets without unbridled trigger amplification, the system achieves substantial but fundamentally limited signal amplification.
The Extragenic Spacer Length Between the 5 ' and 3 ' Ends of the Transgene Expression Cassette Affects Transgene Silencing From Plasmid-based Vectors MOLECULAR THERAPY Lu, J., Zhang, F., Xu, S., Fire, A. Z., Kay, M. A. 2012; 20 (11): 2111-2119
In quiescent tissues, minicircle DNA vectors provide at least 10 times higher sustained levels of transgene expression compared to that achieved with a canonical plasmid containing the same expression cassette. It is not known if there is a specific DNA sequence or structure that is needed for DNA silencing. To directly address this question, we substituted the bacterial plasmid DNA with various lengths of extragenic spacer DNAs between the 5' and 3' ends of the transgene expression cassette and determined the expression profiles using two different reporter expression cassettes. Both the human alphoid repeat (AR) and randomly generated DNA sequences of ≥1 kb in length resulted in transgene silencing while shorter spacers, ≤500 bp exhibited similar transgene expression patterns to conventional minicircle DNA vectors. In contrast, when the ≥1 kb random DNA (RD) sequences were expressed as part of the 3'-untranslated region (UTR) transgene silencing was not observed. These data suggest that the length and not the sequence or origin of the extragenic DNA flanking the expression cassette is responsible for plasmid-mediated transgene silencing. This has implications for the design of nonviral vectors for gene transfer applications as well as providing insights into how genes are regulated.
View details for DOI 10.1038/mt.2012.65
A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality NATURE Buckley, B. A., Burkhart, K. B., Gu, S. G., Spracklin, G., Kershner, A., Fritz, H., Kimble, J., Fire, A., Kennedy, S. 2012; 489 (7416): 447-451
Epigenetic information is frequently erased near the start of each new generation. In some cases, however, epigenetic information can be transmitted from parent to progeny (multigenerational epigenetic inheritance). A particularly notable example of this type of epigenetic inheritance is double-stranded RNA-mediated gene silencing in Caenorhabditis elegans. This RNA-mediated interference (RNAi) can be inherited for more than five generations. To understand this process, here we conduct a genetic screen for nematodes defective in transmitting RNAi silencing signals to future generations. This screen identified the heritable RNAi defective 1 (hrde-1) gene. hrde-1 encodes an Argonaute protein that associates with small interfering RNAs in the germ cells of progeny of animals exposed to double-stranded RNA. In the nuclei of these germ cells, HRDE-1 engages the nuclear RNAi defective pathway to direct the trimethylation of histone H3 at Lys 9 (H3K9me3) at RNAi-targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed short interfering RNAs, which direct nuclear gene silencing in germ cells. In hrde-1- or nuclear RNAi-deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Argonaute protein HRDE-1 directs gene-silencing events in germ-cell nuclei that drive multigenerational RNAi inheritance and promote immortality of the germ-cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.
Amplification of siRNA in Caenorhabditis elegans generates a transgenerational sequence-targeted histone H3 lysine 9 methylation footprint NATURE GENETICS Gu, S. G., Pak, J., Guang, S., Maniar, J. M., Kennedy, S., Fire, A. 2012; 44 (2): 157-164
Exogenous double-stranded RNA (dsRNA) has been shown to exert homology-dependent effects at the level of both target mRNA stability and chromatin structure. Using C. elegans undergoing RNAi as an animal model, we have investigated the generality, scope and longevity of dsRNA-targeted chromatin effects and their dependence on components of the RNAi machinery. Using high-resolution genome-wide chromatin profiling, we found that a diverse set of genes can be induced to acquire locus-specific enrichment of histone H3 lysine 9 trimethylation (H3K9me3), with modification footprints extending several kilobases from the site of dsRNA homology and with locus specificity sufficient to distinguish the targeted locus from the other 20,000 genes in the C. elegans genome. Genetic analysis of the response indicated that factors responsible for secondary siRNA production during RNAi were required for effective targeting of chromatin. Temporal analysis revealed that H3K9me3, once triggered by dsRNA, can be maintained in the absence of dsRNA for at least two generations before being lost. These results implicate dsRNA-triggered chromatin modification in C. elegans as a programmable and locus-specific response defining a metastable state that can persist through generational boundaries.
View details for DOI 10.1038/ng.1039
The Inference of Phased Haplotypes for the Immunoglobulin H Chain V Region Gene Loci by Analysis of VDJ Gene Rearrangements JOURNAL OF IMMUNOLOGY Kidd, M. J., Chen, Z., Wang, Y., Jackson, K. J., Zhang, L., Boyd, S. D., Fire, A. Z., Tanaka, M. M., Gaeta, B. A., Collins, A. M. 2012; 188 (3): 1333-1340
The existence of many highly similar genes in the lymphocyte receptor gene loci makes them difficult to investigate, and the determination of phased "haplotypes" has been particularly problematic. However, V(D)J gene rearrangements provide an opportunity to infer the association of Ig genes along the chromosomes. The chromosomal distribution of H chain genes in an Ig genotype can be inferred through analysis of VDJ rearrangements in individuals who are heterozygous at points within the IGH locus. We analyzed VDJ rearrangements from 44 individuals for whom sufficient unique rearrangements were available to allow comprehensive genotyping. Nine individuals were identified who were heterozygous at the IGHJ6 locus and for whom sufficient suitable VDJ rearrangements were available to allow comprehensive haplotyping. Each of the 18 resulting IGHV│IGHD│IGHJ haplotypes was unique. Apparent deletion polymorphisms were seen that involved as many as four contiguous, functional IGHV genes. Two deletion polymorphisms involving multiple contiguous IGHD genes were also inferred. Three previously unidentified gene duplications were detected, where two sequences recognized as allelic variants of a single gene were both inferred to be on a single chromosome. Phased genomic data brings clarity to the study of the contribution of each gene to the available repertoire of rearranged VDJ genes. Analysis of rearrangement frequencies suggests that particular genes may have substantially different yet predictable propensities for rearrangement within different haplotypes. Together with data highlighting the extent of haplotypic variation within the population, this suggests that there may be substantial variability in the available Ab repertoires of different individuals.
High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Logan, A. C., Gao, H., Wang, C., Sahaf, B., Jones, C. D., Marshall, E. L., Buno, I., Armstrong, R., Fire, A. Z., Weinberg, K. I., Mindrinos, M., Zehnder, J. L., Boyd, S. D., Xiao, W., Davis, R. W., Miklos, D. B. 2011; 108 (52): 21194-21199
The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10(-5), with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.
View details for DOI 10.1073/pnas.1118357109
Wobble base-pairing slows in vivo translation elongation in metazoans RNA-A PUBLICATION OF THE RNA SOCIETY Stadler, M., Fire, A. 2011; 17 (12): 2063-2073
In the universal genetic code, most amino acids can be encoded by multiple trinucleotide codons, and the choice among available codons can influence position-specific translation elongation rates. By using sequence-based ribosome profiling, we obtained transcriptome-wide profiles of in vivo ribosome occupancy as a function of codon identity in Caenorhabditis elegans and human cells. Particularly striking in these profiles was a universal trend of higher ribosome occupancy for codons translated via G:U wobble base-pairing compared with synonymous codons that pair with the same tRNA family using G:C base-pairing. These data support a model in which ribosomal translocation is slowed at wobble codon positions.
View details for DOI 10.1261/rna.02890211
Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated JOURNAL OF EXPERIMENTAL MEDICINE Liao, H., Chen, X., Munshaw, S., Zhang, R., Marshall, D. J., Vandergrift, N., Whitesides, J. F., Lu, X., Yu, J., Hwang, K., Gao, F., Markowitz, M., Heath, S. L., Bar, K. J., Goepfert, P. A., Montefiori, D. C., Shaw, G. C., Alam, S. M., Margolis, D. M., Denny, T. N., Boyd, S. D., Marshal, E., Egholm, M., Simen, B. B., Hanczaruk, B., Fire, A. Z., Voss, G., Kelsoe, G., Tomaras, G. D., Moody, M. A., Kepler, T. B., Haynes, B. F. 2011; 208 (11): 2237-2249
The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non-HIV-1 antigens.
View details for DOI 10.1084/jem.20110363
Competition between ADAR and RNAi pathways for an extensive class of RNA targets NATURE STRUCTURAL & MOLECULAR BIOLOGY Wu, D., Lamm, A. T., Fire, A. Z. 2011; 18 (10): 1094-U24
Adenosine deaminases that act on RNAs (ADARs) interact with double-stranded RNAs, deaminating adenosines to inosines. Previous studies of Caenorhabditis elegans indicated an antagonistic interaction between ADAR and RNAi machineries, with ADAR defects suppressed upon additional knockout of RNAi. This suggests a pool of common RNA substrates capable of engaging both pathways. To define and characterize such substrates, we examined small RNA and mRNA populations of ADAR mutants and identified a distinct set of loci from which RNAi-dependent short RNAs are markedly upregulated. At these same loci, we observed populations of multiply edited transcripts, supporting a specific role for ADARs in preventing access to the RNAi pathway for an extensive population of dsRNAs. Characterization of these loci revealed a substantial overlap with noncoding and intergenic regions, suggesting that the landscape of ADAR targets may extend beyond previously annotated classes of transcripts.
View details for DOI 10.1038/nsmb.2129
'Inc-miRs': functional intron-interrupted miRNA genes GENES & DEVELOPMENT Zhang, H., Maniar, J. M., Fire, A. Z. 2011; 25 (15): 1589-1594
The discovery of microRNAs (miRNAs) lin-4 and let-7 as temporal regulators in Caenorhabditis elegans led to broader searches for novel miRNAs and their biological roles. Unlike protein-coding genes and some long noncoding RNAs, canonical metazoan miRNAs are not known to contain introns within their genomic precursor sequences. Because the short length of miRNAs complicates a statistically definitive assignment of split genes in RNA sequencing data sets, we took an experimental approach toward testing the compatibility of splicing and functional miRNA biogenesis. To definitively evaluate the possibility that miRNAs could derive from interrupted genes, we constructed intron-interrupted variants of C. elegans lin-4 and assayed for their miRNA-encoding capability and biological activity in the developing organism. Our studies indicate that (1) intron-containing miRNAs (inc-miRs) can be efficiently spliced and processed to produce miRNAs with normal termini, and (2) these miRNAs can be functional in full rescue of developmental phenotypes in null mutants lacking endogenous lin-4. This study provides the first evidence to support the ability of intron-interrupted miRNA precursors to produce functional regulators and identifies an additional modality available for metazoan miRNA production.
View details for DOI 10.1101/gad.2058711
Determinants of nucleosome organization in primary human cells NATURE Valouev, A., Johnson, S. M., Boyd, S. D., Smith, C. L., Fire, A. Z., Sidow, A. 2011; 474 (7352): 516-U148
Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.
Up-Regulated Dicer Expression in Patients with Cutaneous Melanoma PLOS ONE Ma, Z., Swede, H., Cassarino, D., Fleming, E., Fire, A., Dadras, S. S. 2011; 6 (6)
MicroRNAs (miRNAs) are small non-coding RNAs (18-24 nucleotides) that have recently been shown to regulate gene expression during cancer progression. Dicer, a central enzyme in the multi-component miRNA biogenesis pathway, is involved in cutting precursor miRNAs to functionally mature forms. Emerging evidence shows that Dicer expression is deregulated in some human malignancies and it correlates with tumor progression, yet this role has not yet been investigated in skin cancers.Using an anti-human monoclonal antibody against Dicer and immunohistochemistry, we compared the expression of Dicer protein among 404 clinically annotated controls and skin tumors consisting of melanocytic nevi (n = 71), a variety of melanomas (n = 223), carcinomas (n = 73) and sarcomas (n = 12). Results showed a cell-specific up-regulated Dicer in 81% of cutaneous, 80% of acrolentiginous and 96% of metastatic melanoma specimens compared to carcinoma or sarcoma specimens (P<0.0001). The expression of Dicer was significantly higher in melanomas compared to benign melanocytic nevi (P<0.0001). In patients with cutaneous melanomas, Dicer up-regulation was found to be significantly associated with an increased tumor mitotic index (P = 0.04), Breslow's depth of invasion (P = 0.03), nodal metastasis (P = 0.04) and a higher American Joint Committee on Caner (AJCC) clinical stage (P = 0.009). Using western blot analysis, we confirmed the cell-specific up-regulation of Dicer protein in vitro. A pooled-analysis on mRNA profiling in cutaneous tumors showed up-regulation of Dicer at the RNA level in cutaneous melanoma, also showing deregulation of other enzymes that participate in the biogenesis and maturation of canonical miRNAs.Increased Dicer expression may be a clinically useful biomarker for patients with cutaneous melanoma. Understanding deregulation of Dicer and its influence on miRNA maturation is needed to predict the susceptibility of melanoma patients to miRNA-based therapy in the future.
View details for DOI 10.1371/journal.pone.0020494
On the nature of in vivo requirements for rde-4 in RNAi and developmental pathways in C-elegans RNA BIOLOGY Blanchard, D., Parameswaran, P., Lopez-Molina, J., Gent, J., Saynuk, J. F., Fire, A. 2011; 8 (3): 458-467
C. elegans RDE-4 is a double-stranded RNA binding protein that has been shown to play a key role in response to foreign double-stranded RNA (dsRNA). We have used diverse tools for analysis of gene function to characterize the domain and organismal foci of RDE-4 action in C. elegans. First, we examined the focus of activity within the RDE-4 protein, by testing a series of RDE-4 deletion constructs for their ability to support dsRNA-triggered gene silencing. These assays indicated a molecular requirement for a linker region and the second dsRNA-binding domain of RDE-4, with ancillary contributions to function from the C and N terminal domains. Second, we used mosaic analysis to explore the cellular focus of action of RDE-4. These experiments indicated an ability of RDE-4 to function non-autonomously in foreign RNA responses. Third, we used growth under stressful conditions to search for evidence of an organismal focus of action for RDE-4 distinct from its role in response to foreign dsRNA. Propagation at high temperatures exposed a conditional requirement for RDE-4 for optimal growth and fertility, indicating at least under these conditions that RDE-4 can serve an essential role in C. elegans.
View details for DOI 10.4161/rna.8.3.14657
EGO-1, a C. elegans RdRP, Modulates Gene Expression via Production of mRNA-Templated Short Antisense RNAs CURRENT BIOLOGY Maniar, J. M., Fire, A. Z. 2011; 21 (6): 449-459
The development of the germline in Caenorhabditis elegans is a complex process involving the regulation of thousands of genes in a coordinated manner. Several genes required for small RNA biogenesis and function are among those required for the proper organization of the germline. EGO-1 is a putative RNA-directed RNA polymerase (RdRP) that is required for multiple aspects of C. elegans germline development and efficient RNA interference (RNAi) of germline-expressed genes. RdRPs have been proposed to act through a variety of mechanisms, including the posttranscriptional targeting of specific mRNAs, as well as through a direct interaction with chromatin. Despite extensive investigation, the molecular role of EGO-1 has remained enigmatic.Here we use high-throughput small RNA and messenger RNA sequencing to investigate EGO-1 function. We found that EGO-1 is required to produce a distinct pool of small RNAs antisense to a number of germline-expressed mRNAs through several developmental stages. These potential mRNA targets fall into distinct classes, including genes required for kinetochore and nuclear pore assembly, histone-modifying activities, and centromeric proteins. We also found several RNAi-related genes to be targets of EGO-1. Finally, we show a strong association between the loss of small RNAs and the rise of mRNA levels in ego-1(-) animals.Our data support the conclusion that EGO-1 produces triphosphorylated small RNAs derived from mRNA templates and that these small RNAs modulate gene expression through the targeting of their cognate mRNAs.
Multimodal RNA-seq using single-strand, double-strand, and CircLigase-based capture yields a refined and extended description of the C. elegans transcriptome GENOME RESEARCH Lamm, A. T., Stadler, M. R., Zhang, H., Gent, J. I., Fire, A. Z. 2011; 21 (2): 265-275
We have used a combination of three high-throughput RNA capture and sequencing methods to refine and augment the transcriptome map of a well-studied genetic model, Caenorhabditis elegans. The three methods include a standard (non-directional) library preparation protocol relying on cDNA priming and foldback that has been used in several previous studies for transcriptome characterization in this species, and two directional protocols, one involving direct capture of single-stranded RNA fragments and one involving circular-template PCR (CircLigase). We find that each RNA-seq approach shows specific limitations and biases, with the application of multiple methods providing a more complete map than was obtained from any single method. Of particular note in the analysis were substantial advantages of CircLigase-based and ssRNA-based capture for defining sequences and structures of the precise 5' ends (which were lost using the double-strand cDNA capture method). Of the three methods, ssRNA capture was most effective in defining sequences to the poly(A) junction. Using data sets from a spectrum of C. elegans strains and stages and the UCSC Genome Browser, we provide a series of tools, which facilitate rapid visualization and assignment of gene structures.
Cell autonomous specification of temporal identity by Caenorhabditis elegans microRNA lin-4 DEVELOPMENTAL BIOLOGY Zhang, H., Fire, A. Z. 2010; 344 (2): 603-610
MicroRNAs provide developing systems with substantial flexibility in posttranscriptional gene regulation. Despite advances made in understanding microRNA structure and function, the relationships between their site-of-synthesis and site-of-action ("autonomy" versus "non-autonomy") remain an open question. Given the well-defined role of microRNA lin-4 in a reproducible series of time-specific developmental switches, lin-4 is an excellent candidate for understanding whether microRNAs and the resulting heterochronic regulatory pathway have the potential to act cell autonomously. By monitoring temporal development and reporter activity in animals where lin-4 is modulated, we have demonstrated that lin-4 acts cell autonomously to specify temporal identity. This work (i) provides an example of cell autonomy in microRNA functions, and (ii) reveals a cell autonomous component of temporal regulation in C. elegans.
View details for DOI 10.1016/j.ydbio.2010.05.018
Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans BMC GENOMICS Sha, K., Gu, S. G., Pantalena-Filho, L. C., Goh, A., Fleenor, J., Blanchard, D., Krishna, C., Fire, A. 2010; 11
Tissue differentiation is accompanied by genome-wide changes in the underlying chromatin structure and dynamics, or epigenome. By controlling when, where, and what regulatory factors have access to the underlying genomic DNA, the epigenome influences the cell's transcriptome and ultimately its function. Existing genomic methods for analyzing cell-type-specific changes in chromatin generally involve two elements: (i) a source for purified cells (or nuclei) of distinct types, and (ii) a specific treatment that partitions or degrades chromatin by activity or structural features. For many cell types of great interest, such assays are limited by our inability to isolate the relevant cell populations in an organism or complex tissue containing an intertwined mixture of other cells. This limitation has confined available knowledge of chromatin dynamics to a narrow range of biological systems (cell types that can be sorted/separated/dissected in large numbers and tissue culture models) or to amalgamations of diverse cell types (tissue chunks, whole organisms).Transgene-driven expression of DNA/chromatin modifying enzymes provides one opportunity to query chromatin structures in expression-defined cell subsets. In this work we combine in vivo expression of a bacterial DNA adenine methyltransferase (DAM) with high throughput sequencing to sample tissue-specific chromatin accessibility on a genome-wide scale. We have applied the method (DALEC: Direct Asymmetric Ligation End Capture) towards mapping a cell-type-specific view of genome accessibility as a function of differentiated state. Taking advantage of C. elegans strains expressing the DAM enzyme in diverse tissues (body wall muscle, gut, and hypodermis), our efforts yield a genome-wide dataset measuring chromatin accessibility at each of 538,000 DAM target sites in the C. elegans (diploid) genome.Validating the DALEC mapping results, we observe a strong association between observed coverage by nucleosomes and low DAM accessibility. Strikingly, we observed no extended regions of inaccessible chromatin for any of the tissues examined. These results are consistent with "local choreography" models in which differential gene expression is driven by intricate local rearrangements of chromatin structure rather than gross impenetrability of large chromosomal regions.
View details for DOI 10.1186/1471-2164-11-465
An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo EPIGENETICS & CHROMATIN Gracey, L. E., Chen, Z., Maniar, J. M., Valouev, A., Sidow, A., Kay, M. A., Fire, A. Z. 2010; 3
The physiological function of eukaryotic DNA occurs in the context of nucleosomal arrays that can expose or obscure defined segments of the genome. Certain DNA sequences are capable of strongly positioning a nucleosome in vitro, suggesting the possibility that favorable intrinsic signals might reproducibly structure chromatin segments. As high-throughput sequencing analyses of nucleosome coverage in vitro and in vivo have become possible, a vigorous debate has arisen over the degree to which intrinsic DNA:nucleosome affinities orchestrate the in vivo positions of nucleosomes, thereby controlling physical accessibility of specific sequences in DNA.We describe here the in vivo consequences of placing a synthetic high-affinity nucleosome-positioning signal, the 601 sequence, into a DNA plasmid vector in mice. Strikingly, the 601 sequence was sufficient to position nucleosomes during an early phase after introduction of the DNA into the mice (when the plasmid vector transgene was active). This positioning capability was transient, with a loss of strong positioning at a later time point when the transgenes had become silent.These results demonstrate an ability of DNA sequences selected solely for nucleosome affinity to organize chromatin in vivo, and the ability of other mechanisms to overcome these interactions in a dynamic nuclear environment.
Individual Variation in the Germline Ig Gene Repertoire Inferred from Variable Region Gene Rearrangements JOURNAL OF IMMUNOLOGY Boyd, S. D., Gaeta, B. A., Jackson, K. J., Fire, A. Z., Marshall, E. L., Merker, J. D., Maniar, J. M., Zhang, L. N., Sahaf, B., Jones, C. D., Simen, B. B., Hanczaruk, B., Nguyen, K. D., Nadeau, K. C., Egholm, M., Miklos, D. B., Zehnder, J. L., Collins, A. M. 2010; 184 (12): 6986-6992
Individual variation in the Ig germline gene repertoire leads to individual differences in the combinatorial diversity of the Ab repertoire, but the study of such variation has been problematic. The application of high-throughput DNA sequencing to the study of rearranged Ig genes now makes this possible. The sequencing of thousands of VDJ rearrangements from an individual, either from genomic DNA or expressed mRNA, should allow their germline IGHV, IGHD, and IGHJ repertoires to be inferred. In addition, where previously mere glimpses of diversity could be gained from sequencing studies, new large data sets should allow the rearrangement frequency of different genes and alleles to be seen with clarity. We analyzed the DNA of 108,210 human IgH chain rearrangements from 12 individuals and determined their individual IGH genotypes. The number of reportedly functional IGHV genes and allelic variants ranged from 45 to 60, principally because of variable levels of gene heterozygosity, and included 14 previously unreported IGHV polymorphisms. New polymorphisms of the IGHD3-16 and IGHJ6 genes were also seen. At heterozygous loci, remarkably different rearrangement frequencies were seen for the various IGHV alleles, and these frequencies were consistent between individuals. The specific alleles that make up an individual's Ig genotype may therefore be critical in shaping the combinatorial repertoire. The extent of genotypic variation between individuals is highlighted by an individual with aplastic anemia who appears to lack six contiguous IGHD genes on both chromosomes. These deletions significantly alter the potential expressed IGH repertoire, and possibly immune function, in this individual.
Ultra-high throughput sequencing-based small RNA discovery and discrete statistical biomarker analysis in a collection of cervical tumours and matched controls BMC BIOLOGY Witten, D., Tibshirani, R., Gu, S. G., Fire, A., Lui, W. 2010; 8
Ultra-high throughput sequencing technologies provide opportunities both for discovery of novel molecular species and for detailed comparisons of gene expression patterns. Small RNA populations are particularly well suited to this analysis, as many different small RNAs can be completely sequenced in a single instrument run.We prepared small RNA libraries from 29 tumour/normal pairs of human cervical tissue samples. Analysis of the resulting sequences (42 million in total) defined 64 new human microRNA (miRNA) genes. Both arms of the hairpin precursor were observed in twenty-three of the newly identified miRNA candidates. We tested several computational approaches for the analysis of class differences between high throughput sequencing datasets and describe a novel application of a log linear model that has provided the most effective analysis for this data. This method resulted in the identification of 67 miRNAs that were differentially-expressed between the tumour and normal samples at a false discovery rate less than 0.001.This approach can potentially be applied to any kind of RNA sequencing data for analysing differential sequence representation between biological sample sets.
Human tRNA-derived small RNAs in the global regulation of RNA silencing RNA-A PUBLICATION OF THE RNA SOCIETY Haussecker, D., Huang, Y., Lau, A., Parameswaran, P., Fire, A. Z., Kay, M. A. 2010; 16 (4): 673-695
Competition between mammalian RNAi-related gene silencing pathways is well documented. It is therefore important to identify all classes of small RNAs to determine their relationship with RNAi and how they affect each other functionally. Here, we identify two types of 5'-phosphate, 3'-hydroxylated human tRNA-derived small RNAs (tsRNAs). tsRNAs differ from microRNAs in being essentially restricted to the cytoplasm and in associating with Argonaute proteins, but not MOV10. The first type belongs to a previously predicted Dicer-dependent class of small RNAs that we find can modestly down-regulate target genes in trans. The 5' end of type II tsRNA was generated by RNaseZ cleavage downstream from a tRNA gene, while the 3' end resulted from transcription termination by RNA polymerase III. Consistent with their preferential association with the nonslicing Argonautes 3 and 4, canonical gene silencing activity was not observed for type II tsRNAs. The addition, however, of an oligonucleotide that was sense to the reporter gene, but antisense to an overexpressed version of the type II tsRNA, triggered robust, >80% gene silencing. This correlated with the redirection of the thus reconstituted fully duplexed double-stranded RNA into Argonaute 2, whereas Argonautes 3 and 4 were skewed toward less structured small RNAs, particularly single-strand RNAs. We observed that the modulation of tsRNA levels had minor effects on the abundance of microRNAs, but more pronounced changes in the silencing activities of both microRNAs and siRNAs. These findings support that tsRNAs are involved in the global control of small RNA silencing through differential Argonaute association, suggesting that small RNA-mediated gene regulation may be even more finely regulated than previously realized.
View details for DOI 10.1261/rna.2000810
Distinct Phases of siRNA Synthesis in an Endogenous RNAi Pathway in C. elegans Soma MOLECULAR CELL Gent, J. I., Lamm, A. T., Pavelec, D. M., Maniar, J. M., Parameswaran, P., Tao, L., Kennedy, S., Fire, A. Z. 2010; 37 (5): 679-689
Endogenous RNA-directed RNA polymerases (RdRPs) are cellular components capable of synthesizing new complementary RNAs from existing RNA templates. We present evidence for successive engagement of two different RdRPs in an endogenous siRNA-based mechanism targeting specific mRNAs in C. elegans soma. In the initiation stage of this process, a group of mRNA species are chosen as targets for downregulation, leading to accumulation of rare 26 nt 5'-phosphorylated antisense RNAs that depend on the RdRP homolog RRF-3, the Argonaute ERGO-1, DICER, and a series of associated ("ERI") factors. This primary process leads to production of a much more abundant class of 22 nt antisense RNAs, dependent on a secondary RdRP (RRF-1) and associating with at least one distinct Argonaute (NRDE-3). The requirement for two RdRP/Argonaute combinations and initiation by a rare class of uniquely structured siRNAs in this pathway illustrate the caution and flexibility used as biological systems exploit the physiological copying of RNA.
Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems PLOS PATHOGENS Parameswaran, P., Sklan, E., Wilkins, C., Burgon, T., Samuel, M. A., Lu, R., Ansel, K. M., Heissmeyer, V., Einav, S., Jackson, W., Doukas, T., Paranjape, S., Polacek, C., dos Santos, F. B., Jalili, R., Babrzadeh, F., Gharizadeh, B., Grimm, D., Kay, M., Koike, S., Sarnow, P., Ronaghi, M., Ding, S., Harris, E., Chow, M., Diamond, M. S., Kirkegaard, K., Glenn, J. S., Fire, A. Z. 2010; 6 (2)
We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.
View details for DOI 10.1371/journal.ppat.1000764
Partitioning the C. elegans genome by nucleosome modification, occupancy, and positioning CHROMOSOMA Guoping, S., Fire, A. 2010; 119 (1): 73-87
We have characterized two post-translational histone modifications in Caenorhabditis elegans on a genomic scale. Micrococcal nuclease digestion and immunoprecipitation were used to obtain distinct populations of single nucleosome cores, which were analyzed using massively parallel DNA sequencing to obtain positional and coverage maps. Two methylated histone H3 populations were chosen for comparison: H3K4 histone methylation (associated with active chromosomal regions) and H3K9 histone methylation (associated with inactivity). From analysis of the sequence data, we found nucleosome cores with these modifications to be enriched in two distinct partitions of the genome; H3K4 methylation was particularly prevalent in promoter regions of widely expressed genes, while H3K9 methylation was enriched on specific chromosomal arms. For each of the six chromosomes, the highest level of H3K9 methylation corresponds to the pairing center responsible for chromosome alignment during meiosis. Enrichment of H3K9 methylation at pairing centers appears to be an early mark in meiotic chromosome sorting, occurring in the absence of components required for proper pairing of homologous chromosomes. H3K9 methylation shows an intricate pattern within the chromosome arms with a particular anticorrelation to regions that display a strong approximately 10.5 bp periodicity of AA/TT dinucleotides that is known to associate with germline transcription. By contrast to the global features observed with H3K9 methylation, H3K4 methylation profiles were most striking in their local characteristics around promoters, providing a unique promoter-central landmark for 3,903 C. elegans genes and allowing a precise analysis of nucleosome positioning in the context of transcriptional initiation.
Measurement and Clinical Monitoring of Human Lymphocyte Clonality by Massively Parallel V-D-J Pyrosequencing SCIENCE TRANSLATIONAL MEDICINE Boyd, S. D., Marshall, E. L., Merker, J. D., Maniar, J. M., Zhang, L. N., Sahaf, B., Jones, C. D., Simen, B. B., Hanczaruk, B., Nguyen, K. D., Nadeau, K. C., Egholm, M., Miklos, D. B., Zehnder, J. L., Fire, A. Z. 2009; 1 (12)
The complex repertoire of immune receptors generated by B and T cells enables recognition of diverse threats to the host organism. In this work, we show that massively parallel DNA sequencing of rearranged immune receptor loci can provide direct detection and tracking of immune diversity and expanded clonal lymphocyte populations in physiological and pathological contexts. DNA was isolated from blood and tissue samples, a series of redundant primers was used to amplify diverse DNA rearrangements, and the resulting mixtures of barcoded amplicons were sequenced using long-read ultra deep sequencing. Individual DNA molecules were then characterized on the basis of DNA segments that had been joined to make a functional (or nonfunctional) immune effector. Current experimental designs can accommodate up to 150 samples in a single sequence run, with the depth of sequencing sufficient to identify stable and dynamic aspects of the immune repertoire in both normal and diseased circumstances. These data provide a high-resolution picture of immune spectra in normal individuals and in patients with hematological malignancies, illuminating, in the latter case, both the initial behavior of clonal tumor populations and the later suppression or re-emergence of such populations after treatment.
View details for DOI 10.1126/scitranslmed.3000540
A Caenorhabditis elegans RNA-Directed RNA Polymerase in Sperm Development and Endogenous RNA Interference GENETICS Gent, J. I., Schvarzstein, M., Villeneuve, A. M., Gu, S. G., Jantsch, V., Fire, A. Z., Baudrimont, A. 2009; 183 (4): 1297-1314
Short interfering RNAs (siRNAs) are a class of regulatory effectors that enforce gene silencing through formation of RNA duplexes. Although progress has been made in identifying the capabilities of siRNAs in silencing foreign RNA and transposable elements, siRNA functions in endogenous gene regulation have remained mysterious. In certain organisms, siRNA biosynthesis involves novel enzymes that act as RNA-directed RNA polymerases (RdRPs). Here we analyze the function of a Caenorhabditis elegans RdRP, RRF-3, during spermatogenesis. We found that loss of RRF-3 function resulted in pleiotropic defects in sperm development and that sperm defects led to embryonic lethality. Notably, sperm nuclei in mutants of either rrf-3 or another component of the siRNA pathway, eri-1, were frequently surrounded by ectopic microtubule structures, with spindle abnormalities in a subset of the resulting embryos. Through high-throughput small RNA sequencing, we identified a population of cellular mRNAs from spermatogenic cells that appear to serve as templates for antisense siRNA synthesis. This set of genes includes the majority of genes known to have enriched expression during spermatogenesis, as well as many genes not previously known to be expressed during spermatogenesis. In a subset of these genes, we found that RRF-3 was required for effective siRNA accumulation. These and other data suggest a working model in which a major role of the RRF-3/ERI pathway is to generate siRNAs that set patterns of gene expression through feedback repression of a set of critical targets during spermatogenesis.
Profiling and Discovery of Novel miRNAs from Formalin-Fixed, Paraffin-Embedded Melanoma and Nodal Specimens JOURNAL OF MOLECULAR DIAGNOSTICS Ma, Z., Lui, W., Fire, A., Dadras, S. S. 2009; 11 (5): 420-429
Archived formalin-fixed, paraffin-embedded human tumors are widely available and represent a unique source of morphologically defined material. Formalin-fixed, paraffin-embedded tissue is known to contain a wealth of molecular information in the form of microRNAs (miRNAs), which could be correlated with clinical outcome for improved prognostication and/or treatment response. miRNAs are endogenous, noncoding RNAs ( approximately 22 nucleotides) and may function as tumor suppressors or oncogenes. A reliable, robust methodology is needed to take full advantage of archived human cancers, especially for those where fresh-frozen tumor banks are unavailable, for example, malignant melanoma. To this end, we applied a simple-to-use protocol for extracting total RNA from various formalin-fixed, paraffin-embedded specimens (colon, liver, prostate, thyroid, uterus, and skin), optimized for small RNA recovery. Using a "poison primer" strategy (ie, primer silencing), we blocked the amplification of ribosomal RNA, enabling the successful sequencing of 17 novel and 53 known miRNAs (including small RNAs) from 10-year-old archived normal skin, cutaneous scalp melanoma, and sentinel lymph nodes (both negative and positive for metastasis) excised from a 52-year-old man. The cloning incidence provided an estimation of the level of specific miRNA expression, which was confirmed by Northern analysis and quantitative real-time polymerase chain reaction. This methodology can therefore be used to facilitate miRNA discovery from archived human cancers.
View details for DOI 10.2353/jmoldx.2009.090041
Caudal-like PAL-1 directly activates the bodywall muscle module regulator hlh-1 in C. elegans to initiate the embryonic muscle gene regulatory network DEVELOPMENT Lei, H., Liu, J., Fukushige, T., Fire, A., Krause, M. 2009; 136 (8): 1241-1249
Previous work in C. elegans has shown that posterior embryonic bodywall muscle lineages are regulated through a genetically defined transcriptional cascade that includes PAL-1/Caudal-mediated activation of muscle-specific transcription factors, including HLH-1/MRF and UNC-120/SRF, which together orchestrate specification and differentiation. Using chromatin immunoprecipitation (ChIP) in embryos, we now demonstrate direct binding of PAL-1 in vivo to an hlh-1 enhancer element. Through mutational analysis of the evolutionarily conserved sequences within this enhancer, we identify two cis-acting elements and their associated transacting factors (PAL-1 and HLH-1) that are crucial for the temporal-spatial expression of hlh-1 and proper myogenesis. Our data demonstrate that hlh-1 is indeed a direct target of PAL-1 in the posterior embryonic C. elegans muscle lineages, defining a novel in vivo binding site for this crucial developmental regulator. We find that the same enhancer element is also a target of HLH-1 positive auto regulation, underlying (at least in part) the sustained high levels of CeMyoD in bodywall muscle throughout development. Together, these results provide a molecular framework for the gene regulatory network activating the muscle module during embryogenesis.
Chromatin-Associated Periodicity in Genetic Variation Downstream of Transcriptional Start Sites SCIENCE Sasaki, S., Mello, C. C., Shimada, A., Nakatani, Y., Hashimoto, S., Ogawa, M., Matsushima, K., Gu, S. G., Kasahara, M., Ahsan, B., Sasaki, A., Saito, T., Suzuki, Y., Sugano, S., Kohara, Y., Takeda, H., Fire, A., Morishita, S. 2009; 323 (5912): 401-404
Might DNA sequence variation reflect germline genetic activity and underlying chromatin structure? We investigated this question using medaka (Japanese killifish, Oryzias latipes), by comparing the genomic sequences of two strains (Hd-rR and HNI) and by mapping approximately 37.3 million nucleosome cores from Hd-rR blastulae and 11,654 representative transcription start sites from six embryonic stages. We observed a distinctive approximately 200-base pair (bp) periodic pattern of genetic variation downstream of transcription start sites; the rate of insertions and deletions longer than 1 bp peaked at positions of approximately +200, +400, and +600 bp, whereas the point mutation rate showed corresponding valleys. This approximately 200-bp periodicity was correlated with the chromatin structure, with nucleosome occupancy minimized at positions 0, +200, +400, and +600 bp. These data exemplify the potential for genetic activity (transcription) and chromatin structure to contribute to molding the DNA sequence on an evolutionary time scale.
View details for DOI 10.1126/science.1163183
High-Throughput Sequencing for Diagnosis, Prognosis and Monitoring of Lymphoid Malignancies. 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Boyd, S. D., Merker, J. D., Zehnder, J. L., Fire, A. Z. AMER SOC HEMATOLOGY. 2008: 1294–94
Transmission Dynamics of Heritable Silencing Induced by Double-Stranded RNA in Caenorhabditis elegans GENETICS Alcazar, R. M., Lin, R., Fire, A. Z. 2008; 180 (3): 1275-1288
Heritable silencing effects are gene suppression phenomena that can persist for generations after induction. In the majority of RNAi experiments conducted in Caenorhabditis elegans, the silencing response results in a hypomorphic phenotype where the effects recede after the F1 generation. F2 and subsequent generations revert to the original phenotype. Specific examples of transgenerational RNAi in which effects persist to the F2 generation and beyond have been described. In this study, we describe a systematic pedigree-based analysis of heritable silencing processes resulting from initiation of interference targeted at the C. elegans oocyte maturation factor oma-1. Heritable silencing of oma-1 is a dose-dependent process where the inheritance of the silencing factor is unequally distributed among the population. Heritability is not constant over generational time, with silenced populations appearing to undergo a bottleneck three to four generations following microinjection of RNA. Transmission of silencing through these generations can be through either maternal or paternal gamete lines and is surprisingly more effective through the male gametic line. Genetic linkage tests reveal that silencing in the early generations is transmitted independently of the original targeted locus, in a manner indicative of a diffusible epigenetic element.
CED-9 and mitochondrial homeostasis in C-elegans muscle JOURNAL OF CELL SCIENCE Tan, F. J., Husain, M., Manlandro, C. M., Koppenol, M., Fire, A. Z., Hill, R. B. 2008; 121 (20): 3373-3382
Mitochondrial homeostasis reflects a dynamic balance between membrane fission and fusion events thought essential for mitochondrial function. We report here that altered expression of the C. elegans BCL2 homolog CED-9 affects both mitochondrial fission and fusion. Although striated muscle cells lacking CED-9 have no alteration in mitochondrial size or ultrastructure, these cells appear more sensitive to mitochondrial fragmentation. By contrast, increased CED-9 expression in these cells produces highly interconnected mitochondria. This mitochondrial phenotype is partially suppressed by increased expression of the dynamin-related GTPase DRP-1, with suppression dependent on the BH3 binding pocket of CED-9. This suppression suggests that CED-9 directly regulates DRP-1, a model supported by our finding that CED-9 activates the GTPase activity of human DRP1. Thus, CED-9 is capable of regulating the mitochondrial fission-fusion cycle but is not essential for either fission or fusion.
View details for DOI 10.1242/jcs.032904
Capped small RNAs and MOV10 in human hepatitis delta virus replication NATURE STRUCTURAL & MOLECULAR BIOLOGY Haussecker, D., Cao, D., Huang, Y., Parameswaran, P., Fire, A. Z., Kay, M. A. 2008; 15 (7): 714-721
The evolutionary origin of human hepatitis delta virus (HDV) replication by RNA-directed transcription is unclear. Here we identify two species of 5'-capped, approximately 18-25-nucleotide small RNAs. One was of antigenomic polarity, corresponding to the 5' end of hepatitis delta antigen (HDAg) mRNA, and interacted with HDAg and RNA polymerase II (Pol II), whereas the other mapped to a structurally analogous region on the genomic RNA hairpin. An HDAg-interaction screen indicated that HDAg interacts with MOV10, the human homolog of the Arabidopsis thaliana RNA amplification factor gene SDE3 and Drosophila melanogaster RISC-maturation factor gene Armitage (armi). MOV10 knockdown inhibited HDV replication, but not HDAg mRNA translation, further supporting a role for MOV10 in RNA-directed transcription. Together, our studies define RNA hairpins as critical elements for the initiation of HDV-related, RNA-directed transcription. The identification of capped small RNAs and the involvement of MOV10 in HDV replication further suggest a conserved mechanism related to RNA-directed transcription in lower eukaryotes.
A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning GENOME RESEARCH Valouev, A., Ichikawa, J., Tonthat, T., Stuart, J., Ranade, S., Peckham, H., Zeng, K., Malek, J. A., Costa, G., McKernan, K., Sidow, A., Fire, A., Johnson, S. M. 2008; 18 (7): 1051-1063
Using the massively parallel technique of sequencing by oligonucleotide ligation and detection (SOLiD; Applied Biosystems), we have assessed the in vivo positions of more than 44 million putative nucleosome cores in the multicellular genetic model organism Caenorhabditis elegans. These analyses provide a global view of the chromatin architecture of a multicellular animal at extremely high density and resolution. While we observe some degree of reproducible positioning throughout the genome in our mixed stage population of animals, we note that the major chromatin feature in the worm is a diversity of allowed nucleosome positions at the vast majority of individual loci. While absolute positioning of nucleosomes can vary substantially, relative positioning of nucleosomes (in a repeated array structure likely to be maintained at least in part by steric constraints) appears to be a significant property of chromatin structure. The high density of nucleosomal reads enabled a substantial extension of previous analysis describing the usage of individual oligonucleotide sequences along the span of the nucleosome core and linker. We release this data set, via the UCSC Genome Browser, as a resource for the high-resolution analysis of chromatin conformation and DNA accessibility at individual loci within the C. elegans genome.
MicroRNA expression signature of human sarcomas ONCOGENE Subramanian, S., Lui, W. O., Lee, C. H., Espinosa, I., Nielsen, T. O., Heinrich, M. C., Corless, C. L., Fire, A. Z., van de Rijn, M. 2008; 27 (14): 2015-2026
MicroRNAs (miRNAs) are approximately 22 nucleotide-long noncoding RNAs involved in several biological processes including development, differentiation and proliferation. Recent studies suggest that knowledge of miRNA expression patterns in cancer may have substantial value for diagnostic and prognostic determinations as well as for eventual therapeutic intervention. We performed comprehensive analysis of miRNA expression profiles of 27 sarcomas, 5 normal smooth muscle and 2 normal skeletal muscle tissues using microarray technology and/or small RNA cloning approaches. The miRNA expression profiles are distinct among the tumor types as demonstrated by an unsupervised hierarchical clustering, and unique miRNA expression signatures were identified in each tumor class. Remarkably, the miRNA expression patterns suggested that two of the sarcomas had been misdiagnosed and this was confirmed by reevaluation of the tumors using histopathologic and molecular analyses. Using the cloning approach, we also identified 31 novel miRNAs or other small RNA effectors in the sarcomas and normal skeletal muscle tissues examined. Our data show that different histological types of sarcoma have distinct miRNA expression patterns, reflecting the apparent lineage and differentiation status of the tumors. The identification of unique miRNA signatures in each tumor type may indicate their role in tumorigenesis and may aid in diagnosis of soft tissue sarcomas.
View details for DOI 10.1038/sj.onc.1210836
Regulation of apoptosis by C-elegans CED-9 in the absence of the C-terminal transmembrane domain CELL DEATH AND DIFFERENTIATION Tan, F. J., Fire, A. Z., Hill, R. B. 2007; 14 (11): 1925-1935
Bcl-2 proteins regulate apoptosis in organisms as diverse as mammals and nematodes. These proteins are often localized at mitochondria by a C-terminal transmembrane domain. Although the transmembrane domain and mitochondrial localization are centrally involved in specific cases of vertebrate Bcl-2 activity, the significance of this localization is not clear for all species. Studying the Caenorhabditis elegans Bcl-2 homolog CED-9, we found that the transmembrane domain was both necessary and sufficient for localization at mitochondrial outer membranes. Furthermore, we found that in our assays, ced-9 transgenes lacking the transmembrane domain, although somewhat less active than equivalent transgenes derived from wild-type ced-9, rescued embryonic lethality of ced-9(lf) animals and responded properly to upstream signals in controlling the fate of Pn.aap neurons. Both of these apoptotic activities were retained in a construct where CED-9 lacking the transmembrane domain was targeted to the cytosolic surface of the endoplasmic reticulum and derived organelles, suggesting that in wild-type animals, accumulation at mitochondria is not essential for CED-9 to either inhibit or promote apoptosis in C. elegans. Taken together, these data are consistent with a multimodal character of CED-9 action, with an ability to regulate apoptosis through interactions in the cytosol coexisting with additional evolutionarily conserved role(s) at the membrane.
View details for DOI 10.1038/sj.cdd.4402215
A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing NUCLEIC ACIDS RESEARCH Parameswaran, P., Jalili, R., Tao, L., Shokralla, S., Gharizadeh, B., Ronaghi, M., Fire, A. Z. 2007; 35 (19)
Multiplexed high-throughput pyrosequencing is currently limited in complexity (number of samples sequenced in parallel), and in capacity (number of sequences obtained per sample). Physical-space segregation of the sequencing platform into a fixed number of channels allows limited multiplexing, but obscures available sequencing space. To overcome these limitations, we have devised a novel barcoding approach to allow for pooling and sequencing of DNA from independent samples, and to facilitate subsequent segregation of sequencing capacity. Forty-eight forward-reverse barcode pairs are described: each forward and each reverse barcode unique with respect to at least 4 nt positions. With improved read lengths of pyrosequencers, combinations of forward and reverse barcodes may be used to sequence from as many as n(2) independent libraries for each set of 'n' forward and 'n' reverse barcodes, for each defined set of cloning-linkers. In two pilot series of barcoded sequencing using the GS20 Sequencer (454/Roche), we found that over 99.8% of obtained sequences could be assigned to 25 independent, uniquely barcoded libraries based on the presence of either a perfect forward or a perfect reverse barcode. The false-discovery rate, as measured by the percentage of sequences with unexpected perfect pairings of unmatched forward and reverse barcodes, was estimated to be <0.005%.
View details for DOI 10.1093/nar/gkm760
Connector Inversion Probe Technology: A Powerful One-Primer Multiplex DNA Amplification System for Numerous Scientific Applications PLOS ONE Akhras, M. S., Unemo, M., Thiyagarajan, S., Nyren, P., Davis, R. W., Fire, A. Z., Pourmand, N. 2007; 2 (9)
We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i.e. "multiplex multiplexing padlocks" (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs.
Distinct ribonucleoprotein reservoirs for microRNA and siRNA populations in C-elegans RNA-A PUBLICATION OF THE RNA SOCIETY Gu, S. G., Pak, J., Barberan-Soler, S., Ali, M., Fire, A., Zahler, A. M. 2007; 13 (9): 1492-1504
MicroRNAs (miRNAs) are regulatory molecules that share both biosynthetic derivation (cleavage from short hairpin precursor RNAs) and functional roles (downregulation of specific mRNAs through targeted degradation and/or translational inhibition). A distinct family of small RNAs, termed siRNAs, have some common characteristics but exhibit distinct modes of biosynthesis and function. In this study, we report procedures for purification of a predominant species of miRNA-containing ribonucleoprotein complexes from Caenorhabditis elegans and demonstrate that this population is distinct from the predominant pool of siRNA-containing ribonucleoprotein complexes. An observed miRNP-associated RNA population consisting predominantly (>95%) of miRNAs supported the unique identity of miRNPs as biological effectors within the cell, provided clean material for analysis of changes in miRNA spectra during development, and provided strong evidence of miRNA character for a number of novel small RNAs. Likewise, the RNA spectrum derived from partial siRNP purification was useful in defining functional characteristics of this more diverse population of small RNAs.
View details for DOI 10.1261/rna.581907
Patterns of known and novel small RNAs in human cervical cancer CANCER RESEARCH Lui, W., Pourmand, N., Patterson, B. K., Fire, A. 2007; 67 (13): 6031-6043
Recent studies suggest that knowledge of differential expression of microRNAs (miRNA) in cancer may have substantial diagnostic and prognostic value. Here, we use a direct sequencing method to characterize the profiles of miRNAs and other small RNA segments for six human cervical carcinoma cell lines and five normal cervical samples. Of 166 miRNAs expressed in normal cervix and cancer cell lines, we observed significant expression variation of six miRNAs between the two groups. To further show the biological relevance of our findings, we examined the expression level of two significantly varying miRNAs in a panel of 29 matched pairs of human cervical cancer and normal cervical samples. Reduced expression of miR-143 and increased expression of miR-21 were reproducibly displayed in cancer samples, suggesting the potential value of these miRNAs as tumor markers. In addition to the known miRNAs, we found a number of novel miRNAs and an additional set of small RNAs that do not meet miRNA criteria.
View details for DOI 10.1158/0008-5472.CAN-06-0561
Distinct populations of primary and secondary effectors during RNAi in C-elegans SCIENCE Pak, J., Fire, A. 2007; 315 (5809): 241-244
RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: "Primary siRNAs" (derived from DICER nuclease-mediated cleavage of the original trigger) and "secondary siRNAs" [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.
Gene silencing by double-stranded RNA (Nobel Lecture). Angewandte Chemie (International ed. in English) Fire, A. Z. 2007; 46 (37): 6966-6984
Gene silencing by double-stranded RNA (Nobel lecture) ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Fire, A. Z. 2007; 46 (37): 6967-6984
View details for DOI 10.1002/anie.200701979
Flexibility and constraint in the nucleosome core landscape of Caenorhabditis elegans chromatin GENOME RESEARCH Johnson, S. M., Tan, F. J., McCullough, H. L., Riordan, D. P., Fire, A. Z. 2006; 16 (12): 1505-1516
Nucleosome positions within the chromatin landscape are known to serve as a major determinant of DNA accessibility to transcription factors and other interacting components. To delineate nucleosomal patterns in a model genetic organism, Caenorhabditis elegans, we have carried out a genome-wide analysis in which DNA fragments corresponding to nucleosome cores were liberated using an enzyme (micrococcal nuclease) with a strong preference for cleavage in non-nucleosomal regions. Sequence analysis of 284,091 putative nucleosome cores obtained in this manner from a mixed-stage population of C. elegans reveals a combined picture of flexibility and constraint in nucleosome positioning. As has previously been observed in studies of individual loci in diverse biological systems, we observe areas in the genome where nucleosomes can adopt a wide variety of positions in a given region, areas with little or no nucleosome coverage, and areas where nucleosomes reproducibly adopt a specific positional pattern. In addition to illuminating numerous aspects of chromatin structure for C. elegans, this analysis provides a reference from which to begin an investigation of relationships between the nucleosomal pattern, chromosomal architecture, and lineage-based gene activity on a genome-wide scale.
View details for DOI 10.1101/gr.5560806
A differential cytolocalization assay for analysis of macromolecular assemblies in the eukaryotic cytoplasm MOLECULAR & CELLULAR PROTEOMICS Blanchard, D., Hutter, H., Fleenor, J., Fire, A. 2006; 5 (11): 2175-2184
We have developed a differential cytolocalization assay (DCLA) that allows the observation of cytoplasmic protein/protein interactions in vivo. In the DCLA, interactions are visualized as a relocalization of a green fluorescent protein-tagged "prey" by a membrane-bound "bait." This assay was tested and utilized in Caenorhabditis elegans to probe interactions among proteins involved in RNA interference (RNAi) and nonsense-mediated decay (NMD) pathways. Several previously documented interactions were confirmed with DCLA, whereas uniformly negative results were obtained in several controls in which no interaction was expected. Novel interactions were also observed, including the association of SMG-5, a protein required for NMD, to several components of the RNAi pathway. The DCLA can be readily carried out under diverse conditions, allowing a dynamic assessment of protein interactions in vivo. We used this property to test a subset of the RNAi and NMD interactions in animals in which proteins central to each mechanism were mutated; several key associations in each machinery that can occur in vivo in the absence of a functional process were identified.
View details for DOI 10.1074/mcp.T600025-MCP200
An antagonistic role for the C-elegans Schnurri homolog SMA-9 in modulating TGF beta signaling during mesodermal patterning DEVELOPMENT Foehr, M. L., Lindy, A. S., Fairbank, R. C., Amin, N. M., Xu, M., Yanowitz, J., Fire, A. Z., Liu, J. 2006; 133 (15): 2887-2896
In C. elegans, the Sma/Mab TGFbeta signaling pathway regulates body size and male tail patterning. SMA-9, the C. elegans homolog of Schnurri, has been shown to function as a downstream component to mediate the Sma/Mab TGFbeta signaling pathway in these processes. We have discovered a new role for SMA-9 in dorsoventral patterning of the C. elegans post-embryonic mesoderm, the M lineage. In addition to a small body size, sma-9 mutant animals exhibit a dorsal-to-ventral fate transformation within the M lineage. This M lineage defect of sma-9 mutants is unique in that animals carrying mutations in all other known components of the TGFbeta pathway exhibit no M lineage defects. Surprisingly, mutations in the core components of the Sma/Mab TGFbeta signaling pathway suppressed the M lineage defects of sma-9 mutants without suppressing their body size defects. We show that this suppression specifically happens within the M lineage. Our studies have uncovered an unexpected role of SMA-9 in antagonizing the TGFbeta signaling pathway during mesodermal patterning, suggesting a novel mode of function for the SMA-9/Schnurri family of proteins.
View details for DOI 10.1242/dev.02476
Unusual DNA structures associated with germline genetic activity in Caenorhabditis elegans GENETICS Fire, A., Alcazar, R., Tan, F. 2006; 173 (3): 1259-1273
We describe a surprising long-range periodicity that underlies a substantial fraction of C. elegans genomic sequence. Extended segments (up to several hundred nucleotides) of the C. elegans genome show a strong bias toward occurrence of AA/TT dinucleotides along one face of the helix while little or no such constraint is evident on the opposite helical face. Segments with this characteristic periodicity are highly overrepresented in intron sequences and are associated with a large fraction of genes with known germline expression in C. elegans. In addition to altering the path and flexibility of DNA in vitro, sequences of this character have been shown by others to constrain DNA::nucleosome interactions, potentially producing a structure that could resist the assembly of highly ordered (phased) nucleosome arrays that have been proposed as a precursor to heterochromatin. We propose a number of ways that the periodic occurrence of An/Tn clusters could reflect evolution and function of genes that express in the germ cell lineage of C. elegans.
Structural analysis of hyperperiodic DNA from Caenorhabditis elegans NUCLEIC ACIDS RESEARCH Moreno-Herrero, F., Seidel, R., Johnson, S. M., Fire, A., Dekker, N. H. 2006; 34 (10): 3057-3066
Several bioinformatics studies have identified an unexpected but remarkably prevalent approximately 10 bp periodicity of AA/TT dinucleotides (hyperperiodicity) in certain regions of the Caenorhabditis elegans genome. Although the relevant C.elegans DNA segments share certain sequence characteristics with bent DNAs from other sources (e.g. trypanosome mitochondria), the nematode sequences exhibit a much more extensive and defined hyperperiodicity. Given the presence of hyperperiodic structures in a number of critical C.elegans genes, the physical characteristics of hyperperiodic DNA are of considerable interest. In this work, we demonstrate that several hyperperiodic DNA segments from C.elegans exhibit structural anomalies using high-resolution atomic force microscopy (AFM) and gel electrophoresis. Our quantitative analysis of AFM images reveals that hyperperiodic DNA adopts a significantly smaller mean square end-to-end distance, hence a more compact coil structure, compared with non-periodic DNA of similar length. While molecules remain capable of adopting both bent and straight (rod-like) configurations, indicating that their flexibility is still retained, examination of the local curvatures along the DNA contour length reveals that the decreased mean square end-to-end distance can be attributed to the presence of long-scale intrinsic bending in hyperperiodic DNA. Such bending is not detected in non-periodic DNA. Similar studies of shorter, nucleosome-length DNAs that survived micrococcal nuclease digestion show that sequence hyperperiodicity in short segments can likewise induce strong intrinsic bending. It appears, therefore, that regions of the C.elegans genome display a significant correlation between DNA sequence and unusual mechanical properties.
View details for DOI 10.1093/nar/gkl397
Nucleic acid structure and intracellular immunity: some recent ideas from the world of RNAi Workshop on Fundamentals of Biomolecular Function - Nucleic Acids, Proteins and Membranes Fire, A. CAMBRIDGE UNIV PRESS. 2005: 303–9
Cells face a constant struggle against unwanted instructions that arrive in the form of viruses and transposons. At the core of this battle are two issues: how can cellular machinery recognize certain informational molecules as 'unwanted' and how can the cell use this recognition to effectively silence malicious genetic activity. While defenses against some specific parasites may be triggered by individual nucleic acid or protein sequences, such sequence-specific mechanisms have the limitation of allowing the parasite to evade following relatively minor evolutionary change. A more general set of defense mechanisms is based on recognition of structural features that are intrinsic aspects of one or more parasitic lifestyle. Recognition of extended regions of double-stranded RNA (dsRNA) provides cells with one such defensive modality. Essentially absent during 'normal' gene expression, long stretches of dsRNA within a cell serve as a dramatic warning that a segment of information may be replicating as RNA. In addition to exemplifying many of the mechanistic issues in genome defense, the cellular response to dsRNA provides several examples of the logic by which organisms attempt to focus their limited immunity resources on the most immediate and dangerous targets.
View details for DOI 10.1017/S0033583505004117
Imprinting capacity of gamete lineages in Caenorhabditis elegans GENETICS Sha, K., Fire, A. 2005; 170 (4): 1633-1652
We have observed a gamete-of-origin imprinting effect in C. elegans using a set of GFP reporter transgenes. From a single progenitor line carrying an extrachromosomal unc-54::gfp transgene array, we generated three independent autosomal integrations of the unc-54::gfp transgene. The progenitor line, two of its three integrated derivatives, and a nonrelated unc-119:gfp transgene exhibit an imprinting effect: single-generation transmission of these transgenes through the male germline results in approximately 1.5- to 2.0-fold greater expression than transmission through the female germline. There is a detectable resetting of the imprint after passage through the opposite germline for a single generation, indicating that the imprinted status of the transgenes is reversible. In cases where the transgene is maintained in either the oocyte lineage or sperm lineage for multiple, consecutive generations, a full reset requires passage through the opposite germline for several generations. Taken together, our results indicate that C. elegans has the ability to imprint chromosomes and that differences in the cell and/or molecular biology of oogenesis and spermatogenesis are manifest in an imprint that can persist in both somatic and germline gene expression for multiple generations.
Cyclin D involvement demarcates a late transition in C-elegans embryogenesis DEVELOPMENTAL BIOLOGY Yanowitz, J., Fire, A. 2005; 279 (1): 244-251
During development, progression through the cell cycle must be coordinately regulated with cellular differentiation. Despite significant progress in identifying genes required independently for each of these processes, the molecules which facilitate this cross talk have for the most part been elusive. Using the six macrophage-like coelomocytes of the nematode Caenorhabditis elegans as a model system to gain insight into the mesodermal differentiation pathway, we have isolated a set of mutants that alter coelomocyte numbers. One of these mutations, cc600, apparently results from a partial loss-of-function in the C. elegans cyclin D gene, cyd-1. The mutant has coelomocyte-specific defects without changes in other lineages. The mutants show that cell growth, terminal differentiation and cellular function proceed in the absence of cyd-1 activity and cell division. The results suggest that certain mesodermal lineages may be uniquely affected by changes in cyd-1 activity.
UNC-39, the C-elegans homolog of the human myotonic dystrophy-associated homeodomain protein Six5, regulates cell motility and differentiation DEVELOPMENTAL BIOLOGY Yanowitz, J. L., Shakir, M. A., Hedgecock, E., Hutter, H., Fire, A. Z., Lundquist, E. A. 2004; 272 (2): 389-402
Mutations in the unc-39 gene of C. elegans lead to migration and differentiation defects in a subset of mesodermal and ectodermal cells, including muscles and neurons. Defects include mesodermal specification and differentiation as well a neuronal migration and axon pathfinding defects. Molecular analysis revealed that unc-39 corresponds to the previously named gene ceh-35 and that the UNC-39 protein belongs to the Six4/5 family of homeodomain transcription factors and is similar to human Six5, a protein implicated in the pathogenesis of type I myotonic dystrophy (DM1). We show that human Six5 and UNC-39 are functional homologs, suggesting that further characterization of the C. elegans unc-39 gene might provide insight into the etiology of DM1.
Inducible systemic RNA silencing in Caenorhabditis elegans MOLECULAR BIOLOGY OF THE CELL Timmons, L., Tabara, H., Mello, C. C., Fire, A. Z. 2003; 14 (7): 2972-2983
Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.
View details for DOI 10.1091/mbc.E03-01-0858
Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi CURRENT BIOLOGY Simmer, F., Tijsterman, M., Parrish, S., Koushika, S. P., Nonet, M. L., Fire, A., Ahringer, J., Plasterk, R. H. 2002; 12 (15): 1317-1319
RNA interference (RNAi) is a broadly used reverse genetics method in C. elegans. Unfortunately, RNAi does not inhibit all genes. We show that loss of function of a putative RNA-directed RNA polymerase (RdRP) of C. elegans, RRF-3, results in a substantial enhancement of sensitivity to RNAi in diverse tissues. This is particularly striking in the nervous system; neurons that are generally refractory to RNAi in a wild-type genetic background can respond effectively to interference in an rrf-3 mutant background. These data provide the first indication of physiological negative modulation of the RNAi response and implicate an RdRP-related factor in this effect. The rrf-3 strain can be useful to study genes that, in wild-type, do not show a phenotype after RNAi, and it is probably the strain of choice for genome-wide RNAi screens.
Rescue of polyglutamine-mediated cytotoxicity by double-stranded RNA-mediated RNA interference HUMAN MOLECULAR GENETICS Caplen, N. J., Taylor, J. P., Statham, V. S., Tanaka, F., Fire, A., Morgan, R. A. 2002; 11 (2): 175-184
RNA interference (RNAi) is a mechanism that appears to control unwanted gene expression in a wide range of species. In Drosophila, RNAi is most effectively induced by double-stranded RNAs (dsRNAs) of over approximately 80 nucleotides (nt) and in mammalian cells an RNAi-like inhibition of gene expression has been shown to be mediated by dsRNAs of approximately 21-23 nt. To test if RNAi can be used to specifically down-regulate a human disease-related transcript we have used Drosophila and human tissue culture models of the dominant genetic disorder spinobulbar muscular atrophy (SBMA). A variety of different dsRNAs were assessed for the ability to inhibit expression of transcripts that included a truncated human androgen receptor (ar) gene containing different CAG repeat lengths (16-112 repeats). In Drosophila cells, dsRNAs corresponding to non-repetitive sequences mediated a high degree of sequence-specific inhibition, whereas RNA duplexes containing CAG repeat tracts only induced gene-specific inhibition when flanking ar sequences were included; dsRNAs containing various lengths of CAG repeats plus ar sequences were unable to induce allele-specific interference. In mammalian cells we tested sequence-specific small dsRNAs of 22 nt; these rescued the toxicity and caspase-3 activation induced by plasmids expressing a transcript encoding an expanded polyglutamine tract. This study demonstrates the feasibility of targeting a transcript associated with an important group of genetic diseases by RNAi.
The T-box factor MLS-1 acts as a molecular switch during specification of nonstriated muscle in C-elegans GENES & DEVELOPMENT Kostas, S. A., Fire, A. 2002; 16 (2): 257-269
We have isolated mutations in a gene mls-1 that is required for proper specification of nonstriated muscle fates in Caenorhabditis elegans. Loss of MLS-1 activity causes uterine muscle precursors to forego their normal fates, instead differentiating as vulval muscles. We have cloned mls-1 and shown that the product is a member of the T-box family of transcriptional regulators. MLS-1 acts as a cell fate determinant in that ectopic expression can transform other cell types to uterine muscle precursors. Uterine muscle patterning is executed by regulation of MLS-1 at several different levels. The mls-1 promoter is activated by the C. elegans orthologs of Twist and Daughterless, but is only active in a subset of the lineage where these two transcription factors are present. mls-1 activity also appears to be regulated by posttranscriptional processes, as expression occurs in both uterine and vulval muscle precursors.
On the role of RNA amplification in dsRNA-triggered gene silencing CELL Sijen, T., Fleenor, J., Simmer, F., Thijssen, K. L., Parrish, S., Timmons, L., Plasterk, R. H., Fire, A. 2001; 107 (4): 465-476
We have investigated the role of trigger RNA amplification during RNA interference (RNAi) in Caenorhabditis elegans. Analysis of small interfering RNAs (siRNAs) produced during RNAi in C. elegans revealed a substantial fraction that cannot derive directly from input dsRNA. Instead, a population of siRNAs (termed secondary siRNAs) appeared to derive from the action of a cellular RNA-directed RNA polymerase (RdRP) on mRNAs that are being targeted by the RNAi mechanism. The distribution of secondary siRNAs exhibited a distinct polarity (5'-->3' on the antisense strand), suggesting a cyclic amplification process in which RdRP is primed by existing siRNAs. This amplification mechanism substantially augments the potency of RNAi-based surveillance, while ensuring that the RNAi machinery will focus on expressed mRNAs.
Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans RNA-A PUBLICATION OF THE RNA SOCIETY Parrish, S., Fire, A. 2001; 7 (10): 1397-1402
RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.
Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Caplen, N. J., Parrish, S., Imani, F., Fire, A., Morgan, R. A. 2001; 98 (17): 9742-9747
Short interfering RNAs (siRNAs) are double-stranded RNAs of approximately 21-25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates. siRNAs have a characteristic structure, with 5'-phosphate/3'-hydroxyl ends and a 2-base 3' overhang on each strand of the duplex. In this study, we present data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice. In each case, the interference by siRNAs was superior to the inhibition of gene expression mediated by single-stranded antisense oligonucleotides. The siRNAs seem to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells. These observations may open a path toward the use of siRNAs as a reverse genetic and therapeutic tool in mammalian cells.
Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C-elegans developmental timing CELL Grishok, A., Pasquinelli, A. E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D. L., Fire, A., Ruvkun, G., Mello, C. C. 2001; 106 (1): 23-34
RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Here we show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation.
Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans GENE Timmons, L., Court, D. L., Fire, A. 2001; 263 (1-2): 103-112
Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.
Overlapping roles of two Hox genes and the exd ortholog ceh-20 in diversification of the C-elegans postembryonic mesoderm DEVELOPMENT Liu, J., Fire, A. 2000; 127 (23): 5179-5190
Members of the Hox family of homeoproteins and their cofactors play a central role in pattern formation of all germ layers. During postembryonic development of C. elegans, non-gonadal mesoderm arises from a single mesoblast cell M. Starting in the first larval stage, M divides to produce 14 striated muscles, 16 non-striated muscles, and two non-muscle cells (coelomocytes). We investigated the role of the C. elegans Hox cluster and of the exd ortholog ceh-20 in patterning of the postembryonic mesoderm. By examining the M lineage and its differentiation products in different Hox mutant combinations, we found an essential but overlapping role for two of the Hox cluster genes, lin-39 and mab-5, in diversification of the postembryonic mesoderm. This role of the two Hox gene products required the CEH-20 cofactor. One target of these two Hox genes is the C. elegans twist ortholog hlh-8. Using both in vitro and in vivo assays, we demonstrated that twist is a direct target of Hox activation. We present evidence from mutant phenotypes that twist is not the only target for Hox genes in the M lineage: in particular we show that lin-39 mab-5 double mutants exhibit a more severe M lineage defect than the hlh-8 null mutant.
Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes MOLECULAR BIOLOGY OF THE CELL Liu, J., Ben-Shahar, T. R., Riemer, D., Treinin, M., Spann, P., Weber, K., Fire, A., Gruenbaum, Y. 2000; 11 (11): 3937-3947
Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs.
Functional anatomy of a dsRNA trigger: Differential requirement for the two trigger strands in RNA interference MOLECULAR CELL Parrish, S., Fleenor, J., Xu, S. Q., Mello, C., Fire, A. 2000; 6 (5): 1077-1087
In RNA-mediated interference (RNAi), externally provided mixtures of sense and antisense RNA trigger concerted degradation of homologous cellular RNAs. We show that RNAi requires duplex formation between the two trigger strands, that the duplex must include a region of identity between trigger and target RNAs, and that duplexes as short as 26 bp can trigger RNAi. Consistent with in vitro observations, a fraction of input dsRNA is converted in vivo to short segments of approximately 25 nt. Interference assays with modified dsRNAs indicate precise chemical requirements for both bases and backbone of the RNA trigger. Strikingly, certain modifications are well tolerated on the sense, but not the antisense, strand, indicating that the two trigger strands have distinct roles in the interference process.
The MADS-Box factor CeMEF2 is not essential for Caenorhabditis elegans myogenesis and development DEVELOPMENTAL BIOLOGY Dichoso, D., Brodigan, T., Chwoe, K. Y., Lee, J. S., Llacer, R., Park, M., Corsi, A. K., Kostas, S. A., Fire, A., Ahnn, J., Krause, M. 2000; 223 (2): 431-440
MEF2 is an evolutionarily conserved MADS (MCM1, Agamous, Deficiens, and serum response factor) box-type transcription factor that plays a critical role in vertebrate and Drosophila melanogaster myogenesis. We have addressed the developmental role of the single MEF2-like factor, CeMEF2, in Caenorhabditis elegans. Using expression assays and two mef-2 deletion alleles, we show that CeMEF2 is not required for proper myogenesis or development. Moreover, a putative null mef-2 allele fails to enhance or suppress the phenotypes of mutants in CeMyoD or CeTwist. Our results suggest that despite its evolutionary conservation of sequence and DNA binding properties, CeMEF2 has adopted a divergent role in development in the nematode compared with Drosophila and vertebrates.
View details for DOI 10.1006/dbio.2000.9758
dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference GENE Caplen, N. J., Fleenor, J., Fire, A., Morgan, R. A. 2000; 252 (1-2): 95-105
RNA interference (RNAi) is a form of post-transcriptional gene silencing that has been described in a number of plant, nematode, protozoan, and invertebrate species. RNAi is characterized by a number of features: induction by double stranded RNA (dsRNA), a high degree of specificity, remarkable potency and spread across cell boundaries, and a sustained down-regulation of the target gene. Previous studies of RNAi have examined this effect in whole organisms or in extracts thereof; we have now examined the induction of RNAi in tissue culture. A screen of mammalian cells from three different species showed no evidence for the specific down-regulation of gene expression by dsRNA. By contrast, RNAi was observed in Drosophila Schneider 2 (S2) cells. Green fluorescent protein (GFP) expression in S2 cells was inhibited in a dose-dependent manner by transfection of dsRNA corresponding to gfp when GFP was expressed either transiently or stably. This effect was structure- and sequence-specific in that: (1) little or no effect was seen when antisense (or sense) RNA was transfected; (2) an unrelated dsRNA did not reduce GFP expression; and (3) dsRNA corresponding to gfp had no effect on the expression of an unrelated target transgene. This invertebrate tissue culture model should allow facile assays for loss of function in a well-defined cellular system and facilitate further understanding of the mechanism of RNAi and the genes involved in this process.
Identification and molecular-genetic characterization of a LAMP/CD68-like protein from Caenorhabditis elegans JOURNAL OF CELL SCIENCE Kostich, M., Fire, A., Fambrough, D. M. 2000; 113 (14): 2595-2606
Lysosome associated membrane proteins (LAMPs) constitute a family of vertebrate proteins located predominantly in lysosomes, with lesser amounts present in endosomes and at the cell surface. Macrosialin/CD68s are similar to LAMPs in their subcellular distribution and amino acid sequence and presumed structure across the carboxyl terminal two thirds of their length. The functions of LAMPs and CD68s are not known. In the present study, a bioinformatics approach was used to identify a Caenorhabditis elegans protein (LMP-1) with sequence and presumed structural similarity to LAMPs and CD68s. LMP-1 appears to be the only membrane protein in C. elegans that carries a GYXX(phi) vertebrate lysosomal targeting sequence at its C terminus (where (phi) is a large, hydrophobic residue). LMP-1 was found to be present from early embryonic stages through adulthood and to be predominantly localized at the periphery of a population of large, membrane-bound organelles, called granules, that are seen throughout the early embryo but in later stages are restricted to the cells of the intestine. Analysis of an LMP-1 deficient C. elegans mutant revealed that LMP-1 is not required for viability under laboratory conditions, but the absence of LMP-1 leads to an alteration in intestinal granule populations, with apparent loss of one type of granule.
Caenorhabditis elegans Twist plays an essential role in non-striated muscle development DEVELOPMENT Corsi, A. K., Kostas, S. A., Fire, A., Krause, M. 2000; 127 (10): 2041-2051
The basic helix-loop-helix (bHLH) transcription factor Twist plays a role in mesodermal development in both invertebrates and vertebrates. In an effort to understand the role of the unique Caenorhabditis elegans Twist homolog, hlh-8, we analyzed mesodermal development in animals with a deletion in the hlh-8 locus. This deletion was predicted to represent a null allele because the HLH domain is missing and the reading frame for the protein is disrupted. Animals lacking CeTwist function were constipated and egg-laying defective. Both of these defects were rescued in transgenic mutant animals expressing wild-type hlh-8. Observing a series of mesoderm-specific markers allowed us to characterize the loss of hlh-8 function more thoroughly. Our results demonstrate that CeTwist performs an essential role in the proper development of a subset of mesodermal tissues in C. elegans. We found that CeTwist was required for the formation of three out of the four non-striated enteric muscles born in the embryo. In contrast, CeTwist was not required for the formation of the embryonically derived striated muscles. Most of the post-embryonic mesoderm develops from a single lineage. CeTwist was necessary for appropriate patterning in this lineage and was required for expression of two downstream target genes, but was not required for the expression of myosin, a marker of differentiation. Our results suggest that mesodermal patterning by Twist is an evolutionarily conserved function.
Recognition and silencing of repeated DNA ANNUAL REVIEW OF GENETICS Hsieh, J., Fire, A. 2000; 34: 187-204
Mechanisms for repetition of DNA pose both opportunities and challenges to a functional genome: opportunities for increasing gene expression by amplification of useful sequences, and challenges of controlling amplification by unwanted sequences such as transposons and viruses. Experiments in numerous organisms have suggested the likely existence of a general mechanism for recognition of repeated character in DNA. This review focuses (a) on the nature of these recognition mechanisms, and (b) on types of chromatin modification and gene silencing that are used to control repeated DNA.
The RING finger/B-box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans GENES & DEVELOPMENT Hsieh, J., Liu, J., Kostas, S. A., Chang, C., Sternberg, P. W., Fire, A. 1999; 13 (22): 2958-2970
Context-dependent gene silencing is used by many organisms to stably modulate gene activity for large chromosomal regions. We have used tandem array transgenes as a model substrate in a screen for Caenorhabditis elegans mutants that affect context-dependent gene silencing in somatic tissues. This screen yielded multiple alleles of a previously uncharacterized gene, designated tam-1 (for tandem-array-modifier). Loss-of-function mutations in tam-1 led to a dramatic reduction in the activity of numerous highly repeated transgenes. These effects were apparently context dependent, as nonrepetitive transgenes retained activity in a tam-1 mutant background. In addition to the dramatic alterations in transgene activity, tam-1 mutants showed modest alterations in expression of a subset of endogenous cellular genes. These effects include genetic interactions that place tam-1 into a group called the class B synMuv genes (for a Synthetic Multivulva phenotype); this family plays a negative role in the regulation of RAS pathway activity in C. elegans. Loss-of-function mutants in other members of the class-B synMuv family, including lin-35, which encodes a protein similar to the tumor suppressor Rb, exhibit a hypersilencing in somatic transgenes similar to that of tam-1 mutants. Molecular analysis reveals that tam-1 encodes a broadly expressed nuclear protein with RING finger and B-box motifs.
The rde-1 gene, RNA interference, and transposon silencing in C-elegans CELL Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A., Timmons, L., Fire, A., Mello, C. C. 1999; 99 (2): 123-132
Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.
RNA-triggered gene silencing TRENDS IN GENETICS Fire, A. 1999; 15 (9): 358-363
Double-stranded RNA (dsRNA) has recently been shown to trigger sequence-specific gene silencing in a wide variety of organisms, including nematodes, plants, trypanosomes, fruit flies and planaria; meanwhile an as yet uncharacterized RNA trigger has been shown to induce DNA methylation in several different plant systems. In addition to providing a surprisingly effective set of tools to interfere selectively with gene function, these observations are spurring new inquiries to understand RNA-triggered genetic-control mechanisms and their biological roles.
Two-color GFP expression system for C-elegans BIOTECHNIQUES Miller, D. M., Desai, N. S., Hardin, D. C., Piston, D. W., Patterson, G. H., Fleenor, J., Xu, S., Fire, A. 1999; 26 (5): 914-?
We describe the use of modified versions of the Aequora victoria green fluorescent protein (GFP) to simultaneously follow the expression and distribution of two different proteins in the nematode, Caenorhabditis elegans. A cyan-colored GFP derivative, designated CFP, contains amino acid (aa) substitutions Y66W, N146I, M153T and V163A relative to the original GFP sequence and is similar to the previously reported "W7" form. A yellow-shifted GFP derivative, designated YFP, contains aa substitutions S65G, V68A, S72A and T203Y and is similar to the previously described "I0C" variant. Coding regions for CFP and YFP were constructed in the context of a high-activity C. elegans expression system. Previously characterized promoters and localization signals have been used to express CFP and YFP in C. elegans. Filter sets designed to distinguish YFP and CFP fluorescence spectra allowed visualization of the two distinct forms of GFP in neurons and in muscle cells. A series of expression vectors carrying CFP and YFP have been constructed and are being made available to the scientific community.
Evolutionary conservation of MyoD function and differential utilization of E proteins DEVELOPMENTAL BIOLOGY ZHANG, J. M., Chen, L. S., Krause, M., Fire, A., Paterson, B. M. 1999; 208 (2): 465-472
The formation of striated muscle in both vertebrates and invertebrates involves the activity of the MyoD family of basic-helix-loop-helix (bHLH) transcription factors. The high degree of evolutionary conservation of MyoD-related proteins, both in the sequence of their bHLH domains and in their general developmental expression patterns, suggests that these factors are also conserved at the level of function. We have addressed this directly using MyoD and E protein factors from vertebrates, Drosophila, and Caenorhabditis elegans. Various MyoD and E factor combinations were tested for their ability to interact in vitro and to function in vivo in the myogenic conversion of 10T12 mouse fibroblasts. We found that the ability of different homo- and heterodimers to bind DNA in vitro was an accurate measure of biological activity in vivo. A second assessment of conserved function comes from the ability of these factors to rescue a C. elegans hlh-1 (CeMyoD) null mutation. We found that both Drosophila and chicken MyoD-related factors were able to rescue a C. elegans CeMyoD loss-of-function mutation. These results demonstrate a remarkable degree of functional conservation of these myogenic factors despite differences in E-protein interactions.
RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Montgomery, M. K., Xu, S. Q., Fire, A. 1998; 95 (26): 15502-15507
Introduction of exogenous double-stranded RNA (dsRNA) into Caenorhabditis elegans has been shown to specifically and potently disrupt the activity of genes containing homologous sequences. In this study we present evidence that the primary interference effects of dsRNA are post-transcriptional. First, we examined the primary DNA sequence after dsRNA-mediated interference and found no evidence for alterations. Second, we found that dsRNA-mediated interference with the upstream gene in a polar operon had no effect on the activity of the downstream gene; this finding argues against an effect on initiation or elongation of transcription. Third, we observed by in situ hybridization that dsRNA-mediated interference produced a substantial, although not complete, reduction in accumulation of nascent transcripts in the nucleus, while cytoplasmic accumulation of transcripts was virtually eliminated. These results indicate that the endogenous mRNA is the target for interference and suggest a mechanism that degrades the targeted RNA before translation can occur. This mechanism is not dependent on the SMG system, an mRNA surveillance system in C. elegans responsible for targeting and destroying aberrant messages. We suggest a model of how dsRNA might function in a catalytic mechanism to target homologous mRNAs for degradation.
View details for PubMedID 9860998
Specific interference by ingested dsRNA NATURE Timmons, L., Fire, A. 1998; 395 (6705): 854-854
Analysis of a Caenorhabditis elegans Twist homolog identifies conserved and divergent aspects of mesodermal patterning GENES & DEVELOPMENT Harfe, B. D., Gomes, A. V., Kenyon, C., Liu, J., Krause, M., Fire, A. 1998; 12 (16): 2623-2635
Mesodermal development is a multistep process in which cells become increasingly specialized to form specific tissue types. In Drosophila and mammals, proper segregation and patterning of the mesoderm involves the bHLH factor Twist. We investigated the activity of a Twist-related factor, CeTwist, during Caenorhabditis elegans mesoderm development. Embryonic mesoderm in C. elegans derives from a number of distinct founder cells that are specified during the early lineages; in contrast, a single blast cell (M) is responsible for all nongonadal mesoderm formation during postembryonic development. Using immunofluorescence and reporter fusions, we determined the activity pattern of the gene encoding CeTwist. No activity was observed during specification of mesodermal lineages in the early embryo; instead, the gene was active within the M lineage and in a number of mesodermal cells with nonstriated muscle fates. A role for CeTwist in postembryonic mesodermal cell fate specification was indicated by ectopic expression and genetic interference assays. These experiments showed that CeTwist was responsible for activating two target genes normally expressed in specific subsets of nonstriated muscles derived from the M lineage. In vitro and in vivo assays suggested that CeTwist cooperates with the C. elegans E/Daughterless homolog in directly activating these targets. The two target genes that we have studied, ceh-24 and egl-15, encode an NK-2 class homeodomain and an FGF receptor (FGFR) homolog, respectively. Twist activates FGFR and NK-homeodomain target genes during mesodermal patterning of Drosophila and similar target interactions have been proposed to modulate mesenchymal growth during closure of the vertebrate skull. These results suggest the possibility that a conserved pathway may be used for diverse functions in mesodermal specification.
Chromatin silencing and the maintenance of a functional germline in Caenorhabditis elegans DEVELOPMENT Kelly, W. G., Fire, A. 1998; 125 (13): 2451-2456
The germline of the nematode Caenorhabditis elegans exhibits a remarkable ability to specifically silence transgenic DNA. We have shown that this silencing mechanism is disrupted in animals mutant for the maternal effect sterile genes mes-2, mes-3, mes-4 and mes-6. The proteins encoded by mes-2 and mes-6 have been shown to be related to the Polycomb Group of transcriptional repressors (Holdeman, R., Nehrt, S. and Strome, S. (1998). Development 125, 2457-2467; Korf, I., Fan, F. and Strome, S. (1998). Development 125, 2469-2478). These results suggest that a genetic silencing process is essential for sustained germline function, and that this silencing is mediated, at least in part, by Polycomb Group proteins.
Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression TRENDS IN GENETICS Montgomery, M. K., Fire, A. 1998; 14 (7): 255-258
MyoD and the specification of muscle and non-muscle fates during postembryonic development of the C-elegans mesoderm DEVELOPMENT Harfe, B. D., Branda, C. S., Krause, M., Stern, M. J., Fire, A. 1998; 125 (13): 2479-2488
Basic-helix-loop helix factors of the myoD/myf5/ myogenin/MRF4 family have been implicated in acquisition and elaboration of muscle cell fates. Here we describe both myogenic and non-myogenic roles for the Caenorhabditis elegans member of this family (CeMyoD) in postembryonic mesodermal patterning. The postembryonic mesodermal lineage in C. elegans provides a paradigm for many of the issues in mesodermal fate specification: a single mesoblast ('M') divides to generate 14 striated muscles, 16 non-striated muscles, and two non-muscle cells. To study CeMyoD function in the M lineage, we needed to circumvent an embryonic requirement for the protein. Two approaches were used: (1) isolation of mutants that decrease CeMyoD levels while retaining viability, and (2) analysis of genetic mosaics that had lost CeMyoD in the M lineage. With either manipulation, we observed a series of cell-fate transformations affecting a subset of both striated muscles and non-muscle cells. In place of these normal fates, the affected lineages produced a number of myoblast-like cells that initially failed to differentiate, instead swelling to acquire a resemblance to sex myoblasts (M-lineage-derived precursors to non-striated uterine and vulval muscles). Like normal sex myoblasts, the ectopic myoblast-like cells were capable of migration and proliferation followed by differentiation of progeny cells into vulval and uterine muscle. Our results demonstrate a cell-intrinsic contribution of CeMyoD to specification of both non-muscle and muscle fates.
Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans NATURE Fire, A., Xu, S. Q., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. 1998; 391 (6669): 806-811
Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
Muscle and nerve-specific regulation of a novel NK-2 class homeodomain factor in Caenorhabditis elegans DEVELOPMENT Harfe, B. D., Fire, A. 1998; 125 (3): 421-429
We have identified a new Caenorhabditis elegans NK-2 class homeobox gene, designated ceh-24. Distinct cis-acting elements generate a complex neuronal and mesodermal expression pattern. A promoter-proximal enhancer mediates expression in a single pharyngeal muscle, the donut-shaped m8 cell at the posterior end of the pharynx. A second mesodermal enhancer is active in a set of eight nonstriated vulval muscles used in egg laying. Activation in the egg laying muscles requires an 'NdE-box' consensus motif (CATATG) which is related to, but distinct from, the standard E-box motif bound by the MyoD family of transcriptional activators. Ectodermal expression of ceh-24 is limited to a subset of sublateral motor neurons in the head of the animal; this activity requires a cis-acting activator element that is distinct from the control elements for pharyngeal and vulval muscle expression. Activation of ceh-24 in each of the three cell types coincides with the onset of differentiation. Using a set of transposon-induced null mutations, we show that ceh-24 is not essential for the formation of any of these cells. Although ceh-24 mutants have no evident defects under laboratory conditions, the pattern of ceh-24 activity is apparently important for Rhabditid nematodes: the related species C. briggsae contains a close homologue of C. elegans ceh-24 including a highly conserved and functionally equivalent set of cis-acting control signals.
Genetically targeted cell disruption in Caenorhabditis elegans PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Harbinder, S., Tavernarakis, N., Herndon, L. A., Kinnell, M., Xu, S. Q., Fire, A., Driscoll, M. 1997; 94 (24): 13128-13133
The elimination of identified cells is a powerful tool for investigating development and system function. Here we report on genetically mediated cell disruption effected by the toxic Caenorhabditis elegans mec-4(d) allele. We found that ectopic expression of mec-4(d) in the nematode causes dysfunction of a wide range of nerve, muscle, and hypodermal cells. mec-4(d)-mediated toxicity is dependent on the activity of a second gene, mec-6, rendering cell disruption conditionally dependent on genetic background. We describe a set of mec-4(d) vectors that facilitate construction of cell-specific disruption reagents and note that genetic cell disruption can be used for functional analyses of specific neurons or neuronal classes, for confirmation of neuronal circuitry, for generation of nematode populations lacking defined classes of functional cells, and for genetic screens. We suggest that mec-4(d) and/or related genes may be effective general tools for cell inactivation that could be used toward similar purposes in higher organisms.
View details for Web of Science ID A1997YJ45600078
The Caenorhabditis elegans NK-2 homeobox gene ceh-22 activates pharyngeal muscle gene expression in combination with pha-1 and is required for normal pharyngeal development DEVELOPMENT Okkema, P. G., Ha, E. J., Haun, C., Chen, W., Fire, A. 1997; 124 (20): 3965-3973
Pharyngeal muscle development in the nematode Caenorhabditis elegans appears to share similarities with cardiac muscle development in other species. We have previously described CEH-22, an NK-2 class homeodomain transcription factor similar to Drosophila tinman and vertebrate Nkx2-5, which is expressed exclusively in the pharyngeal muscles. In vitro, CEH-22 binds the enhancer from myo-2, a pharyngeal muscle-specific myosin heavy chain gene. In this paper, we examine the role CEH-22 plays in pharyngeal muscle development and gene activation by (a) ectopically expressing ceh-22 in transgenic C. elegans and (b) examining the phenotype of a ceh-22 loss-of-function mutant. These experiments indicate that CEH-22 is an activator of myo-2 expression and that it is required for normal pharyngeal muscle development. However, ceh-22 is necessary for neither formation of the pharyngeal muscles, nor for myo-2 expression. Our data suggest parallel and potentially compensating pathways contribute to pharyngeal muscle differentiation. We also examine the relationship between ceh-22 and the pharyngeal organ-specific differentiation gene pha-1. Mutations in ceh-22 and pha-1 have strongly synergistic effects on pharyngeal muscle gene expression; in addition, a pha-1 mutation enhances the lethal phenotype caused by a mutation in ceh-22. Wild-type pha-1 is not required for the onset of ceh-22 expression but it appears necessary for maintained expression of ceh-22.
View details for Web of Science ID A1997YE93400005
Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits JOURNAL OF NEUROSCIENCE Fleming, J. T., Squire, M. D., Barnes, T. M., Tornoe, C., Matsuda, K., Ahnn, J., Fire, A., Sulston, J. E., Barnard, E. A., Sattelle, D. B., Lewis, J. A. 1997; 17 (15): 5843-5857
We show that three of the eleven genes of the nematode Caenorhabditis elegans that mediate resistance to the nematocide levamisole and to other cholinergic agonists encode nicotinic acetylcholine receptor (nAChR) subunits. unc-38 encodes an alpha subunit while lev-1 and unc-29 encode non-alpha subunits. The nematode nAChR subunits show conservation of many mammalian nAChR sequence features, implying an ancient evolutionary origin of nAChR proteins. Expression in Xenopus oocytes of combinations of these subunits that include the unc-38 alpha subunit results in levamisole-induced currents that are suppressed by the nAChR antagonists mecamylamine, neosurugatoxin, and d-tubocurarine but not alpha-bungarotoxin. The mutant phenotypes reveal that unc-38 and unc-29 subunits are necessary for nAChR function, whereas the lev-1 subunit is not. An UNC-29-GFP fusion shows that UNC-29 is expressed in body and head muscles. Two dominant mutations of lev-1 result in a single amino acid substitution or addition in or near transmembrane domain 2, a region important to ion channel conductance and desensitization. The identification of viable nAChR mutants in C. elegans provides an advantageous system in which receptor expression and synaptic targeting can be manipulated and studied in vivo.
View details for Web of Science ID A1997XU91800018
A C-elegans E Daughterless bHLH protein marks neuronal but not striated muscle development DEVELOPMENT Krause, M., Park, M., ZHANG, J. M., Yuan, J., Harfe, B., Xu, S. Q., Greenwald, I., Cole, M., PATERSON, B., Fire, A. 1997; 124 (11): 2179-2189
The E proteins of mammals, and the related Daughterless (DA) protein of Drosophila, are ubiquitously expressed helix-loop-helix (HLH) transcription factors that play a role in many developmental processes. We report here the characterization of a related C. elegans protein, CeE/DA, which has a dynamic and restricted distribution during development. CeE/DA is present embryonically in neuronal precursors, some of which are marked by promoter activity of a newly described Achaete-scute-like gene hlh-3. In contrast, we have been unable to detect CeE/DA in CeMyoD-positive striated muscle cells. In vitro gel mobility shift analysis detects dimerization of CeE/DA with HLH-3 while efficient interaction of CeE/DA with CeMyoD is not seen. These studies suggest multiple roles for CeE/DA in C. elegans development and provide evidence that both common and alternative strategies have evolved for the use of related HLH proteins in controlling cell fates in different species.
View details for Web of Science ID A1997XH38300007
Distinct requirements for somatic and germline expression of a genera expressed Caernorhabditis elegans gene GENETICS Kelly, W. G., Xu, S. Q., Montgomery, M. K., Fire, A. 1997; 146 (1): 227-238
In screening for embryonic-lethal mutations in Caenorhabditis elegans, we defined an essential gene (let-858) that encodes a nuclear protein rich in acidic and basic residues. We have named this product nucampholin. Closely homologous sequences in yeast, plants, and mammals demonstrate strong evolutionary conservation in eukaryotes. Nucampholin resides in all nuclei of C. elegans and is essential in early development and in differentiating tissue. Antisense-mediated depletion of LET-858 activity in early embryos causes a lethal phenotype similar to characterized treatments blocking embryonic gene expression. Using transgene-rescue, we demonstrated the additional requirement for let-858 in the larval germline. The broad requirements allowed investigation of soma-germline differences in gene expression. When introduced into standard transgene arrays, let-858 (like many other C. elegans genes) functions well in soma but poorly in germline. We observed incremental silencing of simple let-858 arrays in the first few generations following transformation and hypothesized that silencing might reflect recognition of arrays as repetitive or heterochromatin-like. To give the transgene a more physiological context, we included an excess of random genomic fragments with the injected DNA. The resulting transgenes show robust expression in both germline and soma. Our results suggest the possibility of concerted mechanisms for silencing unwanted germiline expression of repetitive sequences.
View details for Web of Science ID A1997WW88000017
Repression of gene expression in the embryonic germ lineage of C-elegans NATURE Seydoux, G., Mello, C. C., Pettitt, J., Wood, W. B., Priess, J. R., Fire, A. 1996; 382 (6593): 713-716
The distinction between soma and germline was recognized more than a century ago: somatic cells form the body of an organism, whereas germ cells serve to produce future generations. In Caenorhabditis elegans, the separation of some and germline occurs through a series of asymmetrical divisions, in which embryonic germline blastomeres divide unequally to produce one somatic daughter and one germline daughter. Here we show that after each asymmetrical division, embryonically transcribed RNAs are detected in somatic, but not germline, blastomeres. This asymmetry depends on the activity of the germline specific factor, PIE-1. In the absence of PIE-1, embryonically transcribed RNAs are detected in both somatic and germline blastomeres. Furthermore, ectopic expression of PIE-1 in somatic blastomeres can significantly reduce the accumulation of new transcripts in these cells. Taken together, these results suggest that germ-cell fate depends on an inhibitory mechanism that blocks new gene expression in the early embryonic germ lineage.
View details for Web of Science ID A1996VD33300049
An inductive interaction in 4-cell stage C-elegans embryos involves APX-1 expression in the signalling cell DEVELOPMENT Mickey, K. M., Mello, C. C., Montgomery, M. K., Fire, A., Priess, J. R. 1996; 122 (6): 1791-1798
During the 4-cell stage of C. elegans embryogenesis, the P2 blastomere provides a signal that allows two initially equivalent sister blastomeres, called ABa and ABp, to adopt different fates. Preventing P2 signalling in wild-type embryos results in defects in ABp development that are similar to those caused by mutations in the glp-1 and apx-1 genes, which are homologs of the Drosophila genes Notch and Delta, respectively. Previous studies have shown that GLP-1 protein is expressed in 4-cell stage embryos in both ABa and ABp. In this report, we show that APX-1 protein is expressed in the P2 blastomere and that a temperature-sensitive apx-1 mutant has a temperature-sensitive period between the 4-cell and 8-cell stages. We propose that APX-1 is part or all of the P2 signal that induces ABp to adopt a fate different than ABa.
View details for Web of Science ID A1996UT20700012
ROLLING REPLICATION OF SHORT DNA CIRCLES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fire, A., Xu, S. Q. 1995; 92 (10): 4641-4645
Natural genes and proteins often contain tandemly repeated sequence motifs that dramatically increase physiological specificity and activity. Given the selective value of such repeats, it is likely that several different mechanisms have been responsible for their generation. One mechanism that has been shown to generate relatively long tandem repeats (in the kilobase range) is rolling circle replication. In this communication, we demonstrate that rolling circle synthesis in a simple enzymatic system can produce tandem repeats of monomers as short as 34 bp. In addition to suggesting possible origins for natural tandem repeats, these observations provide a facile means for constructing libraries of repeated motifs for use in "in vitro evolution" experiments designed to select molecules with defined biological or chemical properties.
View details for Web of Science ID A1995QX87600113
Whole-mount in situ hybridization for the detection of RNA in Caenorhabditis elegans embryos METHODS IN CELL BIOLOGY, VOL 48 Seydoux, G., Fire, A. 1995; 48: 323-337
View details for Web of Science ID A1995BE40R00014
DNA transformation METHODS IN CELL BIOLOGY, VOL 48 Mello, C., Fire, A. 1995; 48: 451-482
ELEMENTS REGULATING CELL-SPECIFIC AND STAGE-SPECIFIC EXPRESSION OF THE C-ELEGANS MYOD FAMILY HOMOLOG HLH-1 DEVELOPMENTAL BIOLOGY Krause, M., HARRISON, S. W., Xu, S. Q., Chen, L. S., Fire, A. 1994; 166 (1): 133-148
We investigated the cis-acting sequences regulating expression of the Caenorhabditis elegans gene hlh-1, a homolog of the MyoD family of myogenic regulatory factors. The hlh-1 gene is expressed in mature body wall muscle, in clonal muscle precursors, in a set of early embryonic blastomeres (the MS-granddaughters), and in six glial-like cells called GLRs. The entire natural hlh-1 expression pattern is recapitulated in transgenic animals containing an hlh-1::lacZ fusion with 5.1 kb of hlh-1 sequences beginning upstream of the coding region and extending into the second exon. Deletions and rearrangements in this 5.1-kb sequence were assayed for their effects on reporter gene expression in transgenic animals. Deletions removing a segment 434-550 bp upstream of the coding region resulted in a loss of all aspects of the expression pattern, suggesting that at least one common regulatory factor is required for all expression. Deletions of other regulatory elements affected distinct aspects of the expression pattern; hence, each expression subpattern exhibits a different set of sequence requirements. Interspecies sequence comparison and lacZ expression constructs with the related nematode Caenorhabditis briggsae indicated that the C. briggsae and C. elegans genes contain equivalent sets of control signals. The results from this work implicate the hlh-1 promoter as an integration point for diverse temporal and spatial control signals.
View details for Web of Science ID A1994PT49200011
COMBINATORIAL STRUCTURE OF A BODY MUSCLE-SPECIFIC TRANSCRIPTIONAL ENHANCER IN CAENORHABDITIS-ELEGANS JOURNAL OF BIOLOGICAL CHEMISTRY JANTSCHPLUNGER, V., Fire, A. 1994; 269 (43): 27021-27028
We describe the dissection of a body muscle-specific enhancer sequence contained within the Caenorhabditis elegans myosin heavy chain gene unc-54. A 90-base pair segment that was sufficient for both enhancer function and tissue specificity was subjected to mutational analysis. Several separated sites within this region were required for activity; mutations in these sites led to dramatic decreases in enhancer activity, while substitutions in the intervening regions had minimal effects on activity. The individual sites appear to function as semi-independent and partially interchangeable enhancer subelements, as seen by our ability to create functional enhancers by constructing novel multimers and combinations. Four different enhancer subelements (designated O, I, II, and III) were identified in this way. Although partially interchangeable, some differences between these subelements were evident. In particular, concatamers of site III exhibited the highest levels of activity but had a broader tissue specificity than the intact enhancer, including both hypodermal and muscle tissue. The specificity of the intact enhancer thus reflects a combinatorial function of the specificities of the constituent subelements.
View details for Web of Science ID A1994PQ93100066
SOMA-GERMLINE ASYMMETRY IN THE DISTRIBUTIONS OF EMBRYONIC RNAS IN CAENORHABDITIS-ELEGANS DEVELOPMENT Seydoux, G., Fire, A. 1994; 120 (10): 2823-2834
Early embryogenesis in Caenorhabditis elegans is characterized by a series of unequal cleavages that mark the stepwise separation of somatic and germ lineages. We have developed an in situ hybridization protocol to examine the localization of specific maternal and embryonically transcribed messenger RNAs during these early cleavages. We detected three classes of maternal RNAs: RNAs that are maintained in all cells, RNAs that are maintained in germline cells but are lost from somatic cells, and a population of RNAs that are associated with the germline-specific P granules. We observed embryonically transcribed RNAs in somatic cells as early as the 4-cell stage. These transcripts were not detected in germline cells. These observations suggest that mechanisms which distinguish between soma and germline cause asymmetries in mRNA stability and transcription within the first few cleavages of C. elegans embryogenesis.
View details for Web of Science ID A1994PN07600008
THE CAENORHABDITIS-ELEGANS NK-2 CLASS HOMEOPROTEIN CEH-22 IS INVOLVED IN COMBINATORIAL ACTIVATION OF GENE-EXPRESSION IN PHARYNGEAL MUSCLE DEVELOPMENT Okkema, P. G., Fire, A. 1994; 120 (8): 2175-2186
The pharyngeal muscles of Caenorhabditis elegans are single sarcomere muscles used for feeding. Like vertebrate cardiac and smooth muscles, C. elegans pharyngeal muscle does not express any of the known members of the MyoD family of myogenic factors. To identify mechanisms regulating gene expression in this tissue, we have characterized a pharyngeal muscle-specific enhancer from myo-2, a myosin heavy chain gene expressed exclusively in pharyngeal muscle. Assaying enhancer function in transgenic animals, we identified three subelements, designated A, B and C, that contribute to myo-2 enhancer activity. These subelements are individually inactive; however, any combination of two or more subelements forms a functional enhancer. The B and C subelements have distinct cell type specificities. A duplication of B activates transcription in a subset of pharyngeal muscles (m3, m4, m5 and m7). A duplication of C activates transcription in all pharyngeal cells, muscle and non-muscle. Thus, the activity of the myo-2 enhancer is regulated by a combination of pharyngeal muscle-type-specific and organ-specific signals. Screening a cDNA expression library, we identified a gene encoding an NK-2 class homeodomain protein, CEH-22, that specifically binds a site necessary for activity of the B subelement. CEH-22 protein is first expressed prior to myogenic differentiation and is present in the same subset of pharyngeal muscles in which B is active. Expression continues throughout embryonic and larval development. This expression pattern suggests CEH-22 plays a key role in pharyngeal muscle-specific activity of the myo-2 enhancer.
View details for Web of Science ID A1994PA81400010
A 4-DIMENSIONAL DIGITAL IMAGE ARCHIVING SYSTEM FOR CELL LINEAGE TRACING AND RETROSPECTIVE EMBRYOLOGY COMPUTER APPLICATIONS IN THE BIOSCIENCES Fire, A. 1994; 10 (4): 443-447
The paper describes a digital image archiving system for time-lapse microscopy. The system uses an MS-DOS compatible computer to store video images while simultaneously controlling a stepping motor. In a typical experiment, images might be taken at 30 s intervals in each of 25 consecutive focal planes. A system with 2.5 Gbyte disk capacity can store approximately 18,000 full frame images: 6 h recording at maximum resolution. Once recorded, images series stored on disk can be 'played back' in any order. Generally, images from a single focal plane are displayed consecutively in either forward or reverse time. The focal plane can be shifted during playback, allowing individual cells to be followed as they move between focal planes. To facilitate the annotation and interpretation of the real-time images, a mouse-driven interface allows users to define and follow individual objects (e.g. cells). The recorded image series can be achieved inexpensively using standard digital tape backup hardware. In this laboratory, the system has been particularly useful for tracing embryonic cell lineages and cell migrations. Detailed system specifications, including source code, compiled programs, hardware requirements and users manual are available directly from the author or by anonymous FTP (ciw1.ciwemb.edu).
View details for Web of Science ID A1994PD68000010
THE CAENORHABDITIS-ELEGANS MYOD HOMOLOG HLH-1 IS ESSENTIAL FOR PROPER MUSCLE FUNCTION AND COMPLETE MORPHOGENESIS DEVELOPMENT Chen, L. S., Krause, M., Sepanski, M., Fire, A. 1994; 120 (6): 1631-1641
A family of muscle-specific helix-loop-helix transcription factors (myoD, myogenin, myf-5 and MRF4) has been implicated in the control of vertebrate skeletal myogenesis. Searches for homologues of this family in Caenorhabditis elegans identified a single family member, hlh-1, which is expressed in striated muscles and their clonal precursors. We have isolated a null allele of hlh-1 following chemical mutagenesis. Animals homozygous for the null mutation produce contractile body-wall muscles, although muscle contractions are weak and coordination is defective. In addition to the evident muscle defects, mutant animals fail to complete embryonic elongation and die as larvae or young adults. Ultrastructural analysis of the mutant muscle reveals an apparently normal local lattice of thick and thin filaments, with more global defects in sarcomere organization and muscle cell placement. Mosaic studies using the point mutation and an extrachromosomal transgene indicate that the requirement for hlh-1 is fully zygotic, with no maternal hlh-1 requirement for either muscle development or viability.
View details for Web of Science ID A1994NT55500025
A SCREEN FOR GENETIC-LOCI REQUIRED FOR BODY-WALL MUSCLE DEVELOPMENT DURING EMBRYOGENESIS IN CAENORHABDITIS-ELEGANS GENETICS Ahnn, J., Fire, A. 1994; 137 (2): 483-498
We have used available chromosomal deficiencies to screen for genetic loci whose zygotic expression is required for formation of body-wall muscle cells during embryogenesis in Caenorhabditis elegans. To test for muscle cell differentiation we have assayed for both contractile function and the expression of muscle-specific structural proteins. Monoclonal antibodies directed against two myosin heavy chain isoforms, the products of the unc-54 and myo-3 genes, were used to detect body-wall muscle differentiation. We have screened 77 deficiencies, covering approximately 72% of the genome. Deficiency homozygotes in most cases stain with antibodies to the body-wall muscle myosins and in many cases muscle contractile function is observed. We have identified two regions showing distinct defects in myosin heavy chain gene expression. Embryos homozygous for deficiencies removing the left tip of chromosome V fail to accumulate the myo-3 and unc-54 products, but express antigens characteristic of hypodermal, pharyngeal and neural development. Embryos lacking a large region on chromosome III accumulate the unc-54 product but not the myo-3 product. We conclude that there exist only a small number of loci whose zygotic expression is uniquely required for adoption of a muscle cell fate.
View details for Web of Science ID A1994NM10700013
SEQUENCE REQUIREMENTS FOR MYOSIN GENE-EXPRESSION AND REGULATION IN CAENORHABDITIS-ELEGANS GENETICS Okkema, P. G., HARRISON, S. W., Plunger, V., Aryana, A., Fire, A. 1993; 135 (2): 385-404
Four Caenorhabditis elegans genes encode muscle-type specific myosin heavy chain isoforms: myo-1 and myo-2 are expressed in the pharyngeal muscles; unc-54 and myo-3 are expressed in body wall muscles. We have used transformation-rescue and lacZ fusion assays to determine sequence requirements for regulated myosin gene expression during development. Multiple tissue-specific activation elements are present for all four genes. For each of the four genes, sequences upstream of the coding region are tissue-specific promoters, as shown by their ability to drive expression of a reporter gene (lacZ) in the appropriate muscle type. Each gene contains at least one additional tissue-specific regulatory element, as defined by the ability to enhance expression of a heterologous promoter in the appropriate muscle type. In rescue experiments with unc-54, two further requirements apparently independent of tissue specificity were found: sequences within the 3' non-coding region are essential for activity while an intron near the 5' end augments expression levels. The general intron stimulation is apparently independent of intron sequence, indicating a mechanistic effect of splicing. To further characterize the myosin gene promoters and to examine the types of enhancer sequences in the genome, we have initiated a screen of C. elegans genomic DNA for fragments capable of enhancing the myo-2 promoter. The properties of enhancers recovered from this screen suggest that the promoter is limited to muscle cells in its ability to respond to enhancers.
View details for Web of Science ID A1993LZ43200014
THE NOVEL METALLOTHIONEIN GENES OF CAENORHABDITIS-ELEGANS - STRUCTURAL ORGANIZATION AND INDUCIBLE, CELL-SPECIFIC EXPRESSION JOURNAL OF BIOLOGICAL CHEMISTRY Freedman, J. H., Slice, L. W., Dixon, D., Fire, A., Rubin, C. S. 1993; 268 (4): 2554-2564
Two genes (mtl-1 and mtl-2) that encode the novel metallothioneins (MTs) of Caenorhabditis elegans (CeMTs) were cloned and characterized. Both genes contain a single intron that interrupts codon 6 and short 3'-untranslated regions. However, their promotor regions are distinctively non-homologous. The mtl-2 promoter contains a TATAA box and a single putative metal regulatory element. These elements are absent in the mtl-1 promoter. Nevertheless, both CeMT1 and CeMT2 mRNAs are induced by cadmium and contain precisely initiated, 5'-untranslated sequences. The inducibility and cell type specificity of metallothionein gene expression were investigated in transgenic C. elegans that carry the lacZ (beta-galactosidase) reporter gene under the control of an mtl-1 or mtl-2 promoter sequence. Upon treatment of transgenic C. elegans with cadmium or heat stress, the mtl-2:lacZ fusion gene is abundantly and exclusively expressed in the intestinal cells of larvae and adult animals. Expression is not detected in the absence of metal or heat shock. In contrast, an mtl-1:lacZ construct is constitutively expressed in the pharynx and induced by cadmium and heat shock in the intestinal cells of C. elegans larvae. The metal-inducible expression of the mtl-1:lacZ gene is attenuated in adult transgenic nematodes. Thus, the activity of each mtl promoter is modulated by metals as well as developmental and environmental factors.
View details for Web of Science ID A1993KK81500046
MOLECULAR CHARACTERIZATION OF THE HER-1 GENE SUGGESTS A DIRECT ROLE IN CELL SIGNALING DURING CAENORHABDITIS-ELEGANS SEX DETERMINATION GENES & DEVELOPMENT Perry, M. D., Li, W. Q., Trent, C., Robertson, B., Fire, A., HAGEMAN, J. M., Wood, W. B. 1993; 7 (2): 216-228
We have characterized two transcripts from the male-determining her-1 locus in Caenorhabditis elegans. The larger transcript, which appears more important for male development, is predicted to encode a novel 175-amino-acid, cysteine-rich polypeptide with an apparent amino-terminal signal sequence and potential cleavage and glycosylation sites. Expression of a full-length cDNA construct for the larger transcript driven by a body-wall-myosin promoter causes extensive masculinization of all sexually dimorphic tissues in XX (normally hermaphrodite) animals. This activity is dependent on the presence of the her-1 signal sequence or a substitute synthetic signal sequence in the encoded polypeptide. These results suggest that a secreted product of the her-1 gene dictates male development.
View details for Web of Science ID A1993KM85900006
HISTOCHEMICAL TECHNIQUES FOR LOCATING ESCHERICHIA-COLI BETA-GALACTOSIDASE ACTIVITY IN TRANSGENIC ORGANISMS GENETIC ANALYSIS-BIOMOLECULAR ENGINEERING Fire, A. 1992; 9 (5-6): 151-158
Escherichia coli beta-galactosidase is a commonly used reporter molecule for analyzing gene expression. Recently, beta-galactosidase fusions have been applied to a variety of eukaryotic systems. The techniques for constructing and introducing beta-galactosidase fusion constructs as well as soluble assays for total enzyme function have been described in detail elsewhere. This article describes histochemical techniques for analyzing organisms that contain a functional beta-galactosidase fusion construct. The object is to determine semiquantitatively which cells are expressing the beta-galactosidase fusion protein, as well as the subcellular localization of the protein. Due to its prevalence in the author's laboratory, Caenorhabditis elegans is used as a canonical organism for the detailed methods described.
View details for Web of Science ID A1992KU40600007
BODY-WALL MUSCLE FORMATION IN CAENORHABDITIS-ELEGANS EMBRYOS THAT LACK THE MYOD HOMOLOG HLH-1 SCIENCE Chen, L., Krause, M., Draper, B., Weintraub, H., Fire, A. 1992; 256 (5054): 240-243
The myoD family of DNA binding proteins has been implicated in the control of myogenesis in a variety of organisms. Searches for homologs in the nematode Caenorhabditis elegans yielded only one gene, designated hlh-1, expressed in body-wall muscle cells and their precursors. To assess the role of hlh-1 in C. elegans myogenesis, genetic deficiencies spanning the hlh-1 locus were isolated after gamma irradiation. Embryos homozygous for these deficiencies exhibited extensive body-wall muscle differentiation, including expression of several characteristic myofilament proteins and weak contracile behavior. Thus, zygotic hlh-1 expression was not required for body-wall muscle precursors to adopt muscle cell fates.
View details for Web of Science ID A1992HM89600035
FUNCTIONAL CONSERVATION OF NEMATODE AND VERTEBRATE MYOGENIC REGULATORY FACTORS 1992 SPRING MEETING OF THE BRITISH SOC FOR CELL BIOLOGY : TRANSCRIPTIONAL REGULATION IN CELL DIFFERENTATION AND DEVELOPMENT Krause, M., Fire, A., WHITEHARRISON, S., Weintraub, H., Tapscott, S. COMPANY OF BIOLOGISTS LTD. 1992: 111–115
The Caenorhabditis elegans protein, CeMyoD, is related to the vertebrate myogenic regulatory factors MyoD, myogenin, MRF-4 and Myf-5. Like its vertebrate counterparts, CeMyoD accumulates in the nucleus of striated muscle cells prior to the onset of terminal differentiation. CeMyoD also shares functional similarities with the vertebrate myogenic regulatory factors. Viral LTR driven expression of CeMyoD in mouse 10T1/2 cells can convert this cell line into myoblasts as well as efficiently trans-activate mouse muscle-specific promoters. Furthermore, mouse MyoD expression can activate a CeMyoD-beta-galactosidase reporter construct in a 10T1/2 co-transfection assay.
View details for Web of Science ID A1992KQ21000014
PRODUCTION OF ANTISENSE RNA LEADS TO EFFECTIVE AND SPECIFIC-INHIBITION OF GENE-EXPRESSION IN C-ELEGANS MUSCLE DEVELOPMENT Fire, A., Albertson, D., HARRISON, S. W., Moerman, D. G. 1991; 113 (2): 503-514
We have used an antisense strategy to effectively disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles. DNA segments from the unc-22 and unc-54 genes have been placed in reverse orientation in vectors designed to produce RNA in body wall muscles. When the resulting plasmids are injected into oocytes, progeny with defects in muscle function are produced. These animals have phenotypes consistent with reduction and/or elimination of function of the gene to which antisense RNA has been produced: twitching and disorganization of muscle filaments for the unc-22 antisense constructs and lack of muscle tone, slow movement, and egg laying defects for the unc-54 antisense constructs. A fraction of the affected animals transmit the defective-muscle trait to subsequent generations. In these cases the transforming DNA is present at high copy number and cosegregates with the observed muscle defects. We have examined several of the unc-22 antisense plasmid transformed lines to determine the mechanistic basis for the observed phenotypes. The RNA product of the endogenous unc-22 locus is present at normal levels and this RNA is properly spliced in the region homologous to the antisense RNA. No evidence for modification of this RNA by deamination of adenosine to inosine was found. In affected animals the level of protein product from the endogenous unc-22 locus is greatly reduced. Antisense RNA produced from the transforming DNA was detected and was much more abundant than 'sense' RNA from the endogenous locus. These data suggest that the observed phenotypes result from interference with a late step in gene expression, such as transport into the cytoplasm or translation.
View details for Web of Science ID A1991GM22800011
CEMYOD ACCUMULATION DEFINES THE BODY WALL MUSCLE-CELL FATE DURING C-ELEGANS EMBRYOGENESIS CELL Krause, M., Fire, A., HARRISON, S. W., Priess, J., Weintraub, H. 1990; 63 (5): 907-919
We have cloned a gene from the nematode C. elegans that is closely related to the vertebrate MyoD gene family. The nematode gene product, CeMyoD, is a nuclear protein that is expressed specifically in body wall muscle cells. Antibody staining of early embryos shows that CeMyoD accumulates in early blastomeres that will subsequently produce only body wall muscle cells. CeMyoD is not detected in pharyngeal muscle cells or in nonmyogenic lineages. A CeMyoD-beta-galactosidase fusion gene is accurately expressed in myogenic cells that also express CeMyoD. In addition, the beta-galactosidase reporter is expressed as early as the 28 cell stage of embryogenesis in specific blastomeres prior to their clonal commitment to body wall muscle. This early fusion gene activity reveals that part of the specificity for CeMyoD transcription can arise very early in development and that subsequently, negative events may restrict CeMyoD expression in progeny cells not destined to become muscle.
View details for Web of Science ID A1990EL75900006
A MODULAR SET OF LACZ FUSION VECTORS FOR STUDYING GENE-EXPRESSION IN CAENORHABDITIS-ELEGANS GENE Fire, A., HARRISON, S. W., Dixon, D. 1990; 93 (2): 189-198
We describe a series of plasmid vectors which contain modular features particularly useful for studying gene expression in eukaryotic systems. The vectors contain the Escherichia coli beta-galactosidase (beta Gal)-encoding region (the lacZ gene) flanked by unique polylinker segments on the 5' and 3' ends, and several combinations of a variety of modules: a selectable marker (an amber suppressor tRNA), a translational initiation region, a synthetic intron segment, the early polyadenylation signal from SV40, and 3' regions from two nematode genes. A segment encoding the nuclear localization peptide from the SV40 T antigen is incorporated into many of the constructs, leading to beta Gal accumulation in nuclei, which can facilitate identification of producing cells in complex tissues. To make functional beta Gal fusions to secreted proteins, we constructed plasmids with an alternate module encoding a synthetic transmembrane domain upstream from lacZ. This domain is designed to stop transfer of secreted proteins across the membrane during secretion, allowing the beta Gal domain of the fusion polypeptide to remain in the cytoplasm and thus function in enzymatic assays. We have used the vectors to analyze expression of several genes in the nematode Caenorhabditis elegans, and have demonstrated in these studies that lacZ can be expressed in a wide variety of different tissues and cell types. These vectors should be useful in studying gene expression both in C. elegans and in other experimental systems.
View details for Web of Science ID A1990ED35800004
VECTORS FOR LOW COPY TRANSFORMATION OF C-ELEGANS NUCLEIC ACIDS RESEARCH Fire, A., Kondo, K., Waterston, R. 1990; 18 (14): 4269-4270
View details for Web of Science ID A1990DR45400051
PROPER EXPRESSION OF MYOSIN GENES IN TRANSGENIC NEMATODES EMBO JOURNAL Fire, A., Waterston, R. H. 1989; 8 (11): 3419-3428
Caenorhabditis elegans has four genes which encode skeletal myosin heavy chain isoforms. We have re-introduced clones of two of these genes, myo-3 and unc-54 at low copy number into the germline of C. elegans. The resulting loci behave as functional copies of the genes by two genetic criteria: (i) they can result in phenotypic rescue of strains carrying inactivating myo-3 or unc-54 mutations, and (ii) their presence in strains with wild-type copies of the endogenous myosin loci has genetic consequences similar to duplicating the endogenous loci. The re-introduced genes function at a level close to that of the endogenous loci. Monoclonal antibodies specific for the different isoforms have been used to localize the expressed proteins. The re-introduced genes express in precisely the same cell types as the endogenous genes, and the myosin products produced assemble into filament structures as in wild-type. Unexpectedly, we have found in the course of this work that very high copy numbers of the unc-54 gene lead to a disruption of muscle structure which may result from overexpression of the protein product.
View details for Web of Science ID A1989AV30800032
5,6-DICHLORO-1-BETA-D-RIBOFURANOSYLBENZIMIDAZOLE INHIBITS TRANSCRIPTION ELONGATION BY RNA POLYMERASE-II INVITRO JOURNAL OF BIOLOGICAL CHEMISTRY Chodosh, L. A., Fire, A., Samuels, M., Sharp, P. A. 1989; 264 (4): 2250-2257
The purine nucleoside analog 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is a selective inhibitor of transcription by RNA polymerase II. Although a wealth of in vivo studies have suggested that DRB inhibits transcription by enhancing the premature termination of elongating polymerase molecules, in vitro studies to date have been interpreted to suggest that DRB acts at the level of transcription initiation. We have analyzed the mechanism of DRB-mediated transcription inhibition in vitro both in HeLa whole cell extracts and in a partially purified transcription system. The results indicate that the extent to which DRB inhibits the synthesis of a RNA transcript is directly proportional to its length. For example, DRB was found to preferentially inhibit transcription in vitro of promoter-distal relative to promoter-proximal portions of the adenovirus major late transcription unit. A factor potentially involved in mediating this inhibitory effect is identified. We conclude that the mechanism of DRB inhibition of transcription in vivo and in vitro are similar.
View details for Web of Science ID A1989T106200055
INTEGRATIVE TRANSFORMATION OF CAENORHABDITIS-ELEGANS EMBO JOURNAL Fire, A. 1986; 5 (10): 2673-2680
A technique for introducing exogenous DNA into the chromosomes of the nematode Caenorhabditis elegans is presented. A cloned C. elegans amber suppressor tRNA gene, sup-7, is used as a selectable marker. The activity of this amber suppressor is selected for by injecting worms which carry an amber termination mutation in a gene (tra-3) whose function is required for fertility. Transient expression of sup-7 is evidenced by the presence of fertile (rescued) animals in the generation after injection. In a fraction of cases, these fertile animals give rise to stable suppressor lines (eight have been characterized so far). Each of the stable suppressor lines carries injected DNA sequences. The suppressor activities have been mapped to chromosomal loci, indicating that the exogenous DNA has integrated into the genome. This technique has been used to introduce a chimeric gene containing a Drosophila heat shock promoter element fused to coding sequences from the Escherichia coli beta-galactosidase gene. This chimeric gene functions and is heat inducible in the resulting stably transformed lines.
View details for Web of Science ID A1986E419000035
INTERACTIONS BETWEEN RNA POLYMERASE-II, FACTORS, AND TEMPLATE LEADING TO ACCURATE TRANSCRIPTION JOURNAL OF BIOLOGICAL CHEMISTRY Fire, A., Samuels, M., Sharp, P. A. 1984; 259 (4): 2509-2516
Accurate transcription by RNA polymerase II has been shown to require multiple factors in addition to the purified polymerase. In this study, we use a reconstituted transcription system, consisting of purified RNA polymerase II and three essential HeLa cell chromatographic fractions, to study events leading to transcription from the adenovirus major late promoter. A preincubation-pulse-chase protocol resolves the reaction into events occurring before and after nucleotide addition. Preincubation of template with a mixture of RNA polymerase II and factors allows formation of "activated" complexes, which are defined by the ability to rapidly commence accurate transcription when presented nucleotides. Maximal activation requires that polymerase, template, and each of the three HeLa fractions be present during preincubation. The activated complexes are template associated, as shown by their inability to exchange onto a second template added during further preincubation. Similar protocols are used to define functional intermediates leading to the activated complex. A template-associated functional complex is formed during the preincubation of template with just two of the HeLa fractions. Polymerase can associate with this intermediate complex in the absence of the third HeLa fraction. In the accompanying paper, we describe a direct analysis of initiation by "activated" complexes.
View details for Web of Science ID A1984SE62900077
DINUCLEOTIDE PRIMING OF TRANSCRIPTION MEDIATED BY RNA POLYMERASE-II JOURNAL OF BIOLOGICAL CHEMISTRY Samuels, M., Fire, A., Sharp, P. A. 1984; 259 (4): 2517-2525
Mammalian RNA polymerase II was shown to utilize dinucleoside monophosphates for priming of promoter specific RNAs. In a reconstituted system containing purified polymerase and HeLa cell fractions, dinucleotides were incorporated by complementarity with template sequences at the in vivo cap sites of the adenovirus major late and adenovirus early region IV promoters. Incorporation was shown by label transfer experiments and by determining the size of 5'-terminal RNase T1-resistant oligonucleotides. All 16 dinucleotides were tested for priming of RNA chains at the major late promoter. RNA polymerase II initiated with various primers over a contiguous region of 9 bases, centered around the in vivo initiation site. We suggest that the polymerase drifts or oscillates over this region. Using a dinucleotide challenge protocol, the rate of initiation at the major late promoter was measured following preincubation of the template DNA with RNA polymerase II and factors. Initiation with ATP was 90% complete within the 1st min after addition of nucleotide triphosphates. Stimulation of transcription by dinucleotides was not observed, due to this rapid initiation. The 5'-hydroxyl terminus of dinucleotide-primed RNAs remained unmodified. Although transcripts initiated with ATP were rapidly capped in whole cell extracts, ATP-primed RNA synthesized in the reconstituted system retained free 5'-terminal phosphates. Thus, capping was not essential for synthesis of long runoff RNAs.
Characterization of tRNA precursor splicing in mammalian extracts. journal of biological chemistry Laski, F. A., Fire, A. Z., RajBhandary, U. L., Sharp, P. A. 1983; 258 (19): 11974-11980
Transcription of a Xenopus laevis tRNATyr gene and splicing of the transcript have been studied in HeLa cell extracts. This tRNATyr gene has a 13-base intervening sequence and is expressed as mature tRNA when transfected into mammalian cells. The tRNATyr gene is transcribed under conditions of low concentrations of magnesium and ATP, but is processed by splicing only when both of these cofactors are added at higher concentrations. The endonucleolytic activity of the tRNA-splicing system in the HeLa extract produces exons with 3'-phosphate and 5'-hydroxyl groups. The 3'-phosphate is retained during the ligation reaction and forms the phosphodiester bond in the mature tRNA. Retention of the 3'-phosphate during tRNA splicing differs from the more extensively studied process in yeast extracts where a phosphate group from an ATP cofactor is used to form the phosphodiester bond joining the exons. Thus, eucaryotic organisms can splice tRNA precursors by at least two distinguishable mechanisms.
INVITRO TRANSCRIPTION - WHOLE-CELL EXTRACT METHODS IN ENZYMOLOGY Manley, J. L., Fire, A., Samuels, M., Sharp, P. A. 1983; 101: 568-582
View details for Web of Science ID A1983RD90000036
CHARACTERIZATION OF TRANSFER-RNA PRECURSOR SPLICING IN MAMMALIAN EXTRACTS JOURNAL OF BIOLOGICAL CHEMISTRY Laski, F. A., Fire, A. Z., RajBhandary, U. L., Sharp, P. A. 1983; 258 (19): 1974-1980
View details for Web of Science ID A1983RL31600088
Separation and characterization of factors mediating accurate transcription by RNA polymerase II. journal of biological chemistry Samuels, M., Fire, A., Sharp, P. A. 1982; 257 (23): 14419-14427
A whole cell extract of HeLa cells was resolved through two successive chromatographic steps using an extension of the procedure of Matsui et al. (Matsui, T., Segall, J., Weil, P. A., and Roeder, R. G. (1980) J. Biol. Chem. 255, 11992-11996). RNA polymerase II and three of the resulting fractions were necessary and sufficient for accurate transcription of the adenovirus major late promoter. This accurate transcription was quantitated as a function of each of the required fractions, polymerase, and DNA. A linear range of response was observed in each case. Using the linear ranges for assay, it was possible to calculate net purifications and yields for each of the required transcriptional activities after chromatography. These activities were each shown to sediment with a distinct peak on sucrose gradients. The effects of variations in salt concentration, magnesium concentration, temperature, and reaction time were determined. High resolution analysis of runoff transcripts showed that the reconstituted system initiated transcription precisely at the adenovirus major late and early region IV promoters.
SEPARATION AND CHARACTERIZATION OF FACTORS MEDIATING ACCURATE TRANSCRIPTION BY RNA POLYMERASE-II JOURNAL OF BIOLOGICAL CHEMISTRY Samuels, M., Fire, A., Sharp, P. A. 1982; 257 (23): 4419-4427
INVITRO TRANSCRIPTION OF ADENOVIRUS JOURNAL OF VIROLOGY Fire, A., Baker, C. C., Manley, J. L., Ziff, E. B., Sharp, P. A. 1981; 40 (3): 703-719
A series of recombinants of adenovirus DNA fragments and pBR322 was used to test the transcriptional activity of the nine known adenovirus promoters in a cell-free extract. Specific initiation was seen at all five early promoters as well as at the major late promotor and at the intermediate promoter for polypeptide IX. The system failed to recognize the two other adenovirus promoters, which were prominent in vivo only at intermediate and late stages in infection. Microheterogeneity of 5' termini at several adenovirus promoters, previously shown in vivo, was reproduced in the in vitro reaction and indeed appeared to result from heterogeneous initiation rather than 5' processing. To test for the presence of soluble factors involved in regulation of nRNA synthesis, the activity of extracts prepared from early and late stages of infection was compared on an assortment of viral promoter sites. Although mock and early extracts showed identical transcription patterns, extracts prepared from late stages gave 5- to 10-fold relative enhancement of the late and polypeptide IX promoters as compared with early promoters.
View details for Web of Science ID A1981MT86000009
INHIBITION OF TRANSCRIPTION FACTOR ACTIVITY BY POLIOVIRUS CELL Crawford, N., Fire, A., Samuels, M., Sharp, P. A., Baltimore, D. 1981; 27 (3): 555-561
To study the poliovirus-induced inhibition of host-cell RNA synthesis, we prepared transcription extracts from mock-infected and poliovirus-infected HeLa cells. In contrast with the control extracts, poliovirus-infected cell extracts prepared 3 hr after infection were unable to transcribe specifically DNA templates recognized by RNA polymerase II. Accurate transcription by RNA polymerase III, however, was only slightly reduced. Supplementation of the infected cell extract with a crude preparation of transcription factors (S100) restored its ability to transcribe a polymerase II template specifically; supplementation with purified polymerase II had no effect. When the S100 was fractionated on a phosphocellulose column, the restoration activity eluted between 0.35 M and 1 M KCl. When we tested infected extracts for inhibitory activity by mixing uninfected and infected cell extracts, no in vitro inhibition of polymerase II transcription by the uninfected extract was evident. These results indicate that at least one factor required for specific transcription by polymerase II is deficient in extracts from poliovirus-infected cells.
View details for Web of Science ID A1981MW20000017
Regulation of adenovirus mRNA synthesis. Annals of the New York Academy of Sciences Sharp, P. A., Manley, J., Fire, A., Gefter, M. 1980; 354: 1-15
The lytic cycle of adenovirus is a tightly regulated sequence of stages. When this regulation is studied at the level of mRNA production, the most significant step in controlling gene expression is initiation of transcription. Thus in preceding from one stage of expression to another, viral factors seem to turn on transcription of new sets of genes. At the moment, it is thought that viral mRNA synthesis involves initiation of transcription at ten different promoter sites. It is likely that in some manner the frequency of an initiation of transcription at nine of these sites is affected by one or more viral gene products. With the recent development of soluble in vitro transcription systems that respond to exogenously added DNA, it should be possible to begin to study regulation of gene expression at this stage of transcription. At present, these systems yield the paradoxical observation that extracts prepared from uninfected human cells more efficiently recognize the late promoter as compared to the early promoter of adenovirus. As more is learned about regulation of synthesis of viral mRNAs, examples will surely be found where RNA processing and RNA turnover play a critical role in determining the level of mRNAs. Such cases are more likely to appear in the balancing of synthesis of different mRNAs derived from one transcriptional unit. Few experiments have been directed to this possibility and the study of adenovirus molecular biology is only now entering the age of maturity where these experiments are feasible.
DNA-DEPENDENT TRANSCRIPTION OF ADENOVIRUS GENES IN A SOLUBLE WHOLE-CELL EXTRACT PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Manley, J. L., Fire, A., Cano, A., Sharp, P. A., Gefter, M. L. 1980; 77 (7): 3855-3859
We have developed a cell-free system for studying the synthesis of mRNA in mammalian cells. The system consists of a dialyzed and concentrated whole-cell extract derived from HeLa cells, small molecules and cofactors needed for transcription, and exogenously added DNA. Accurate transcription by RNA polymerase II is entirely dependent upon addition of promoter-containing eukaryotic DNA. At optimal DNA and extract concentrations, transcription initiation from the adenovirus serotype 2 late promoter is readily detectable, and specific transcripts over 4000 nucleotides in length are observed. The RNA synthesized in vitro contains the same 5' capped RNase T1 undecanucleotide as does the in vivo transcript. RNA synthesis also initiates accurately at both an early and an intermediate adenovirus promoter site.
View details for Web of Science ID A1980KB88900020