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The Mapping of Predicted Triplex DNA:RNA in the Drosophila Genome Reveals a Prominent Location in Development- and Morphogenesis-Related Genes
View ORCID ProfileClaude Pasquier, Sandra Agnel and Alain Robichon
G3: Genes, Genomes, Genetics July 1, 2017 vol. 7 no. 7 2295-2304; https://doi.org/10.1534/g3.117.042911
Claude Pasquier
*Université Côte d'Azur, CNRS, I3S, France
ORCID record for Claude Pasquier
For correspondence: claude.pasquier@unice.fr
Sandra Agnel
†Université Côte d'Azur, INRA, CNRS, ISA, France
Alain Robichon
Double-stranded DNA is able to form triple-helical structures by accommodating a third nucleotide strand. A nucleic acid triplex occurs according to Hoogsteen rules that predict the stability and affinity of the third strand bound to the Watson–Crick duplex. The "triplex-forming oligonucleotide" (TFO) can be a short sequence of RNA that binds to the major groove of the targeted duplex only when this duplex presents a sequence of purine or pyrimidine bases in one of the DNA strands. Many nuclear proteins are known to bind triplex DNA or DNA:RNA, but their biological functions are unexplored. We identified sequences that are capable of engaging as the "triplex-forming oligonucleotide" in both the pre-lncRNA and pre-mRNA collections of Drosophila melanogaster. These motifs were matched against the Drosophila genome in order to identify putative sequences of triplex formation in intergenic regions, promoters, and introns/exons. Most of the identified TFOs appear to be located in the intronic region of the analyzed genes. Computational prediction of the most targeted genes by TFOs originating from pre-lncRNAs and pre-mRNAs revealed that they are restrictively associated with development- and morphogenesis-related gene networks. The refined analysis by Gene Ontology enrichment demonstrates that some individual TFOs present genome-wide scale matches that are located in numerous genes and regulatory sequences. The triplex DNA:RNA computational mapping at the genome-wide scale suggests broad interference in the regulatory process of the gene networks orchestrated by TFO RNAs acting in association simultaneously at multiple sites.
Hoogsteen rules
gene networks
triplex DNA:RNA
Noncanonical DNA structures such as the triplex have been the subject of extensive studies in biophysics using simple synthetic templates. These structures may also involve RNA as a third strand to achieve tertiary structures (Cheng and Pettitt 1992; Escudé et al. 1993; Frank-Kamenetskii and Mirkin 1995; Morgan and Wells 1968; Plum 1997; Roberts and Crothers 1992, 1996; Sun et al. 1996). These combinatorial assemblages have been confirmed by thermodynamic analyses that enabled a determination of the constants of association and dissociation of the third strand to double-stranded DNA depending on pH and its length (Cheng and Pettitt 1992; Escudé et al. 1993; Frank-Kamenetskii and Mirkin 1995; Morgan and Wells 1968; Plum 1997; Roberts and Crothers 1992, 1996; Sun et al. 1996). Although the measured affinities were able to indicate the stability of triplexes, their presence and biological functions in vivo have been poorly explored due to technical limitations. However, we have solid and indirect evidence of their putative roles and existence from the finding that a few transcription factors and other DNA-binding proteins show high-affinity binding to some triplexes (Buske et al. 2011). Their roles in the genomic context of regulation of gene networks remain elusive.
Basically, strict sequence alignments are required for stable triple-helix formation via hydrogen bonds between the bases of a DNA duplex and a third RNA strand according to Hoogsteen base-pairing rules. The sequences in the DNA duplex must show a succession of pyrimidine bases [T, C] in one strand and a consequent succession of purine base repeats [G or A] in the opposite strand. On the RNA side, the sequences composed of pyrimidine bases [U, C] must be the copy of one DNA strand and bind parallel to the DNA duplex, or the sequences of the purine bases [G, A] must be the copy of one DNA strand and bind antiparallel to the DNA duplex. A third possibility is the sequence of purine-pyrimidine bases [G, T, or A] in the RNA strand that binds antiparallel and in which T or A can indistinctly bind to the A:T pair in the DNA strand (Cheng and Pettitt 1992; Escudé et al. 1993; Frank-Kamenetskii and Mirkin 1995; Morgan and Wells 1968; Plum 1997; Roberts and Crothers 1992, 1996; Sun et al. 1996). The minimal requirement for the third strand to form a DNA:RNA triplex is a length of nine bases; the strand is typically 10–30 nt in length (Buske et al. 2011; Cheng and Pettitt 1992; Escudé et al. 1993; Frank-Kamenetskii and Mirkin 1995; Knauert and Glazer 2001; Morgan and Wells 1968; Plum 1997; Roberts and Crothers 1992, 1996; Sun et al. 1996). However, the affinity between the strands when the matches are perfect increases with the length of the third strand. The large number of compatible sequences over 20 bases in length in the RNA presenting the potential to form triplexes in the genome strongly suggests that these structures might have a large spectrum of biological functions under their control, which are unknown to date.
We used Triplexator (Buske et al. 2011) to investigate the putative triple helix according to the Hoosgteen rules, involving a double-stranded nucleic acid and a single-stranded RNA on a genome-wide scale and hypothesizing that these structures might constitute a new component of epigenetic regulation in gene imprinting, developmental biology, and environmental adaptation. The melting curves in biophysics studies have led to the general acceptance that a higher affinity is obtained when the third strand is RNA instead of DNA (Cheng and Pettitt 1992; Escudé et al. 1993; Morgan and Wells 1968; Roberts and Crothers 1992). More importantly, many authors have attempted in vivo investigations in diverse organisms to determine the roles of such structures in relation to chromosome architecture, gene regulation, and human pathologies, such as cancer or hereditary neurological disorders (Buske et al. 2011). Furthermore, the aim of investigating the putative extent of these structures in genomes was inspired by the fact that we found that a large panel of nuclear proteins have been reported to bind triplex structures. For instance, the high mobility group proteins HMG1 and HMG2 are chromatin-associated proteins and are able to bind triplexes (Buske et al. 2011; Suda et al. 1996). Their roles seem to involve the higher architecture of chromosomes and the distribution of euchromatin/heterochromatin (Buske et al. 2011; Suda et al. 1996). As another example, the GAGA transcription factor is reported to bind a pyrimidine triplex motif with the same affinity and specificity as for a duplex (Jiménez-García et al. 1998). These triplex structures inhibit the expression of genes located downstream (Jiménez-García et al. 1998). The GAGA DNA motif and GA factor in Drosophila are strongly involved in developmental patterning of the body by regulating the homeotic bithorax genes in the Antennapedia complex (Granok et al. 1995; Lehmann 2004). Moreover, GA factors are known to participate in the epigenetic maintenance of heterochromatin and the silencing of genes embedded in this structure (Granok et al. 1995; Lehmann 2004). As another example, CDP1 (centromere binding factor) binds to a purine motif triplex with high affinity (Kd 5 nM), whereas the pyrimidine motif triplex along with the duplex shows a low affinity (5 µM) (Musso et al. 2000). CDP1 mutants show defects in chromosome segregation during mitosis and a high level of chromosome fragmentation (Musso et al. 2000). Moreover, the dihydrofolate reductase (DHFR) promoter is able to engage the triplex structure with some RNAs, which prevents transcription factor binding (Buske et al. 2011; Martianov et al. 2007). Finally, on a health-related topic, Friedreich ataxia is caused by the insertion of GAA/TTC repeats located in the first intron of the FXN gene, creating triplex structures (Soragni et al. 2008). The histones surrounding the ataxia triplex structures show hypoacetylation and hyper-methylation (Soragni et al. 2008). Altogether, these examples highlight that pieces of RNA presenting sequences that are compatible with Hoogsteen rules might have a widespread role in epigenetically regulating a large number of genes.
We chose to work on the simple genetic model Drosophila melanogaster because of the availability of genome annotation information with substantial descriptions of mutant phenotypes in multiple databases. We investigated whether the two classes of RNA (lncRNA and mRNA) sheltered compatible sequences to engage the triplex structure with DNA, raising the idea that they might have a widespread role in chromosome 3D architecture and gene regulation. lncRNAs are known to specifically and restrictively recruit promoters, resulting in downregulation or overexpression of targets genes (Mercer et al. 2009; Rinn and Chang 2012). Many lncRNAs are composed of a few hairpin structures that form higher order spatial architecture by their association with hairpin-bound proteins. Each hairpin motif is supposed to bind tightly to a specific nuclear protein involved in the transcription machinery and 3D chromatin organization (Chen 2016; Goff and Rinn 2015; Li et al. 2016; Quinn and Chang 2015; Rinn and Chang 2012; Tsai et al. 2010). These multimeric associations form extensive networks of ribonucleoprotein (RNP) complexes that target genomic locations (Chen 2016; Goff and Rinn 2015; Li et al. 2016; Quinn and Chang 2015; Rinn and Chang 2012; Tsai et al. 2010). Furthermore, lncRNAs appear to produce alternative splice variants acting as modular scaffolds for variable RNP complexes that specify chromatin states (Chen 2016; Goff and Rinn 2015; Li et al. 2016; Mercer et al. 2009; Quinn and Chang 2015; Rinn and Chang 2012; Tsai et al. 2010). lncRNAs are noncoding and defined as being larger than 200 nt in length (Chen 2016; Goff and Rinn 2015; Quinn and Chang 2015; Rinn and Chang 2012). Despite these advances, most lncRNAs remain partially or not at all characterized. A large fraction of these lncRNAs are predominantly or strictly localized to the nucleus, although a few seem exclusively present in the cytoplasm (Cabili et al. 2015; Chen 2016; Geisler and Coller 2013).
While many lncRNAs are rapidly degraded after or during the completion of their transcription, a significant fraction of the lncRNAs are stable nuclear transcripts that are 5′-capped, spliced, and polyadenylated similar to mRNAs (Chen 2016; Goff and Rinn 2015; Li et al. 2016; Quinn and Chang 2015; Rinn and Chang 2012; Wilusz et al. 2009). While all these structural modifications are known to stimulate the nuclear export of protein-coding transcripts, this is not the case for lncRNAs, most of which remain surprisingly locked inside the nuclear perimeter (Cabili et al. 2015; Chen 2016; Geisler and Coller 2013).
Because some lncRNAs are located inside the introns of genes, this finding raises the possibility that pieces of intronic RNA in the mRNA collection may have widespread regulatory functions beyond translation. The TFOs (triplex-forming oligonucleotides) that were found in the RNA (pre-lncRNA and pre-mRNA) were blasted against the entire genome, and then, location analysis was performed. The genes to which the TFO matched were submitted to wide-scale genome GO enrichment analysis in order to find networks of genetic interactions. The results unambiguously suggest that genes involved in development and morphogenesis might be a preferential target for TFO binding.
Data for the D. melanogaster genome are stored in the FTP directory of the FlyBase website that stores FASTA sequences. We downloaded FASTA sequences of genes, introns, exons, transcripts (mRNAs and lncRNAs), and chromosomes. Our work was based on version 6.06 of the data, released on June 26, 2015.
Triplex identification
There are several algorithms dedicated to the identification of triplexes. Triplexator (Buske et al. 2012) is the first computational framework that integrates all aspects of triplex formation. It allows the detection of triplex-forming oligonucleotides (TFOs), triplex-target sites (TTSs), and the localization of TFO:TSS matchings that are potential sites for triplex formation. LongTarget (He et al. 2015) includes similar features plus the ability to determine the best TFO:TTS matchings and to evaluate triplex stability. Unfortunately, the software is no longer available for download. Trident (Paugh et al. 2016) is dedicated to the identification of microRNAs favoring triplex formation. The tool cannot be used to detect TFOs in long RNA sequences. In our study, we used Triplexator, which has all the features that we needed.
The FASTA sequences of genes encoding lncRNAs and mRNAs were processed to identify TFOs of a minimum length of 20 bases that complied strictly to the triplex formation rules. The following motifs were searched:
the pyrimidine motif, [TC], where thymines and cytosines bind parallel;
the purine motif, [GA], where guanines and adenines bind antiparallel; and
the purine-pyrimidine motif, [GT], where guanines and thymines bind either parallel or antiparallel with respect to the purines in the duplex.
This search led to the identification of 896 TFOs in pre-lncRNAs and 12,898 TFOs in pre-mRNAs.
Using the files available in FlyBase, we distinguished the sequences corresponding to the introns and the sequences corresponding to the exons. We show the number of TFOs corresponding to each of these categories in Table 1.
Table 1 Distribution of TFOs
The program Triplexator was also used to process the FASTA sequences of the chromosomes, genes, and exons in order to identify TTSs and to detail their distribution. For this search, no mismatches were allowed, and a minimum length of 20 bases was also used. Using these parameters, the program identified 9702 target sites on the chromosomes.
Potential triplex sites were also computed with the Triplexator program using perfect matches between TFOs and TTSs and a minimum length of 20 bases. Normally, each TFO was potentially able to form a triplex with the TSS located at the same position on the complementary strand. These systematic auto-matches did not bring anything to the study of the importance of the TFO-TSS matches on a genome-wide scale. We decided not to take into account these self matches. Accordingly, approximately one quarter of the TFOs, whose only matches are with the TSSs located at the same position on the reverse strand, were removed. However, most of the time, each TFO could target TTSs at numerous sites, including matches that differed in a single nucleotide (this could be due to a simple shift or because the region suitable for triplex formation included one of a smaller size). The analysis of raw outputs generated by the program has led to a count of 2,249,658 potential triplexes with TFOs originating from pre-lncRNAs and 29,339,437 potential triplexes for TFOs originating from pre-mRNAs. We filtered these results by considering that two sites that overlap over >10 bases constituted the same triplex. This processing allowed a reduction in the number of potential triplexes to 354,963 and 4,317,708 for pre-lncRNAs and pre-mRNAs, respectively. We performed the same processing for TFOs originating from introns and TFOs originating from exons.
All statistics are calculated from results obtained by setting the number of maximum mismatches to zero. We used this parameter because allowing some errors increased the computation time and the number of identified sequences. For example, with a maximum of one error every 20 bases (an error rate of 5%), the processing time was multiplied by 35 (12 hr vs. 20 min), and the sequences to analyze were much more numerous (3,083,421 triplexes originating from pre-lncRNAs vs. 354,963 and >38 million triplexes originating from pre-mRNAs). However, at least for a proportion of the errors that did not exceed 5%, this consideration did not change the overall repartition of patterns, as seen by comparing Supplemental Material, Table S17 in File S4 with Table 2.
Table 2 Distribution of TTSs and triplexes on the genome
Estimation of the presence of paired double helixes in TFOs
We searched the RNAs for every sequence of at least four bases that could fold back on itself to form a loop that varied in size from 4 to 8 bases long. In addition, the presence of long-loop regions, such as those occurring in long-range pseudoknots, were identified by searching for the RNA:RNA interactions of at least 10 bases separated by a loop region up to 500 bases. We did a systematic search of the pre-RNAs (for both lncRNAs and mRNAs) and mature forms. The presence of the intermolecular interactions in the mature RNA TFOs was estimated at 15.5 and 16.4% for lncRNAs and mRNAs, respectively. The presence of these potential paired double helices in the pre-RNA TFOs was estimated at 25.7 and 21.9% for pre-lncRNAs and pre-mRNAs, respectively.
Gene ontology enrichment analysis
The distributions of potential triplexes by genes formed with pre-lncRNA TFOs and pre-mRNA TFOs highlight the fact that a limited number of genes concentrate most of the triplexes (Tables S12 and S13 in File S4 list the genes most targeted by the pre-lncRNA TFOs and pre-mRNA TFOs, respectively). However, the contribution of some genes to the formation of the triplexes was more due to their above-average lengths than to their enrichment in TSS zones. To highlight triplex-enriched genes, we normalized the count of the triplexes by taking into account the lengths of the genes. As a baseline, we used all the matching combinations between TFO and TSS divided by the sum of gene lengths. On average, we could count 32 TFO-TSS matches every 1000 bases (File S1 contains lists of genes targeted by pre-lncRNA TFOs and pre-mRNA TFOs sorted by the density of the TFO-TSS matches). For Gene Ontology enrichment analysis, we used the lists of genes that accounted for 75% of the total number of triplexes and that grouped a count of TFO-TSS matches per 1000 bases above the average. This list represented 35 genes targeted by pre-lncRNA TFOs and 447 genes targeted by pre-mRNA TFOs.
Transcripts that contributed to the formation of TFO-TTS matches had similar distributions (Tables S8 and S9 in File S4 list the pre-lncRNAs and pre-mRNAs that contributed the most to the formation of triplexes). For Gene Ontology enrichment analysis, we used the transcripts that contributed to the formation of >75% of the triplexes with a density of TFO-TSS matches above the average. These transcripts represented 381 pre-mRNAs and 15 pre-lncRNAs (File S1 contains lists of pre-lncRNAs and pre-mRNAs that contributed to the formation of triplexes sorted by the density of the TFO-TSS matches). For genes encoding lncRNAs that had few annotations, no significant enrichment was found.
Gene Ontology enrichment analysis was performed with the online service provided on the Gene Ontology Consortium website: http://geneontology.org/.
Analysis of genetic interactions
Genetic interactions for D. melanogaster were downloaded from the FTP directory of the FlyBase website that stored the precomputed files. Our analysis was based on release "FB2015_03."
The 35 genes that were most targeted by pre-lncRNA TFOs are included in the list of genes that were most targeted by pre-mRNA TFOs. We used the list of 447 genes enriched in the triplexes to build a network showing the genetic interactions that existed between two different genes on the list. After this filtering, we obtained a list of 76 interacting genes. From this list, 24 genes belonged to disconnected groups of two or three interacting genes. The 52 remaining genes shaped a large network. Network topology was analyzed by Cytoscape (Shannon et al. 2003) and the plugin ClusterViz (Wang et al. 2014). The EAGLE algorithm (Shen et al. 2009) in ClusterViz was used to identify three network modules containing 8–34 genes. The EAGLE clustering method, included in ClusterViz, was able to find spanning clusters, meaning that in our case, some genes were common to several clusters. Gene Ontology enrichment analyses were performed on the list of genes composing these modules and used to annotate the graph presented in Figure 3.
Isolation of the triplex structures with a biotinylated probe from the cleaved genome
The genomic DNA extraction was performed with the Isolate II Genomic DNA kit from Bioline. Drosophila were directly ground in lysis buffer and incubated for 3 hr. Double-stranded genomic DNA fragments were produced using dsDNA Fragmentase from New England Biolabs. The incubation was performed for 20 min in order to obtain 200–300 bp fragments. After the digestion, the DNA was left at room temperature for 1 hr to let the triplex reform. The fragments were incubated with TO biotin (5 µl of TO for 50 µl of DNA) for 30 min at 25°. Then we added TO (without biotin) in excess to compete for the "nonspecific" DNA duplex binding, at a concentration of 100 µg/µl. The incubation was performed for 30 min at 25°. Then the DNA-TO biotin complexes were isolated using a Dynabeads MyOne Streptavidin C1 Kit from Invitrogen. The beads were used in excess (10 µg for 50 µl of DNA). Two washes at room temperature were performed according to the kit instructions. The beads were then placed at 90° for 30 min to break the binding between the streptavidin and biotin, and the supernatant was collected. A measurement by Nanodrop showed a very low amount of DNA. To amplify these few fragments, we used a High-Sensitivity DNA Library Preparation Kit from Abcam. This kit included a DNA end polishing, an adaptor ligation, an amplification by PCR, and a clean-up of the library. The fragments were cloned into the pCR 2.1-TOPO vector using a TOPO TA Cloning Kit from Invitrogen, with an incubation of 30 min. TOP10 chemically competent bacteria were transformed with the resulting plasmids. The bacteria were plated on LB agar petri dishes prepared with kanamycin and X-gal to discriminate bacteria with recombinant plasmids. Approximately 300 colonies were analyzed by Sanger sequencing.
More detailed results (3 figures and 18 tables) are available within File S1, File S2, and File S3. Complete versions of data listed in Tables S8 and S9 in File S4 and Tables S12 and S13 in File S4 are available in File S1 while File S2 contains the complete versions of data listed in Tables S10 and S11 in File S4 and the complete GO annotations used to build Tables S14–S16 in File S4. All data associated with Figure 3 are listed in File S3: list of genes on the graph, composition of each cluster, GO enrichments obtained for the whole graph, and the three identified clusters.
We analyzed the pre-lncRNA and pre-mRNA sequences of D. melanogaster to identify the putative sites of triplex formation for at least 20 ribonucleotides that comply strictly to the triplex formation rules. Of the 2470 pre-lncRNA and the 13919 pre-mRNA sequences stored in FlyBase, a notable proportion of them, between 10 and 20% (269 and 2888, respectively), contain triplex-forming oligonucleotide (TFO) patterns. Most of them include only one TFO, but several transcripts contain more (Table S1 in File S4 lists the transcripts including >50 TFOs).
The pre-lncRNAs contain 896 TFOs of lengths ranging from 20 to 58 ribonucleotides with a mean of 24 (the distribution of TFOs shorter than 46 bases is shown in Figure 1A). Only seven sequences with lengths over 45 bases have been identified in seven pre-lncRNAs (Table S2 in File S4). All the genes encoding these lncRNAs have unknown functions.
Distribution of TFOs by length and type. Distribution of TFOs of length <46 identified in lncRNAs. Bars show, for each length, the number of TFOs corresponding to the different motifs. (A) Distribution of the TFOs identified in lncRNAs (TFOs longer than 45 bases are listed in Table S2 in File S4). (B) Distribution of the TFOs of lengths <46 identified in pre-mRNAs (TFOs longer than 100 bases are listed in Table S3 in File S4).
Pre-mRNAs containing 12,898 TFOs with an average length of 24 bases but with longer motifs, up to 460 ribonucleotides in length, were identified (the distribution of TFOs shorter than 46 bases is shown in Figure 1B, while TFOs longer than 100 bases are listed in Table S3 in File S4).
Detailed analysis showed that the TFOs are mainly located inside introns, with a proportion of 70% for both pre-lncRNAs and pre-mRNAs (Table 1).
To verify that most TFOs are not located in regions that might not be accessible for RNA:DNA triplex formation, we determined a rough estimate of the number of TFOs potentially included in the paired double helix (see Materials and Methods). At the most, the number of potentially not accessible TFOs does not exceed 25%.
A search for DNA sequences theoretically able to bind a third strand identified 9702 unique triplex-target sites (TTS) along the genome. Some sites are extensive, with a length of up to 490 nucleotides (the list of TTSs that are longer than 100 bases is presented in Table S4 in File S4, while the distribution of TTSs shorter than 51 bases is presented in Figure S1 in File S4). Detailed analysis revealed that 7486 TTSs (60% of the total number of sites) are located inside open reading frames (ORFs) with 5801 of them (77%) located inside introns (TTS column in Table 2). Many TTSs consist of repeated sequences along the chromosomes, strongly suggesting that a particular TFO might target many sites far beyond the gene that generates it.
The number of TTSs by gene ranges from 1 to 93 (Table S5 in File S4 lists the genes that contain 30 TTSs or more). Consequently, 2471 different genes contain triplex-target sites.
An assessment of potential triplex sites was performed by looking at the perfect matches between the TFOs and TTSs. Although the number of identified TFOs and TSSs is not very large (∼14,000 TFOs and <10,000 TSSs), the number of potential TFO:TSS matches is considerable. As an example, the 12,898 TFOs found in the mRNA genes can bind the 9702 TTSs identified on the genome in over 20 million different ways. In addition, due to their sequences being composed of repetitive patterns, each TFO can often target a TTS at numerous sites. To reduce this enumeration bias, we considered that a set of potential triplexes that overlapped over >10 bases constituted the same site. Despite this consideration, the combinatorics between the TFOs and TTSs still had a multiplier effect that was difficult to estimate. Discarding the matches that overlapped over >10 bases allowed a reduction in the number of potential triplexes by a factor of 5. However, exploring the matching of the triplex patterns found on the RNA sequences and the whole genome of D. melanogaster still revealed millions of potential combinations that can be formed along the genome.
In summary, 354,963 and 4,317,708 potential triplex sites have been found in the full genome for pre-lncRNA and pre-mRNA, respectively, and three fourths of them are localized inside genes (columns pre-lncRNA and pre-mRNA of Table 2). The localization of the triplex sites in introns massively overwhelms their presence in exons, which amounts to ∼10% for the two classes of RNA (Table 2). The distribution/localization of putative triplexes on the chromosomes is not a uniform distribution, as shown in Figure 2 and Table S6 in File S4. Chromosome X, for example, contains numerous potential triplexes: more than double those for other chromosomes of equivalent size. On each chromosome, we can see some important hot spots that group a large number of triplexes. The most remarkable concentration of triplexes occurs in positions that contain ∼21.5 million base pairs on chromosome 2L. This zone contains a cluster of histone genes (His1, His2B, His2A, His4, and His3). The putative triplexes are mainly located between His4 and His3. Several other concentrations of triplexes are detailed in Table S7 in File S4.
Distribution/localization of putative triplexes on chromosomes. Chromosomes were divided into bins of 200 kbp. Each bar on the plot represents the number of triplexes belonging to each bin. The histograms highlight some important hot spots that group a large number of triplexes. The most remarkable concentrations of triplexes occur on chromosome X at ∼5 Mbp, on chromosome 2L at ∼21 Mbp, and on chromosomes 3L and 2L at ∼10 Mbp.
As we could expect, TFOs located in introns are the ones that contribute the most to the formation of triplexes. However, the localization on chromosomes of triplexes originating from introns and exons is very similar (Figures S2 and S3 in File S4).
Gene Ontology (GO) enrichment analysis of the mRNAs that mostly contribute to the formation of triplexes highlighted an overrepresentation of genes involved in receptor activity and molecular binding. These genes are also involved in a multitude of developmental processes, as revealed by the enrichment of corresponding annotations with p-values ranging from 10−10 to 10−40 (File S1, and Tables S8 and S9 in File S4 list the transcripts that contribute the most to the formation of triplexes, while the GO enrichment analyses are presented in File S2 and Tables S10 and S11 in File S4). GO enrichment analysis of the genes that are most targeted by lncRNA TFOs revealed an important overrepresentation of genes involved in molecular binding. Functions such as "histone binding," "protein dimerization activity," or "DNA binding" are enriched in the targets of both lncRNA TFOs and mRNA TFOs with incredibly low p-values (of the order of 10−41, 10−24, and 10−17, respectively, for the lncRNA TFO targets and 10−17, 10−17, and 10−18, respectively, for the mRNA TFO targets). Processes related to metabolism and cell organization are strongly emphasized in genes targeted by lncRNA TFOs with p-value scores exceeding 10−30 for "chromatin assembly," "DNA packaging," and "DNA-templated transcription." The same biological processes are also overrepresented in genes targeted by mRNA TFOs but more modestly (with p-values still reaching 10−10). The specificity of genes targeted by mRNA TFOs over those targeted by lncRNA TFOs shows their important involvement in development and morphogenesis processes. A broad spectrum of processes, highlighted by very significant p-values, includes the following: "appendage development" (10−29), "axon development" (10−20), "cell differentiation" (10−31), "generation of neurons" (10−29), "metamorphosis" (10−26), "nervous system development" (10−26), "postembryonic development" (10−26), "tissue morphogenesis" (10−32), "tube morphogenesis" (10−30), and "axonogenesis" (10−19) (File S1 and Tables S12 and S13 in File S4 list the genes that are most targeted by TFOs, and GO enrichment analyses are summarized in File S2 and Tables S14–S16 in File S4). This high specific targeting strongly suggests that triplex DNA:RNA might constitute an unexpected layer of regulation in developmental biology.
The search for genetic interactions among the genes predictably targeted by lncRNA and mRNA TFOs identified a set of genes shaping a large interacting network (Figure 3). Not surprisingly, the GO enrichment analysis performed on this network highlighted annotations mainly related to development and morphogenesis (File S3). Interestingly, we distinguish three subnetworks corresponding to different biological processes very clearly on the graph of genes putatively targeted by lncRNA and mRNA TFOs (Figure 3 and File S3).
Network of genetic interactions between the genes that were most targeted by TFOs. The search for genetic interactions among the genes mostly targeted by pre-lncRNA and pre-mRNA TFOs identified a set of genes shaping a large interacting network. On the graph, red arrows represent a relation of suppression, green arrows represent a relation of enhancement, and gray arrows identify the cases when there is both a relation of suppression and a relation of enhancement. The graph can be divided into three subnetworks. The GO enrichment analysis performed on the genes composing the subnetworks produces an overrepresented annotation corresponding to different processes. (A) The 13 genes in this subnetwork are enriched with terms related to the regulation of cellular process, organ development, and morphogenesis. (B) This large cluster of 34 genes is enriched with terms related to morphogenesis and tissue development. (C) A cluster of eight genes related to axon guidance, locomotion, and the response to external stimulus.
To support the bioinformatics data on a genome-wide scale, we attempted to address the question of a minimal biological validation that might corroborate the predictive analysis. We took advantage of thiazole orange-biotin (TO-biotin), which binds triplexes and quadruplexes with a higher affinity than DNA duplexes (Granzhan and Ihmels 2006; Lubitz et al. 2010; Ren and Chaires 2000), thus allowing us to perform pull-down experiments using the enzymatically cleaved genome of Drosophila under conditions that minimized duplex binding. Several runs of PCR amplification allowed us to sequence >300 colonies in which few sequences were highly represented. The results appear to show some variability between repeats with an evident affinity of TFO to streptavidin. The proportion of sequences corresponding to the triplex structure were constantly enriched compared to the ratio in the initial genomic fragments although they likely result from the combination TO/streptavidin isolation (93 compatible triplex structures that were at least 10 bases long and 24 that were at least 13 bases long originated from few genomic sequences). These results are even more significant if we accept 10% mismatches in the canonical patterns (100 with minimal 10-base lengths and 30 with minimal 20-base lengths) (see Table S18 in File S4). Optimizing and fine tuning the procedure with full genomic material is a reasonable goal due to the strong affinity of triplex made of synthetic oligonucleotides to the TO probe that has been reported in the literature (Granzhan and Ihmels 2006; Lubitz et al. 2010; Ren and Chaires 2000).
The properties of RNA, which are extensively documented in the literature, show their multi-functionality, such as binding specifically to proteins involved in chromatin remodeling and numerous regulatory transcriptional complexes (Elliot and Ladomery 2016). A less studied property is their capacity to engage in DNA:RNA triplex formation according to the Hoogsteen base pairing rules. We report that developmental genes are highly represented in the TTS, which corroborates the hypothesis of unexpectedly large-scale genome regulation mediated by the triplex DNA:RNA tertiary structure. In this regard, we investigated two classes of RNA, lncRNAs, and mRNA introns because their regulatory roles in gene expression have been extensively reported.
Many sequences of lncRNAs are derived from transposable elements, inactive pseudogenes, and intronic sequences and present few conserved features (Chen 2016; Goff and Rinn 2015; Li et al. 2016; Quinn and Chang 2015; Rinn and Chang 2012). lncRNAs operate through trans- and cis-acting functions that nucleate the assembly of RNA/DNA/proteins structures by recruiting proteins close to the site where they are transcribed (cis) or to remote loci (trans) (Chen 2016; Quinn and Chang 2015; Tsai et al. 2010). This mechanism explains lncRNA-mediated epigenetic control of cell fate through ordering the three-dimensional context of the nucleus. For instance, some lncRNAs operate sequentially in a temporal and spatial order to regulate homeobox (hox) genes (Mercer et al. 2009; Rinn and Chang 2012; Wilusz et al. 2009) by recruiting chromatin remodeling proteins that specify whether the zone is active or silent (Mercer et al. 2009; Rinn and Chang 2012; Wilusz et al. 2009). This is also the case for the lncRNA Xist, which is crucial for the epigenetic regulation observed in X chromosome inactivation (Mercer et al. 2009; Rinn and Chang 2012; Wilusz et al. 2009). Regarding the human DHFR gene, a lncRNA whose gene is located upstream of the DHFR promoter represses the expression DHFR. This inhibition acts by forming an RNA–DNA triplex structure with the DHFR promoter (Martianov et al. 2007). The modENCODE project (the consortium that built the encyclopedia of complete RNA elements on the genome scale available on the FlyBase public resource) has markedly highlighted the complex and unexpected dynamic of the spatial and temporal expression of the different RNA categories. Singularly, this finding has led to an unexpectedly large number of lncRNAs, although we are far from attributing biological functions to this newly emerged population of molecules.
Regarding the mRNA population, strong interest in intron functions has emerged over the past decade. A few examples of introns have shown induction of higher expression than that of the promoter itself for the same gene (Chorev and Carmel 2012; Juneau et al. 2006; Rose 2008; Shabalina et al. 2010). Many genes presenting intact coding sequences and promotors are not expressed without their main intron (Chorev and Carmel 2012; Juneau et al. 2006; Rose 2008; Shabalina et al. 2010). The transcript level in the intron-bearing gene can be boosted in a variable scale from 30 to a few 100 times more than the intronless counterpart (Chorev and Carmel 2012; Juneau et al. 2006; Rose 2008; Shabalina et al. 2010). It has also been reported that for many genes, the introns direct the tissue specificity of the expression pattern (Chorev and Carmel 2012; Juneau et al. 2006; Rose 2008; Shabalina et al. 2010). As expected, introns were shown to constitute repositories of cis elements, which participate in the regulation of transcription and genome organization. These cis elements include enhancers, silencers, or other elements that modulate the function of the upstream promoter. Interestingly, introns are also well known for hosting numerous lncRNAs.
More importantly, the dogma stipulates that the fragments of degraded cytosolic mRNA do not reenter by retro transfer into the nucleus, in contrast with the retention/accumulation of intronic RNAs in this organelle. However, most of the unspliced and flawed mRNAs are known to be trapped in the nucleus. These trapped transcripts likely include a considerable number of nascent transcripts, which leads to their concomitant in situ degradation (Gudipati et al. 2012; Houseley et al. 2006; Jackson et al. 2000; Kilchert et al. 2015). Therefore, the probable scenario is that the TFOs coming from exons of mRNA are produced in situ likely because of the blockage of the export of mature mRNAs to the cytoplasm resulting from quality check failure (Houseley et al. 2006). We observed that the introns appear to be the most important putative providers of the TFO, which makes sense due to their exclusive nuclear localization. The multi-subunit protein exosome complex that degrades aberrantly processed RNA generated by error in polymerase pausing, inefficient or erroneous splicing, a deficit in 5′ capping, and failure to add the poly(A) tail should provide a large pool of fragments with TFO motifs. These TFO motifs might have functional epigenetic roles by promoting TFO/TTS association and subsequent nucleation of DNA/protein complexes.
The presented genome-wide scale predictive analysis of triplex DNA:RNA according to Hoogsteen rules has amazingly shown that (i) 10% of the lncRNA and mRNA populations present perfect TFOs, (ii) the number of perfect TTSs on the genome is at an unexpectedly high level, and (iii) the triplexes appear to be predominantly localized to the introns of genes compared to other genome localizations. This finding strongly suggests that a few fragments of RNA might have broad actions on multiple loci on the chromosomes to realize a genome-wide scale of epigenetic control. We present theoretical evidence that a particular TFO in either lncRNA or mRNA might engage triplexes in thousands of sites embedded in the chromosomes. In this regard, we found that TFO-compatible motifs are mostly located between and/or outside the hairpin double strands in spliced lncRNAs. These TFOs could anchor lncRNAs at specific genomic sites and nucleate a higher order architecture of ribonucleoproteins to these locations. Furthermore, the introns of lncRNAs and mRNAs and the exons of mRNAs show equally low proportions of TFOs located in hairpins. Our data reveal a new approach for what is likely a new layer of genetic regulation by fragments of RNAs that have the potential to form triplex structures. This finding leads to the paradigm that some genes, through the expression of intronic RNAs and relevant lncRNAs, might potentially regulate, from a distance, numerous topologically distinct loci through DNA:RNA triplexes. The still poorly studied specific binding of a growing number of proteins to triplex DNA:RNA appears to be an unappreciated layer of regulation of gene expression that confers heritability (Matzke and Mosher 2014). We reasoned that chromosomes "bathe" in a soup of RNAs coming indistinctly from transfer, ribosomal, micro, lnc, and messenger RNAs along with their multiple products of degradation. We postulate that some of these molecules conforming to the Hoogsteen rules might have unknown and powerful structuring effects on the genome. To date, nothing is known or little is studied regarding placing the triplex structure in the context of developmental biology, the emergence of phenotypes, and the epigenetic variability of full organisms.
Our data led to the astonishing observation that triplexes match with very significant p-values to genes of the morphogenesis process. The large number of TTSs in development- and morphogenesis-related genes and their overwhelming selective matches with TFOs that originated from introns and lncRNAs strongly suggest that refined and poorly known regulation of expression operates through these tertiary structures. The discovery that a particular TFO in a lncRNA or intron can theoretically associate with a large number of TTSs at different locations of the genome was amazingly completed with the finding that the putative targets are restrictively associated with development- and morphogenesis-related genes. This finding strengthens the hypothesis of a new layer of on/off regulation of genes that are tightly controlled in a temporal and spatial order.
As a presage, many publications have reported the potential use of oligomers to form an in situ triplex as a tool in cancer or other human pathologies (Hélène 1991; Maher et al. 1991; Schleifman et al. 2008). The use of sequencing technology and bioinformatics tools along with public resources such as FlyBase will allow rapid progress in the exploration of this genome-wide scale potentiality.
We thank Professor Peter Unrau, Simon Frazer University, Burnaby, Canada for the gracious gift of TO-biotin. This work was supported by an ANR grant "methylclonome" ANR-12-BSV6-006-01 attributed to Alain Robichon and Claude Pasquier.
Author contributions: C.P. and A.R. conceived the study. C.P. carried out the computational analysis. C.P. and A.R. wrote the manuscript. S.A. performed the molecular experiments.
Supplemental material is available online at www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.042911/-/DC1.
Communicating editor: B. Oliver
Received February 2, 2017.
Copyright © 2017 Pasquier et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Since the beginning of recorded history, the moon has been a source of creative inspiration. Our interpretations of the moon have developed over time across different cultures, some of those representations include a divine being, a resting place for the gods or a heavenly body. One constant feature has been the moon's unattainability, marked by its sheer distance from us. Now, the 'moon' is closer than ever, and we are able to see its infamous 'dark side' illuminated inside Durham Cathedral.
Spanning seven meters in diameter, the 'Museum of the Moon' is a touring exhibition, currently residing in Durham Cathedral. The sculpture's creator, Luke Jerram, has used NASA lunar imagery at an approximate scale of 1:500,000. This means that each centimetre of the sculpture represents 5km of the moon's surface. A musical soundtrack, composed by BAFTA, Ivor Novello and Dan Jones, accompanies the exhibition, and further transports the exhibition viewer.
I visited the Moon exhibition at the end of September, on one of the evening viewings, and it was a wholly enjoyable experience. Upon seeing the sculpture, I was shocked at the sheer size of it. I found walking around and underneath the moon a mesmerising experience, and it allowed me to appreciate the high level of detail. The exhibition helped me to reflect on our current relationship with the moon, a connection that was so important to past generations, but often brushed away in modern metropolitan society.
Looking back on its representations in the history of art, the moon has been ever-present as a source of artistic inspiration. On the limestone interiors of the Lascaux caves in France (c. 15,000 BCE), there are several drawings with constellations of dots, which scientists have identified as representing the moon's comings and goings. This highlights the significance of the Moon at the beginning of image-making. It is significant to note that in ancient mythology and religious belief systems, the moon was perceived as an untouchable divine being and represented as such. For example, in Ancient Egyptian iconography, the Moon is often found balanced on the head of the god 'Khonsu', one of the most important figures of Egyptian mythology.
Within the Roman belief system, Diana, the virginal goddess of the hunt, was the goddess of the moon, and during the transition to Christianity, the moon kept its association with the 'immaculate'. In Western European art, in particular, in Christian art of the 16th-17th century, the moon became a prominent icon. Raphael's 'Mond Crucifixion' (1502-3) shows the sun and moon as divine witnesses to Christ's painful suffering. Furthermore, there was a renewed interest in nature and the sublime during the Romantic period, which abounded in a plethora of literature, poetry and music which depicted the moon. This period marks a shift in the Western European art canon, from moon iconography being used largely in Christian pieces to more secular pieces. Caspar David Friedrich's 'Two Men Contemplating the Moon' (1825-30) encapsulates the increasingly secular fascination in the cosmos.
Finally, an overview of the representations of the moon in art could not be complete without mentioning one of Van Gogh's most famous works, 'Starry Night' (1889). Many critics have argued that the spiral-like night sky reflects the emotional intensity and troubled consciousness of its artist. This painting was created at a time of public fascination with astronomy and astrology, whilst contemporaries such as Jules Verne wrote about travelling to the moon.
Over its voyage through the ages, from ancient history to the present, the moon has changed and developed in our imaginations and art forms- and I haven't even had time to touch upon representations of the moon in science fictional artworks in the early 20th century and the space age! One constant between the ages, connecting us to our earliest predecessors, is a sense of wonder and contemplation when viewing the moon, and its representations, which I hope that you can experience when visiting the exhibition. *
*= The Moon exhibition at Durham Cathedral is open to all during normal Cathedral opening hours, and is available until Thursday 11th November 2021.
From banana bread to painting, new lockdown hobbies and what having more time means.
With galleries, cinemas and theatres closed for months during Lockdown throughout COVID19 this year, the future of the visual arts has been looking pretty bleak. With life now taking place in a new virtual sphere, we may be questioning if the timeless experience of seeing the arts live or in the flesh will ever be the same again. The more optimistic among us, however, will hopefully agree that this sort of spectatorship could never be digitally replaced with enough success and these establishments will live on despite some considerable projected losses.
However, on a scaled down level, we do not need to look too far for a positive to come out of this momentary pause on life. The curious trends that swept across the nation, from making banana bread to tie-dying clothes and revamping the garden seemed to entice even the self-proclaimed 'non-creatives'. For me this begged reflection on the nature of creativity. In the hectic everyday life of work, downtime is fleeting and to be cherished and the easy Sunday morning is followed all too quickly by the Sunday blues before Monday morning. In other words, time, and being bored, is a luxury – one that most of us were overindulged with this year. The feeling that we had completed Netflix and there was no more feed left to scroll is what pushed us all to try new things that we would not have in any other circumstance. What would happen, then, if we had more time on our hands? And what has it meant? This shift has moved a lot of us from being consumers to creators – making bread when we couldn't buy any, making clothes when the shops were closed, and for some who were desperately bored, making TikToks instead of watching them.
Will we see a sort of Renaissance then, after the effects of this pandemic? With the rise of technology more is definitely possible, and who knows, maybe some of these hobbies have given some people a taste of the creative lifestyle and sparked enough interest to continue. One thing this time at home has made us all realise is the importance of winding down time and de-stressing and hopefully people will continue to make time in their busy schedule for these liberating activities. Let's watch this space then to see some of the results of the big contenders.
Classic but Contemporary: What would Botticelli's The Birth of Venus look like with today's standards of beauty?
One of the most recognisable paintings of the Renaissance, Botticelli's The Birth of Venus depicts the arrival of the classical Goddess of love and sex, and with it the entrance of divine beauty into the world. The face of Venus in this painting has been considered one of the most beautiful throughout the history of art, but how do the Renaissance ideals of long flowing blonde hair, voluptuous curves and a large forehead translate to what the notion of female beauty means today? The possession of a large forehead today has its place in old tales of big brains and intelligence, but correct me if I'm wrong in saying that it isn't a particular foothold on the scale of attractiveness nowadays.
Held up as a deity in this painting Venus herself is considered to be the idealised version of a woman, and Botticelli was known for his appreciation and expression of the perfect woman. Pair them together and we are presented with the '[1]original blonde bombshell,' but what later becomes strikingly obvious is the impossibility of it all (aside from the birth of a full grown woman in a shell and the Wind God Zephyr who blows her to shore, that is). Her typical contrapposto pose does not have the correct centre of weight for it to be possible for her to stand this way, there are no shadows underneath the figure, and her beauty distracts from her unnaturally long neck and the steep fall of her shoulders. In sacrificing realism for a graceful outline of harmony, the pedestal upon which Botticelli places Venus with this depiction of perfection immediately enters the realm of the imaginary and pertains even in 1485, to the unrealistic expectations that still haunt the idea of feminine beauty today.
Some of the more learned among us may cast aside the novels of Ian Fleming overlooking their academic capability, however in his novel Dr. No., Fleming alludes to Botticelli's painting when the figure of Honey Ryder emerges from the sea. Realised in the film by Ursula Andress, we are presented with a more contemporary embodiment of this idealised perfection. Although still blonde, the actress' tanned skin and more slender figure redefine the upheld object of the male gaze (we won't try to tackle in this short article the shift from Botticelli's melancholic modesty to Honey's dagger at the hip in this series of films where women are highly sexualised). It seems then that what we describe as a 'Botticelli figure' today was not part of the perfect woman in the 60s, the decade of Twiggy and a Vogue diet that consisted of hard-boiled eggs, black coffee and white wine (preferably chablis). If painted in these years, Venus would have most likely been an unrecognisable version of herself, but with the recent body positivity movement (woo girl power) perhaps these ideals will begin to shift back to how they were in the 1400s? Despite hard work it is taking time to debunk the wide circulated rumour that gingers have no souls, but with the recent anti-racist movement and embracing of all colours of skin, the ideal of blonde hair has rather been lost in translation. As for the length of her hair, quite frankly I commend anyone who has the patience to maintain this Rapunzel level mane in this day and age and I think we will put the appeal of a large forehead down to personal preference.
[1] Sotheby's Most Famous Artworks in the World series, https://www.youtube.com/watch?v=_dYAa5hjEZg
How will the arts world be affected by the coronavirus pandemic?
A wave of financial crises is looming, and according to predictions, the arts sector will be among the most adversely affected. Museums and galleries worldwide have lain shuttered for almost three months, and while digitization projects have allowed for their collections to be viewed online, such initiatives do not bring in nearly as much money as ticket sales.
Yet despite the distress unleashed by the pandemic, some people believe it could have a positive impact on the arts in the long term.
Museums and galleries were reopened in Asia some time ago, with many in Europe recently following suit. As a new age dawns upon the world of the arts, it seems an auspicious time to explore how it will be affected by the pandemic.
There is no doubt that the sector has taken a hard hit financially. Research conducted by the Network of European Museum Organisations indicates that, during the pandemic, 13% of the 650 museums surveyed lost up to €30,000 per week, and a further 5% lost almost €50,000 per week. Amsterdam's world-famous Rijksmuseum, which would normally receive up to 12,000 visitors daily, recorded losses of between €100,000 and €600,000 per week. Though some museums have yet to disclose the magnitude of their losses, the slew of redundancies they have been issuing indicates the extent of their financial struggles. In late March, the UCLA Hammer Museum laid off 150 part-time employees, and the Massachusetts Museum of Contemporary Art announced plans to dismiss 72% of its workforce.
It is unlikely that museums' ticket sales will bounce back to normality when they reopen. Both Philip Tinari and Eike Schmidt, directors of Bejing's UCCA Center for Contemporary Art and Florence's Uffizi Gallery respectively, recently announced plans to admit half as many visitors as usual owing to social distancing regulations. The decision is a costly one for Schmidt; the Uffizi, Italy's most visited gallery in 2016, already lost over €10 million in revenue during lockdown. Moreover, people might be deterred from visiting art institutions for fear of putting their health at risk. Others might be put off by the lengthy entry procedures; before entering the UCCA, visitors are required to get their temperature taken, scan a QR code twice and show staff their green code.
If the pandemic will cost institutions as prestigious as New York's Metropolitan Museum $100 million, as its executives recently projected, it is likely to threaten the very existence of smaller artistic institutions. The Comitié professionnel des galeries announced at the start of April that a third of French art galleries could be forced to shut by the end of 2020. The Indianapolis Contemporary, which first opened in 2001 and appointed a new director in January of this year, has already fallen victim to the pandemic. Announcing plans to close the museum permanently, board president Casey Cronin said "the impact of the coronavirus is certain to exacerbate economic hardships" and pointed out that "we are not alone as other arts institutions struggle in this crisis."
Smaller artistic institutions could also struggle to attract visitors because people might assume their health and safety measures are inadequate. Some people believe that institutions must use expensive equipment to ensure visitors abide by hygiene and distancing regulations, such as gadgets which emit an alarm when visitors aren't social distancing. The Duomo in Florence, an institution which is unlikely to suffer from great financial difficulty, distributes such devices free of charge to its visitors.
Amid the financial uncertainty posed by the pandemic, artists might also abandon smaller galleries in favour of larger, more established institutions. According to Vulture, even before the pandemic "artists were leaving smaller galleries in droves for megagalleries."
The pandemic will no doubt deliver a serious financial blow to artists. Art fairs, which Alessandro Lupi, the owner of Bologna's Labs Gallery recently described as "extremely important events where collectors discover artists' projects and buy their work", have been postponed or cancelled across the world. Jerry Saltz points out in Vulture that Covid-induced restrictions on mobility might even spell the end for such events.
Governments across the world have responded differently to artists' financial struggles. While President Emmanuel Macron has promised to provide intermittent support for French artists, Togo's Minister of Culture, Kossivi Egbetonyo, recently released a statement encouraging artists who have been hard-hit by the pandemic to temporarily change occupation. Many Togolese citizens were offended by his comments, and felt they were symptomatic of the government's lack of respect for the arts.
However governments choose to treat artists, one thing is for certain: their work is the fuel that keeps the arts industry moving. Without them, the arts world would not be nearly as rich and diverse as it is today.
It is from this cornerstone that one positive impact of lockdown emerges. Many of the world's most famous artworks were produced in isolation. As Waldemar Januszczak writes in The Sunday Times, the 12 months Vincent van Gogh spent in an asylum in Provence proved to be the "single most productive year of his career." Other masters such as Pablo Picasso and Richard Dadd have shown us that isolation stimulates rather than stifles creativity.
What's more, many great artworks have been born out of crises. As Frances Morris, the director of Tate Modern, recently put it, "if you look at the great traumatising events of the past – world wars, global emergencies of different kinds – artists have always responded." We might in fact be on the cusp of a new artistic movement, driven by artists committed to helping us navigate our way out of isolation and make sense of what we have just lived through.
There is another reason, however, why many people believe the pandemic might have a positive impact on the arts world. The amount of money swilling about in the sector has long been a subject of contention among artists and curators. Works by industry heavyweights including Banksy and Maurizio Cattelan reflect their frustration with the obscene circus that the arts world now represents. Stuart Shave, owner of London's Modern Art, recently admitted that, prior to the pandemic, his schedule was filled with days when he would make a 6,000-mile round trip for an opening and a dinner.
Yet the social and financial implications of the pandemic could very possibly put an end to the industry's large-scale operations, since they will make it harder for gallerists, art works and devotees to travel around the world. The pace of exhibitions will no doubt be slower, as will the rate at which blockbuster shows are churned out. According to Rebecca Salter, President of London's Royal Academy of Arts, the appetite for blockbuster shows might even find itself sated by "smaller, more intimate encounters with artists we might not know." Many people are hopeful that the pandemic will also open the floodgates to more serious discussion about crowd control. Jan Dalley writes in the Financial Times: "perhaps in future a visit to the Louvre or the British Museum or the Uffizi won't be a touristic given but more like going to the theatre — seeing the 'Mona Lisa' could be like getting a ticket for Hamilton."
While so much about the post-corona arts world remains a mystery, one thing is for certain: art galleries and museums will be far emptier upon opening. Visitors will have more space, more time and, perhaps, a more rewarding experience. And who knows, perhaps this rewarding experience will remind industry leaders why they were drawn to work in the sector in the first place, and prompt them to precipitate changes that artists and curators have been seeking for so long.
Sleeping Beauty? The impact of Covid-19 on the arts
Although the impact of the current pandemic is severe in many areas of society, the arts are certainly among the worst affected. It is unfortunately in the nature of this sector that it involves crowds – and now, concerts, theatre and opera performances, dance festivals, exhibitions, and even book fairs have been cancelled and postponed indefinitely. Millions of artists, dancers, singers, producers and musicians are left in limbo, without an income and without a set date for the return of normality to comfort them.
Nevertheless, as everyone else, those working and creating in the arts are counting on a swift conclusion of the current situation, and for the meantime, they´re continuing to do what they do best – being creative. Yes, art requires to be seen, yet for a temporary timespan a surprising quantity of it can be experienced online or in more intimate formats than big concert halls or theatres. Musicians are livestreaming themselves performing – at home, of course – on Instagram or Facebook, sometimes even collaborating. Art galleries and museums are making their collections available online to take virtual tours. Opera singers are delighting their neighbourhoods with free concerts from their balconies, or, like Andrea Bocelli, from an eerily empty Duomo in Milan. Ballet companies around the world offer online streams of past performances, such as the Bolshoi Ballet from Moscow or the Ballet Opéra de Paris.
All these developments are both generous – many of them are for free to brighten everyday life for the millions of people quarantined or in strict lockdown situations – and important, as art is particularly necessary in times of uncertainty. It offers both refuge from the flood of tragic news and hope in this time of crisis, something that is perhaps shown by the popularity of reading among tips for surviving self-isolation and quarantine. Literature is currently presumably the only creative category that will not suffer as extensively as the rest; reading has always been a mostly solitary enjoyment.
However, there will be a day when every past performance has been streamed and when artists and creators need to return to the less glamourous necessity of generating an income for themselves and their industry. This, however, can only be done the traditional way. Most areas of the arts rely on live audiences and on the rhythm of performance seasons that involve people coming together to create their art. One day, concerts and performances will have to take place again. When this day will be remains uncertain; until then, the sector can only rely on the support of governments and financial solidarity shown by the public. The Czech Republic for instance, is leading by example: people there are buying tickets for cancelled cultural events in order to support the arts scene and its institutions.
While this article does not intend to downplay the seriousness of the situation – the economic consequences of the pandemic are already predicted to be astronomically high, and will thus also affect the arts severely – it is important to remember that there is hope: this is not the first pandemic in the history of humanity that the arts have survived. Art, in some form, has accompanied us from almost the very beginning of our existence as a species, as is visible for instance in discoveries of pre-civilizational instruments and cave drawings in Lascaux or Altamira. Although we don´t often realize, art is as necessary to most of us as is a regular supply of food – or toilet paper.
By Cristina Coellen
Italy's cultural heritage is crumbling away and not even the Monuments Men can save it
By Francesca Halliwell
Italy. The country of Raphael and Botticelli, Giotto and Veronese, da Vinci, Michelangelo, even the Galleria Borghese. Home to some of the Renaissance's most important artists, not to mention a magnificent ancient civilisation, Europe's 'bel paese' boasts a treasure trove of artistic masterpieces that many nations could only dream of possessing.
Owing to recent theft reports, however, the truth seems somewhat different.
Less than a fortnight ago, European police arrested 23 people on charges of trafficking tens of thousands of archaeological artefacts. The objects, which reportedly date back to the third and fourth centuries B.C., were looted from Calabria before being smuggled out of Italy and auctioned off across Europe.
This isn't the first time Italian artefacts have been looted and sold off abroad. According to the Carabinieri's most recent stolen artworks bulletin, 8,405 items have gone missing from Italy in the past year alone.
Nor is the phenomenon a recent one. It dates back as far as 1969, when a specialised police squad – the Command for the Protection of Cultural Heritage – was created by the Italian government to track down stolen paintings and statues. Though this first-of-its-kind art corps has allegedly recovered over three million objects since its foundation, its mission is still far from complete: more than one million Italian artworks remain missing or stolen.
The scale of their task is quite monumental. In 2014, these so-called Monuments Men were tasked with tracking the theft of Sicilian treasures worth over 40 million euros. The ensuing investigation culminated in a series of Europe-wide arrests, in addition to the recovery of over 20,000 objects and the dismantling of a criminal organisation that had been set up solely to smuggle Sicilian archaeological goods.
Italy has fallen victim to art crime more often than any other country. Many of its missing artworks are now deemed untraceable, having been stolen long before the art squad was even established. Something of a miracle is required for their recovery, especially when owners don't even recognise the importance of their artefacts. This was the case in October 2017, when a couple living in New York discovered they had been using a 2,000-year-old mosaic that once decorated Emperor Caligula's ship as a coffee table in their apartment.
The thought that lingers? Italy's artistic heritage is fading into obscurity. And in this newly globalised age where materialism is rife, many seem blinkered to the severity of the situation.
Some believe museum directors are too often blinded by the short-term financial benefits of selling Italian art abroad. Only eight of Leonardo da Vinci's 24 major extant works remain in Italy, while the largest collections of Titian's works and Raphael's drawings are housed abroad: the former in Madrid, the latter in Oxford's Ashmolean Museum.
To make matters worse, an Italian court recently ruled that da Vinci's drawing Vitruvian Man could be shown at an exhibition in Paris. The decision was a controversial one, not least vis-à-vis the possible implications for Italy's cultural heritage. Specialists also contended it was too fragile to travel and risked being damaged by the lighting in the Louvre display. Upon hearing the ruling, heritage conservation group Italia Nostra released a statement saying, "today is not a good day for protection in Italy."
When the Louvre announced plans to hold a da Vinci exhibition, Italian politicians instantly ramped up nationalist rhetoric. "Leonardo is Italian, he only died in France," announced Lucia Borgonzoni, an undersecretary at Italy's Ministry of Culture.
At least the whereabouts of the Vitruvian Man are being closely monitored. Plenty of prestigious Italian works have disappeared without a trace, including da Vinci's Salvator Mundi, which was recently sold via an intermediary to the Saudi Arabian Crown Prince, Mohammed bin Salman. Many believe the painting to have vanished, with speculation abounding that it is on display in his superyacht, which he bought for 500 million Euros in 2015.
The whereabouts of both Caravaggio's Nativity with St Francis and St Lawrence and Raphael's Portrait of a Young Man also remain a mystery. While the former was stolen from an oratory in Palermo in 1969, the latter was snatched by the Nazis in Poland during World War II.
Natural disasters and climate change have brought another dimension to Italy's cultural crisis. Geographical instability has long ravaged the peninsula, leaving nothing but damage and destruction in its wake. Hardly anything is spared, not least Italy's cultural capital. When disaster strikes, basilicas are flooded, monuments are torn to the ground and paintings disintegrate beyond all recognition.
Take for example the Basilica of St. Benedict in Norcia, which was destroyed by an earthquake in 2016, as well as half the façade of the 15th-century church of Sant'Agostino in Amatrice. Quake-induced cracks have even appeared on the Colosseum in recent years.
Just under a month ago, the city of Venice – an artistic treasure in itself – was submerged by three major floods. Images of the inundated St. Mark's Basilica sent shockwaves around the world, illustrating the damage caused to its tiling and mosaics. The floods are the worst to have hit Venice in more than half a century, and according to scientists, they are set to continue.
Damages incurred will take years to repair, and what is most frustrating is that they could have been avoided. In the 1980s, city authorities launched a project to build hydraulic barriers and improve sea defences, though delays and budget overruns impeded its progress. Had it been fully functioning by 13th November, the disaster described by Prime Minister Conte as "a blow to the art of Italy" would have been narrowly avoided. It seems there may be a grain of truth in Paul Lechat's observation that "the Italian has no use for the long-term" after all.
Yet for the plethora of criticisms that have been levelled against Italians, it would be wholly unjust to view Italy's cultural crisis as a product of their propensity for chaos and disorder.
Art crimes and natural disasters are gnawing away at Italy's cultural identity, and the tragedy at stake here must not be undermined. To quote leading art historian Tomaso Montanari, "in Italy the stones, the buildings, the churches and the artworks are the backbone of the country. The loss of our roots means we have lost the future."
French Chic
Is the image of French chic that abounds in popular culture nothing more than a misleading stereotype?
Picture a 'French girl,' and the image which inevitably springs to mind is that of Brigitte Bardot's effortless beauty in Et Dieu créa la femme; Anna Karina's mesmerising allure in Vivre sa Vie; Catherine Deneuve's impeccable wardrobe in Belle de Jour.
The 'French girl' is the epitome of cool; she exudes natural perfection and sophistication. She has infiltrated our wardrobe (I am wearing Breton stripes as I write this), she has Pinterest boards devoted to her: she has captivated us with her "je ne sais quoi." The media churns out articles on an almost daily basis instructing us how to wear our hair, which clothes to wear and what attitude to adopt in order to achieve 'French girl style.'
Yet the 'cool French girl' maintains a sense of careless and effortless style while adhering to strict fashion rules. She is thin, but eats croissants for breakfast and is never seen without a baguette under her arm. She is elegant and casual, all at the same time.
The 'cool French girl' is a paradox.
This idea of effortless perfection poses problems: the pursuit of perfection is an all-consuming, and ultimately futile quest, and the stereotype of a thin, white woman does not represent today's France. The image which we all recognise and idolise is not accommodating of other skin tones, body types or genders.
This is an issue fashion journalist Alice Pfeiffer has picked up on in her book Je ne suis pas Parisienne, in which she aims to deconstruct the myth of the 'French girl.' Through the book, Pfeiffer shares her experiences of being held to impossibly high standards, expected to maintain a simultaneous image of carefully cultivated perfection and insouciance. She shares insulting comments made about her weight and her style, and ultimately declares the society's preconceived idea of the 'French girl' to be sexist, elitist and unachievable.
But, is the image of the French girl changing? Today, the body positivity movement is thriving, and more and more marginalised groups are taking to social media and using technology to create their own platforms through which they are able to take control of their own narratives.
Amandine Gay is a French-African filmmaker, journalist and activist based in Montreal. Through her directorial debut Ouvrir la voix (Speak Up), a documentary in which women of African descent discuss their identity as African women, Gay aims to "occupy public space and explain why the intersection of discriminations faced by black women in France and Belgium is as problematic as it is political."
Yet there still remain positive aspects of the image of 'French chic' that we can use as sources of inspiration. In 80s supermodel Ines de Fressange's book Parisian Chic: A Style Guide, she writes that, to be a true Parisian, a woman must never neglect herself for the sake of adhering to convention or trends. This idea can be interpreted to promote the idea of self-acceptance, as can the trope of minimal makeup associated with the French, which appears to be making a resurgence today.
The 'French girl,' then, must be reclaimed. We do not need to abolish the image completely: it is an iconic souvenir of the history of French literature and visual culture, but the stereotype needs, however, to shift into accordance with modern-day French society.
Isabelle Adams
BP or not BP? - A dilemma facing the Art World in the Extinction Rebellion era
On October 2nd of this year, the aptly named campaign group BP or not BP?, whose mission was to end the oil firm BP's sponsorship of the Royal Shakespeare Company, celebrated a hard-earned victory. After seven years of consistent campaigning, alongside a boycott of the company from high-profile individual actors such as Mark Rylance, the RSC decided to cut short their five-year contract with BP at the end of this year. In response, BP or not BP? issued this statement:
We're over the moon to see the RSC understanding and acting on the reality of the climate emergency. Oil companies like BP have no place in our museums, galleries and theatres.
They are not the only activist group opposed to fossil-fuel companies' continued sponsorship of the arts, nor is the RSC by any means the only cultural institution coming under fire from protestors for accepting such donations. The call for more ethical corporate sponsorship is being echoed throughout the arts world. The British Museum, the National Portrait Gallery and the Royal Opera House are just a handful of the prestigious bastions of the British art establishment under pressure to review their corporate sponsorship partnerships with oil and gas firms. In 2016, BP announced plans to invest £7.5 million in British art institutions from 2018, but in the era of Extinction Rebellion, these institutions could find that such partnerships leave them out of touch with the values of their visitors and facing a pressing moral dilemma.
The resistance to such sponsorship from British artists has been increasingly relentless, collaborative and, as expected, creative. One protest group with a particularly high-profile artistic output is Liberate Tate. Founded in 2010, the group of activists mobilised a spirit of 'creative disobedience' to complicate the Tate institution's relationship with BP and push them into dropping the sponsorship agreement, which did in fact come to an end in May 2016. Their unsanctioned performances have seen the installation of a 16.5 metre wind turbine blade in the Tate Modern's turbine gallery, and a simulated oil spill at the institution's summer gala in response to the environmentally disastrous Deepwater Horizon Oil spill in 2010.
Such activist groups accuse oil companies of using cultural sponsorship to essentially purchase the 'social licence to operate'. Stripping back the PR veneer of this phrase, this entails boosting their brand's reputation through the association with cultural goods and the establishment of influential relationships, a practice which allows them free reign to legitimately expand environmentally damaging operations in a time of climate crisis. However, seen in another light the very nature of their controversial commercial activity prompts oil and gas companies to donate more generously to cultural institutions, since they perhaps serve to benefit more from such positive marketing than other 'cleaner' enterprises.
Indeed, the relationship between environmental sustainability and financial decision-making in the arts remains a vexed one. Government spending in the arts saw a cut of almost 30% between 2010 and 2016, and the question remains whether innovation in the arts can be sustained without this backing. Britain's cultural institutions are renowned for their pioneering and creative stance, and the artistic output is second to none. If we remove oil and gas firms from the equation, are there alternative sources of sponsorship with the capacity or desire to replace these environmentally damaging corporations?
Examining these questions in light of the controversy around the National Portrait Gallery's BP Portrait Award is revealing. Marketing itself as the most prestigious portrait award in the world, the sought-after accolade recently celebrated is 40th year at the Gallery and its 30th year of BP sponsorship. At his presentation of the 2019 award, the CEO of BP Bob Dudley addressed the current sponsorship controversy:
"We respect different opinions and welcome discussion, even debate, about our involvement. But, let's also remember there are many different points of view."
Yet these different points of view are becoming more polarised in tandem with increasing public awareness of the climate crisis. BP asserts that its financial support to the gallery has enabled five million people to enjoy the award free of charge since 1990 and has given artists the necessary support to pursue their passion. However, prior to BP taking up the sponsorship for this award, it was sponsored by none other than British American Tobacco. Obviously such a partnership would be unthinkable these days. In 2016, there was outcry from medical experts against the art establishment's lingering ties with tobacco companies, and it seems that the same fate could await big oil firms.
Public opinion and government priorities can change dramatically over 30 years, and the day could be approaching where government policy regarding the promotion of oil and gas declares it to be morally unacceptable. Until that day however, cultural institutions that declare themselves in favour of environmental sustainability while accepting donations from companies whose practices actively harm the environment, face accusations of hypocrisy. In an age of global climate protest and social media activism, these accusations cannot be brushed away lightly. Many agree that the art arena is one in which the corporate world should not be at the forefront of the story. However, these protests against corporate sponsorship will most likely gain more mainstream attention, particularly from young visitors, a consideration that should be taken seriously by cultural entities if they wish to remain relevant and respected in the arts world and in today's society.
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Review: Robert Horrocks,Masculinity In Crisis: 'a hard man is good to find!' (Mae West) ★★★★
First, might I profess with dismay that I do not know what it is like to be a man, nor will I ever fully understand life as a man. However, what I can contribute to the ever-topical discussion of masculinity is an ode to the penis and a push to embrace the body you've been given, all from the mind of a feminist.
'Penis', a small segment from Robert Horrocks' book Masculinity in Crisis, delves into the relationship between men and their 'rhythm sticks', emphasising the need for self-acceptance and pride. Big-headed or big-shafted, curved or straight, men we've seen them all. And, with about 40% of the male population with penises that don't always function the way they should, it's about time someone gave the little-man a lot of praise.
"The penis is the source of a man's greatest vulnerability, and his greatest feeling of power. It is a place from where he expresses love, hate, fear, tenderness, contempt, friendship, disgust, and also a place where he can become completely expressionless, without feeling. It is used as a battering ram against women; as a weapon of revenge; as an expression of love and adoration; it is also a little boy's willy, a dick, a shlong, the python, prick, rhythm stick, ding-a-ling.
It can be anything. We might say: the penis is the man. One cannot talk about penises separate from their owners, just as we cannot talk about 'sexuality' in abstract, or talk about 'sexual problems' separate from the problems that people have in relationships.
So men feel about their penises as they feel about themselves: proud, shy, afraid, disgusted, ignorant, loving, hating, angry – the whole gamut of human feeling is potentially found between a man and his penis.
Sexually a penis enables a man to penetrate deeply into a woman (or a man) and caress her (him) there. Thus the penis in this respect is bound up with relating to others: it is a means of contact. But as a baby or a boy, the male finds the penis as a source of comfort for himself, maybe a substitute for the breast, something he can manipulate at will. He discovers masturbation, which again can mean so many things to different men- comfort, company, desolation, lust, fear, love.
Thus the penis is, as it were, poised between narcissism and altruism. It can be used to relate to oneself, or to others. Quite often these two directions get confused, and one hears a woman angrily accuse a man of masturbating inside her during sex. So one can go through the motions of relating sexually but in fact remain completely in a world of one's own. In that world, the penis may loom large as a kind of fantasy friend, or lover, or mother or father.
The penis is also concerned with performance. It gets erect, remains erect during sexual intercourse and then subsides. But what anxiety is aroused in men about this! The erection becomes a fetish, that measures one's manhood, virility, athleticism, or whatever. How afraid many men are not to have an erection, when in fact that often signifies something emotionally important: I don't want to make love, I don't want to relate to you, I feel afraid, I feel childlike, and so on.
'Keep your pecker up' men say to each other – reflecting the desperate need to keen the damn thing erect. Never mind if you feel depressed or sad or lonely, or bored or angry or uninterested – keep the flag flying, make the python do its tricks, hoist the top-sail, expand the gland- what a mythology surrounds penis performance! Mae West celebrated and mocked it in her famous gibe: 'Is that a six-gun in your pocket, or are you just pleased to see me?'".
Roger Horrocks Penis (162 – 166).
There is something majestic about the penis; a mix of adoration, fear and fascination that accompanies it. Indeed, Roger Horrocks recounts his own phallic experiences. From school P.E days where he and his comrades were forced to swim naked, where they'd spend the full hour comparing the circumcised with the uncircumcised (which 'ding-a-ling' was superior?)…to discovering a freckle on the foreskin of his penis and deciding there and then it was cancer: Embrace death now and enjoy masturbation whilst the penis is still there?
The penis is "a trail blazing-experience", a source of ready-made comfort. Though, the difficulty comes from claiming the grandeur of the penis; the fear that this in itself is sexist. Ludicrous as it might be because the act of sex is inherently at its best when enjoyed by both the man and woman. A so-called 'animal beauty', a power that our civilisation unrightfully shuns. After all, the penis is inescapably animal, primitive. Think with your head all day and wake up early morning and it's hard. I bet you're unable to think your way out of that one!
Men today need to reclaim this sense of beauty and power about the penis. Accept the animal qualities within us. Freud says we all yearn for the sheer animal side of being alive, so let's reclaim it.
Review: Khaled Khalifa, No Knives in the Kitchens of this City ★ ★ ★ ★ ★
Aleppo circa December 2016.)
Ben Smart
In recent years, Syria has repeatedly made headlines in the West, detailing the horrific events of the civil war. Khaled Khalifa's No Knives in the Kitchens of this Citynarrates the long tale of the Syrian city Aleppo. Though the novel's main historical setting is between the 1960s and 2000s, flashbacks to earlier times mean the novel unravels Aleppo's tale right from the First World War up until the time of the 2003 invasion of Iraq. What No Knives in the Kitchens of this Cityshows us is Syria's slow degeneration under the Assad regime. We can see that the war's outbreak was not a spontaneous event but rather had been a long time in coming.
With Aleppo at its centre, the novel follows the lives of an exceedingly large cast of characters, all told through the voice of a single narrator who, though a real character in the story, remains a barely felt, almost invisible, presence. The author, Khaled Khalifa, has made it a recurring trope in his novels to explore the influence of a single emotion on his characters. In his previous novel, In Praise of Hatred, he explored the way his characters were motivated by their hate. In No Knives in the Kitchens of this City, the focus is now on shame. The narrator's mother is ashamed of her youngest daughter, Suad, because of her disability, and so she locks her away, waiting for her to die. Sawsan is ashamed of her past sexual encounters, and so undergoes hymen reconstruction surgery and throws herself into a devoutly religious lifestyle. Nizar's family are ashamed of his homosexuality, and so have him sent to prison. The only character who seems capable of resisting the all-pervading shame in the novel is in fact Nizar himself, who does not attempt to hide or conceal his past. The non-judgmental Nizar's acceptance and compassion lead him to act as an emotional confidante for many of the other characters in the novel.
On a more societal level, shame cripples both the regime and resistance to the regime. The character Jean's so-called 'theory of historical shame' details the inseparable link between shame and fear, and the debilitating societal effects this entails. Jean 'sketched out the inhabitants of a city, who shared the air of that city but were afraid of each other.' The theory is one of universal, mutual fear with 'minority sects afraid of majorities, and the many afraid of the despotism of the few', and 'races and religions and sects afraid of the President and his mukhabarat' and 'the President afraid of his aides and his own guard'.
The hopelessness of Aleppo's stagnation is exemplified in the eponynmous scene where an Aleppian man set his wife and four children on fire, then committed suicide with a kitchen knife as he screamed at his neighbors, who were watching dispassionately, that dying in a fire was more honorable than waiting to starve. He asked them bitterly,
"Are there no knives in the kitchens of this city?"
The novel makes for interesting and evocative reading both as a lesson in Syrian history and also as an insight into the circumstances preceding the civil war.
Book Review: The Good Immigrant
Deimantas Narkevičius
Divines (2016)
Doubled Youth
How 'belle' Really Was France's Belle Époque?
Language Trivia: Five Fun Facts
Nikolai Erman's 'The Suicide'
Paul Theroux Kowloon Tong
Review: All Quiet On The Western Front
Review: Rose Remain 'The Gustav Sonata'
Street Art Of Barranco
The Black Square: The Only Painting You Need
World AIDS Day Film Review: Asmaa

redpajama_set_name: RedPajamaCommonCrawl


The Friday, July 25, 2014, 8:30 a.m. Special Meeting of the Urban Forestry Council was cancelled due to a lack of quorum at the meeting. No action items were heard at this meeting. Public Comment was heard, presentations given, and discussions held on Items 6 and 8.
The next meeting of the Urban Forestry Council is scheduled for Tuesday, August 26, 2014 at 6:00 p.m., Room 416, San Francisco City Hall.


In 20 years "there will be enough Muslim voters in the U.S. to elect the president."
There's been a "20 percent increase in young people coming back to Buffalo after a decade where we lost 10 percent of our young people."
West Virginia has "more veterans per capita" than any state.
Does a PA congressional candidate want to tax large families?