Drosophila SWR1 and NuA4 complexes are defined by DOMINO isoforms
Nucleosomes , the fundamental units of chromatin , are inherently stable and organized in polymeric fibers of variable compactness ( Baldi et al . , 2018; Erdel and Rippe , 2018 ) . The dynamic properties of the fiber required for gene regulation are implemented by several broad principles . ATP-dependent nucleosome remodeling factors slide or evict nucleosomes ( Clapier et al . , 2017 ) , chemical modifications of histones create new interaction surfaces ( Bowman and Poirier , 2015; Zhao and Garcia , 2015 ) and histone variants furnish nucleosomes with special features ( Talbert and Henikoff , 2017 ) . The very conserved H2A variant H2A . Z accounts for ~5–10% of the total H2A-type histone pool in vertebrates ( Redon et al . , 2002; Thatcher and Gorovsky , 1994 ) and flies ( Bonnet et al . , 2019 ) . H2A . Z is primarily found at active promoters and enhancers , where it is thought to be important to regulate transcription initiation and early elongation ( Adam et al . , 2001; Weber et al . , 2010; Weber et al . , 2014 ) . In Saccharomyces cerevisiae , H2A . Z is introduced into chromatin by the SWR1 complex ( SWR1 . C ) , a multi-subunit ATP-dependent chromatin remodeler with the INO80-type ATPase SWR1 at its core ( Mizuguchi et al . , 2004; Ranjan et al . , 2013; Wang et al . , 2018a; Willhoft et al . , 2018; Wu et al . , 2005 ) . In humans , the two SWR1 orthologs EP400 and SRCAP may also be involved in H2A . Z incorporation ( Greenberg et al . , 2019; Pradhan et al . , 2016 ) . In Drosophila melanogaster , where H2A . Z is named H2A . V ( Baldi and Becker , 2013; van Daal and Elgin , 1992 ) , only one gene codes for a SWR1 ortholog: domino ( dom ) ( Ruhf et al . , 2001 ) . The first biochemical characterization revealed the presence of a multi-subunit complex composed of 15 proteins associated with the DOM ATPase ( Kusch et al . , 2004 ) . While many of the interactors identified are orthologous to the yeast SWR1 . C subunits , additional interactors were found . Surprisingly , they showed similarity to components of a distinct yeast complex , the Nucleosome Acetyltransferase of H4 ( NuA4 . C ) ( Kusch et al . , 2004 ) . NuA4 . C is a histone acetyltransferase ( HAT ) complex with the histone H4 N-terminal domain as a primary target ( Allard et al . , 1999; Doyon et al . , 2004; Wang et al . , 2018a; Xu et al . , 2016 ) . The DOM complex ( DOM . C ) appeared then to be a chimera , a fusion between two complexes with different biochemical activities . It has been proposed that both enzymatic activities of DOM . C , histone acetylation and histone variant exchange , are required for H2A . V turnover during DNA damage response ( Kusch et al . , 2004 ) . It is unclear , however , if this model of DOM . C action could be generalized to other processes , such as transcription regulation . Furthermore , it is still not known how H2A . V is incorporated globally into chromosomes while , at the same time , enriched at promoters . It is long known that the dom transcripts are alternatively spliced to generate two major isoforms , DOM-A and DOM-B ( Ruhf et al . , 2001 ) . We and others previously found that the two splice variants play non-redundant , essential roles during development with interesting phenotypic differences ( Börner and Becker , 2016; Liu et al . , 2019 ) . In this work , we systematically characterized the molecular context and function of each DOM splice variant in D . melanogaster cell lines and assessed their contribution to the activity of the DOM . C in the context of transcription . We discovered the existence of two separate , isoform-specific complexes with characteristic composition . Both are involved in transcription regulation , but through different mechanisms . On the one hand , we found that the DOM-B . C is the main ATP-dependent remodeler for H2A . V , responsible for its deposition across the genome and specifically at active promoters . On the other hand , we discovered that DOM-A . C is not involved in bulk H2A . V incorporation , despite the presence of an ATPase domain and many shared subunits with DOM-B . C . Rather , we realized that DOM-A . C might be the ‘missing’ acetyltransferase NuA4 . C of D . melanogaster , which specifically targets lysine 12 of histone H4 ( H4K12 ) , the most abundant and yet uncharacterized H4 acetylation in flies ( Feller et al . , 2015 ) . Surprisingly , our data also suggest that the ATPase activity of DOM-A is dispensable for H4K12 acetylation by the DOM-A . C , a principle that might be conserved across metazoans . Our work illustrates how alternative splicing generates functional diversity amongst chromatin regulators .
The isoforms of the DOMINO ATPase , DOM-A and DOM-B , are identical for the first 2008 amino acids , but alternative splicing diversifies their C-termini ( Figure 1A ) . Both proteins share an N-terminal HSA domain and a central , INO80-like ATPase domain . DOM-A has a longer C-terminus characterized by a SANT domain and a region rich in poly-glutamine stretches ( Q-rich ) . The shorter C-terminus of DOM-B , instead , folds in no predictable manner . Given these differences , we wondered if the interaction partners of the two isoforms might differ . To avoid artefactual association of DOM isoforms with proteins upon overexpression , we inserted a 3XFLAG tag within the endogenous dom gene in D . melanogaster embryonic cell lines using CRISPR/Cas9 . The sites were chosen such that either DOM-A ( DOM-RA ) or DOM-B ( DOM-RE ) would be tagged at their C-termini . Of note , the editing of DOM-A C-terminus results in the additional tagging of a longer , DOM-A-like isoform ( DOM-RG , which compared to DOM-RA has an insertion of 35 residues at its N-terminus starting from residue 401 ) , but leaves a second DOM-A-like isoform untagged ( DOM-RD , 16 residues shorter than DOM-RA at the very C-terminus ) . We obtained three different clonal cell lines for each isoform ( 3 homozygous clones for DOM-A , 2 homozygous and 1 heterozygous clone for DOM-B ) ( Figure 1—figure supplement 1A , B ) . The dom gene editing resulted in the expression of 3XFLAG-tagged proteins of the correct size and with similar expression levels across clones ( Figure 1B ) . To identify the strongest and most stable interactors , we enriched the isoforms and associated proteins from nuclear extracts by FLAG-affinity chromatography under very stringent conditions . Mass-spectrometry analysis revealed 13 and 12 strongly enriched interactors ( FDR < 0 . 05 and log2 fold-change >0 ) for DOM-A and DOM-B , respectively ( Figure 1C , Supplementary file 1 ) . Of those , 7 are common between the two isoforms and were previously characterized as DOM interactors ( Kusch et al . , 2004 ) . Two of the expected subunits , PONT and REPT , associated more strongly with DOM-B than with DOM-A under these conditions ( log2 DOM-A IP/CTRL = 1 . 19 and 1 . 16 , FDR = 0 . 373 and 0 . 338 ) . A newly identified DOM-B interactor , HCF , also interacts less strongly DOM-A ( log2 DOM-A IP/CTRL = 0 . 70 , FDR = 0 . 466 ) . The unique interactors revealed interesting differences between DOM-A and DOM-B ( Figure 1D ) . Three of the proteins that specifically associate with DOM-A [ING3 , E ( Pc ) and TIP60] share extensive homology with the acetyltransferase module of the yeast NuA4 complex . Another component of the yeast NuA4 . C , NIPPED-A , specifically associates with DOM-A . Additionally , we found two transcription factors , XBP1 and CG12054 , amongst the DOM-A specific interactors . On the DOM-B side , only ARP6 and PPS appear to be specific interactors of this isoform . While ARP6 was not described before in Drosophila , its yeast homolog is essential for H2A . Z remodeling by the SWR1 . C ( Wu et al . , 2005 ) . To validate the DOM-A/TIP60 interaction , we raised monoclonal antibodies against TIP60 . Co-immunoprecipitation confirmed that TIP60 interacts with DOM-A and not with DOM-B ( Figure 1E and Figure 1—figure supplement 1C ) . The immunoprecipitation of DOM-A appears to be more efficient when probing with the anti-FLAG antibody compared to the anti-DOM-A polyclonal antibody . This difference might be explained by the presence of one of the DOM-A-like isoforms ( DOM-RD ) , which was left untagged . This isoform is therefore not immunoprecipitated by the anti-FLAG antibody , but it is recognized by the DOM-A specific antibody . Importantly , the same co-immunoprecipitation showed that DOM-A and DOM-B do not interact with each other under these conditions . Taken together , these findings document the existence of two distinct DOM complexes: DOM-A . C and DOM-B . C . Previous observation in flies suggested that DOM-A and DOM-B have different , non-redundant functions during Drosophila development ( Börner and Becker , 2016; Ruhf et al . , 2001 ) . Isoform-specific depletion by RNA interference ( RNAi ) of either DOM variant in a Drosophila embryonic cell line did not lead to depletion of the other isoform ( Figure 2A ) . Interestingly , knock-down of DOM-A ( but not DOM-B ) led to a strong reduction of TIP60 protein levels . tip60 mRNA levels were unchanged ( Figure 2—figure supplement 1A ) , indicating that TIP60 requires DOM-A for stability ( Figure 2A ) . This suggests that most of TIP60 resides in the DOM-A complex in Drosophila cells . The yeast SWR-1 and NuA4 complexes are both implicated in transcription ( Morillo-Huesca et al . , 2010; Searle et al . , 2017 ) . We therefore explored the functional differences of the two DOM isoforms on transcription by RNAseq . In our analysis , we also included knock-downs of H2A . V and TIP60 . Knock-down of either DOM-A , DOM-B , TIP60 or H2A . V individually resulted in significant perturbation of transcription , with notable differences ( Figure 2—figure supplement 1B , Supplementary file 2 ) . Principal Components Analysis ( PCA ) revealed clearly different transcriptional responses upon loss of DOM-A or DOM-B ( Figure 2B ) , which can be visualized by comparing their log2 fold-changes relative to control ( Figure 2C ) . The correlation value of 0 . 45 indicates that many genes are regulated similarly by both ATPases , but a significant number of genes are also differentially affected upon specific depletion of either DOM-A or DOM-B ( Figure 2—figure supplement 1B ) . As expected , the transcriptional effects of DOM-A knock-down , but not of DOM-B , resemble the ones caused by knock-down of TIP60 ( Figure 2B , D ) . Depletion of H2A . V led to a global reduction of transcription , only observable by normalization to spiked-in D . virilis RNA ( see methods ) ( Figure 2—figure supplement 1C ) . The effects of H2A . V depletion were better correlated to DOM-B ( r = 0 . 51 ) than to DOM-A knock-down ( r = 0 . 25 ) ( Figure 2B , D ) . Many effects of DOM-B depletion may be explained by its H2A . V deposition function , but the ATPase also affects transcription through different routes . In summary , we found that the depletion of the two DOM isoforms in cells caused specific transcriptional perturbations . The partially overlapping responses upon DOM-B and H2A . V depletions motivated a more in-depth analysis of the relationship between DOM-B and H2A . V levels in the genome . Both SWR1-type ATPases , DOM-A and DOM-B may contribute to H2A . V incorporation and turnover . We explored global changes in H2A . V levels upon isoform-specific RNAi in nuclear extracts containing chromatin and soluble nuclear proteins . We found a strong H2A . V reduction upon DOM-B depletion ( Figure 3A , Figure 3—figure supplement 1A ) , while H2A . V mRNA level was unchanged ( Figure 3—figure supplement 1B ) . Among the interactors found in our mass-spectrometry analysis , only RNAi against the DOM-B . C-specific subunit ARP6 reduced H2A . V levels to a similar extent ( Figure 3—figure supplement 1C , D ) . H2A . V was not affected by the knock-down of DOM-A , TIP60 or other DOM-A . C-specific subunits ( Figure 3A , Figure 3—figure supplement 1A , C , D ) . While western blots reveal global changes , they are not sufficiently sensitive to detect changes of H2A . V occupancy at specific sites in chromatin . We therefore employed a more sensitive chromatin immunoprecipitation ( ChIP-seq ) approach , in which we included D . virilis spike-in cells to quantify global changes in H2A . V levels . As expected , we scored dramatic effects on H2A . V levels along the entire genome upon depletion of DOM-B , including promoters and transcriptional termination sites ( Figure 3B , C , Figure 3—figure supplement 1E ) . Depletion of DOM-A did not affect chromosomal H2A . V at any of these sites ( Figure 3B , C ) . These observations support the notion that the DOM-B . C , and not the DOM-A . C , is the remodeler dedicated to H2A . V incorporation . Since SWR1-type remodelers bind and hydrolyze ATP to incorporate H2A . Z variants ( Hong et al . , 2014; Willhoft et al . , 2018 ) , we wanted to confirm the ATP-requirement for in vivo incorporation of H2A . V by DOM-B . We devised an RNAi-based complementation strategy in which we rescued the effects of depleting endogenous dom-B mRNA by expression of RNAi-resistant dom-B transgenes . The functional complementation involved wild-type DOM-B or a mutant predicted to be deficient in ATP-binding ( K945G ) ( Hong et al . , 2014; Mizuguchi et al . , 2004; Figure 3D ) . GFP-tagging of the DOM-B proteins allowed to selectively monitor the H2A . V levels by immunofluorescence microscopy in the cells in which the transgenes are expressed . We detected higher levels of H2A . V in cells complemented with a wild-type DOM-B transgene , indicating the expected rescue ( Figure 3E ) . Conversely , the remodeling-defective mutant transgene did not increase the residual H2A . V levels ( Figure 3F ) . Comparing the mean H2A . V signal between cells that express the transgene ( GFP+ ) and cells that don’t ( GFP- ) , revealed once more that only the wild-type could restore H2A . V levels ( Figure 3G , Figure 3—figure supplement 1F ) . The data suggest that the DOM-B . C is responsible for the incorporation of H2A . V in an ATP-dependent manner . Despite the presence of an ATPase domain identical to DOM-B , DOM-A does not seem to be responsible for H2A . V incorporation in steady state . Therefore , we considered other functions for DOM-A . C . The striking correlation between transcriptional responses upon TIP60 and DOM-A depletion suggests a unique association with functional relevance . Our mass-spectrometry analysis had identified several proteins that are homologous to corresponding subunits of the yeast NuA4 HAT complex . The core NuA4 . C subunit EAF1 is a small protein with prominent N-terminal HSA and C-terminal SANT domains . DOM-A also features similarly arranged domains , but they are separated by the long ATPase domain ( Figure 4—figure supplement 1A ) . This raises the question whether DOM-A might serve as the central subunit of a NuA4-type complex in Drosophila . The existence of such a complex with functional and structural similarity to the well-studied yeast complex has not been reported so far . Since NuA4 . C is responsible for histone acetylation , we looked at H3 and H4 acetylation changes upon DOM isoform-specific knock-down by targeted mass-spectrometry . The hypothesis of a Drosophila NuA4 . C poses the acetyltransferase TIP60 as the main effector of DOM-A . C . This is supported by the earlier finding that TIP60 is unstable in the absence of DOM-A ( Figure 2A ) . We therefore included TIP60 knock-down for our targeted mass-spectrometry . Our analysis showed that RNAi against DOM-A , but not against DOM-B , specifically reduces H4K12ac ( average 28 . 9% reduction ) and , to a lesser extent , H4K5ac ( average 23 . 1% reduction ) ( Figure 4A , Figure 4—figure supplement 1B ) . Importantly , unsupervised clustering shows that depleting DOM-A or TIP60 lead to very similar changes in histone acetylation patterns: depletion of TIP60 also reduces H4K12ac , by on average 36 . 3% and H4K5ac by 16 . 4% ( Figure 4A , Figure 4—figure supplement 1B ) . Interestingly , we detected a decrease in monomethylation and increase of trimethylation at H3K27 by DOM depletion , a bit stronger for DOM-A or TIP60 ( Figure 4—figure supplement 1C , D ) . H4 methylation was unchanged ( Figure 4—figure supplement 1E ) . The H4K12 seems to be the most prominent chromatin target of the DOM-A/TIP60 complex . We sought to confirm the mass spectrometric result by an orthogonal ChIP-seq experiment . We found the H4K12ac signal reduced in many regions of the genome , including promoters and transcriptional termination sites , upon TIP60 RNAi and to a lesser extent upon DOM-A RNAi ( Figure 4B , C , Figure 4—figure supplement 1F , H , I ) , but the results suffer from variability , probably due to a low ChIP efficiency of the H4K12ac antibody ( Figure 4—figure supplement 1F ) . Comparison between H4K12ac and transcription showed that genes downregulated in DOM-A or TIP60 knock-down tend to have higher basal levels of H4K12ac at promoters ( Figure 4—figure supplement 1G ) . The reduction of H4K12ac by DOM-A or TIP60 knock-down , however , is global and does not affect some genes specifically ( Figure 4—figure supplement 1H ) . Remarkably , depletion of DOM-B caused an unexpected increase in H4K12 acetylation at many chromosomal regions that lose the mark upon DOM-A ablation ( Figure 4B , C ) . Depletion of H2A . V also causes a small H4K12ac increase similarly to DOM-B knock-down , as if the absence of this remodeler and/or its substrate allowed more DOM-A activity at promoters . The yeast NuA4 . C does not contain a functional ATPase at its core . To explore whether the acetylation of H4K12 catalyzed by the DOM-A . C depends on ATP-dependent nucleosome remodeling activity , we employed the same RNAi-based complementation strategy we had used for DOM-B ( Figure 3D ) . DOM-A was depleted and RNAi-resistant DOM-A wild-type or ATPase mutant derivatives were tested for their ability to rescue the loss of H4K12ac . As expected , the wild-type DOM-A transgene restored H4K12 acetylation ( Figure 4D ) . Remarkably , this acetylation did not depend on a functional DOM-A ATPase . Comparison of the mean H4K12ac signals in cells that do or do not express the transgene confirmed that both , the wild-type and the mutant transgenes , could restore H4K12ac levels ( Figure 4E , Figure 4—figure supplement 1J ) .
Our mass-spectrometry analysis of endogenously expressed DOM isoforms purified under stringent conditions revealed two separate complexes . A DOM . C was previously reported after overexpression and affinity-purification of tagged PONTIN ( Kusch et al . , 2004 ) , which yielded a mixture of DOM-A and DOM-B complexes and may be contaminated with the dINO80 complex , which also contains PONTIN ( Klymenko et al . , 2006 ) . In light of our results , we think the model for H2A . V exchange during DNA damage response proposed in this early work ( Kusch et al . , 2004 ) should be re-visited accounting for the contribution of both DOM-A . C , DOM-B . C and possibly dINO80 . C . It will be interesting to define the role of each complex on the recognition and restoration of damaged chromatin , especially at the level of H2A . V remodeling and acetylation-based signaling . We previously showed that DOM-B , and not DOM-A or TIP60 , affects H2A . V levels during fly oogenesis ( Börner and Becker , 2016 ) . In addition to confirming this finding in a different system and with complementary experimental approaches , we now also showed that the DOM-B . C is responsible for H2A . V incorporation into chromatin . The reaction requires ATP , like SWR1 . C-mediated H2A . Z incorporation . We also discovered a previously unidentified subunit , ARP6 , which is necessary for the maintenance of H2A . V global levels , just like DOM-B . In yeast SWR1 . C and human SRCAP . C , the ARP6 orthologs are indispensable for nucleosome remodeling since they couple the ATPase motor to productive nucleosome sliding ( Matsuda et al . , 2010; Willhoft et al . , 2018; Willhoft and Wigley , 2020; Wu et al . , 2005 ) . The Drosophila DOM-B . C is likely to employ a similar remodeling mechanism . Knock-down of DOM-B affects transcription , but the effects overlap only partially with those that follow H2A . V depletion . This discrepancy could be explained in several ways . First , the reduction of H2A . V levels upon DOM-B knock-down is not as extensive as the one caused by direct depletion of H2A . V . The residual levels of H2A . V upon DOM-B depletion may suffice to regulate transcription at many promoters . Second , we cannot exclude that DOM-B . C also impacts transcription independently of H2A . V incorporation . Third , the global increase of H4K12ac at promoters upon DOM-B knock-down might indirectly compensate for the loss of H2A . V at some specific genes . The DOM-A . C , surprisingly , did not affect H2A . V incorporation under physiological conditions in any of our assays , in agreement with what has been observed for the DOM-A isoform during oogenesis ( Börner and Becker , 2016 ) . DOM-A . C lacks the ARP6 subunit that is a mechanistic requirement for nucleosome remodeling by INO80-type remodelers ( Willhoft and Wigley , 2020 ) . Because the ATPase domain of DOM-A is identical to the one in DOM-B , it is possible that DOM-A utilizes ATP under circumstances that we did not monitor in our study . It is also possible that DOM-A . C-specific subunits have an inhibitory effect on DOM-A ATPase activity through allosteric regulation . Of note , the recombinant human ortholog of DOM-A , EP400 , can incorporate H2A . Z ( Park et al . , 2010 ) , but H2A . Z levels are unaffected if EP400 is depleted in vivo , where it resides in a multi-subunit complex ( Pradhan et al . , 2016 ) . Regulation of nucleosome remodeling through autoinhibitory domains or associated subunits is a widespread mechanism ( Clapier et al . , 2017 ) . Our data suggest that the DOM-A . C is the functional equivalent of the yeast NuA4 . C , which acetylates the H4 N-terminus ( Kuo et al . , 2015 ) and possibly other proteins . Depletion of DOM-A . C causes a significant reduction of H4K12ac at a global level . Some genomic regions that still retain a high H4K12ac ChIP signal in the absence of DOM-A may be explained by the presence of additional acetyltransferases targeting H4K12 , such as CHAMEAU ( Feller et al . , 2015; Peleg et al . , 2016 ) . The function of H4K12ac is still largely unknown in Drosophila , although it has been implicated in aging ( Peleg et al . , 2016 ) . We speculate that H4K12ac may participate in transcriptional regulation since knock-down of DOM-A or TIP60 perturb the transcriptional program in a very similar manner . Genes down-regulated upon DOM-A and TIP60 RNAi show high H4K12ac around their TSS , but the H4K12ac is not specifically reduced at their promoters . We speculate that these genes might rely more on H4K12ac for their expression or be more sensitive to changes in acetylation . Given that H4K12ac is the most abundant H4 acetylation ( Feller et al . , 2015; Peleg et al . , 2016 ) , we also cannot exclude that some of these effects are due to global and aberrant chromosomal condensation . The increase in H4K12ac at promoter observed upon DOM-B RNAi appears to be partially phenocopied by the knock-down of H2A . V . It is possible that H2A-containing nucleosomes are a better substrate for the DOM-A . C compared to the ones containing only H2A . V . The loss of the variant might therefore result in higher H4K12ac catalyzed by TIP60 . In the absence of DOM-A , TIP60 is unstable suggesting that the DOM-A . C is the major form of TIP60 , at least in D . melanogaster cells . It also suggests that during evolution a HAT module and some components of the SWR1 . C became stably associated in a new functional complex , the dNuA4 . C , as it has been proposed for the human EP400 complex ( Auger et al . , 2008 ) . An intermediate case is found in C . albicans , where acetylation of EAF1 by TIP60 mediates a reversible association between the NuA4 . C and SWR1 . C ( Wang et al . , 2018b ) . We found that the DOM-A . C H4K12 HAT activity does not need the DOM-A ATPase activity . In yeast , inserting the ATPase domain of the Drosophila DOM between the HSA and SANT domains of EAF1 , the central subunit of the yeast NuA4 , does not affect its function ( Auger et al . , 2008 ) . Such a situation could also apply to DOM-A . Lastly , our mass-spectrometry analysis revealed new , uncharacterized interactors for DOM-A . Of those , the transcription factor CG12054 has been found as a potential DOM partner in a previous screen ( Rhee et al . , 2014 ) . Its human ortholog , JAZF1 , appears to be involved in transcriptional repression ( Nakajima et al . , 2004 ) and has been associated to endometrial stromal tumors ( Koontz et al . , 2001 ) . Its function in flies is unknown . Division of labor between chromatin modifying enzymes is key to ensure efficient regulation of nuclear processes . During evolution , genome duplications and genetic divergence expand and diversify activities . The case of DOM illustrates beautifully how evolution can take different routes to achieve what must be assumed as an important functional specification ( Figure 4F , Supplementary file 3 ) . In yeast , the SWR1 and NuA4 complexes are entirely separate entities . In humans , whose genomes underwent duplication events , the paralogous SRCAP and EP400 protein ATPases each organize different complexes that may serve distinct , conserved functions . In Drosophila a similar specialization was achieved by alternative splicing . Surprisingly , the gene orthologs of dom in honeybee ( A . mellifera , LOC413341 ) , jewel wasp ( N . vitripennis , LOC100115939 ) , Jerdon’s jumpin ant ( H . saltator , LOC105183375 ) , red flour beetle ( T . castaneum , LOC656538 ) and even in the common house spider ( P . tepidariorum , LOC107448208 ) undergo alternative splicing to generate at least two isoforms with different C-termini . The mode of specification of SWR1 and NuA4 through splice variants might therefore not be limited to Drosophila , but more wide-spread throughout the Arthropoda phylum .
D . melanogaster embryonic Kc167 cell line was obtained from the Drosophila Genomic Resource Center ( https://dgrc . bio . indiana . edu/Home ) . D . melanogaster S2 ( subclone L2-4 ) cell line was a kind gift of P Heun ( Villa et al . , 2016 ) . D . virilis 79f7Dv3 cell line was a kind gift of BV Adrianov ( Albig et al . , 2019 ) . The identity of cell lines was verified by high-throughput sequencing . Cells were subjected to mycoplasma testing . Cells were maintained at 26°C in Schneider’s Drosophila Medium ( Thermo-Fischer , Cat . No 21720024 ) supplemented with 10% FBS ( Kc167 and S2 ) or 5% FBS ( 79f7Dv3 ) and 1% Penicillin-Streptomycin solution ( Sigma-Aldrich , Cat No P-4333 ) . gRNAs targeting exon 14 ( DOM-A and DOM-G , Flybase transcripts dom-RA and dom-RG ) or exon 11 ( DOM-B , Flybase transcript dom-RE ) were initially designed using GPP sgRNA designer ( https://portals . broadinstitute . org/gpp/public/analysis-tools/sgrna-design; Doench et al . , 2016 ) . gRNA candidates were checked for off-targets using flyCRISPR Target Finder ( https://flycrispr . org/target-finder/ , guide length = 20 , Stringency = high , PAM = NGG only ) ( Gratz et al . , 2014 ) . Two gRNAs each for DOM-A and DOM-B were selected ( Supplementary file 4 ) . The 20 bp gRNAs were fused to a tracrRNA backbone during synthesis and cloned downstream of the Drosophila U6 promoter . These constructs were synthesized as gBlocks ( Integrated DNA Technologies ) and PCR-amplified before transfection using Q5 polymerase ( New England Biolabs , Cat No . M0491S ) . To generate the repair template , the sequence encoding for 3XFLAG tag , including a stop codon , was inserted between two homology arms of 200 bp each by gene synthesis ( Integrated DNA Technologies ) ( Supplementary file 4 ) . The repair templates were cloned in pUC19 to generate repair plasmids . For CRISPR editing , one million S2 cells ( subclone L2-4 ) in 500 µL medium were seeded in each well of a 24-well plate . After 4 hr , cells were transfected with 110 ng of gRNAs ( 55 ng each ) , 200 ng of repair plasmid and 190 ng of pIB_Cas9_Blast ( encoding SpCas9 and carrying Blasticidin resistance , kind gift of P Heun ) using X-tremeGENE HP DNA Transfection Reagent ( Roche , Cat . No 6366236001 ) . 24 hr after transfection , medium was replaced with 500 µL of fresh medium containing 25 µg/ml Blasticidin ( Gibco , Cat . No A1113903 ) . Three days after selection the cells were collected and seeded into 6 cm tissue culture dishes at three different concentrations ( 1000 , 2000 and 5000 cells/well ) and allowed to attach for 1–2 hr . Medium was then removed and cells carefully overlaid with 2 . 5 mL of a 1:1 mix of 2X Schneider’s Medium ( prepared from powder , Serva , Cat . No 35500 ) + 20% FBS + 2% Penicillin-Streptomycin and 0 . 4% low-melting agarose equilibrated to 37°C . The dishes were sealed with parafilm , inserted into 15 cm dishes together with a piece of damp paper and sealed once more with parafilm . After 2 to 3 weeks , individual cell colonies were picked using a pipet , suspended in 100 µL of Schneider’s Drosophila Medium + 10% FBS + 1% Penicillin-Streptomycin and plated into 96-well plates . Clones were expanded for 1–2 weeks and further expanded into 48-well plates . For PCR-testing of clones , 50 µL of cells were collected , 50 µL water was added and DNA was purified using Nucleospin Gel and PCR Cleanup ( Macherey-Nagel , Cat . No 740609 . 250 ) . Extracted DNA was PCR-amplified to check the insertion of the 3XFLAG tag . The PCR product of DOM-A clone #2 results larger due to the presence of an insertion 29 bp downstream of the stop codon ( Figure 1—figure supplement 1B ) . Selected clones were further expanded and stored in liquid nitrogen in 90% FBS + 10% DMSO . For nuclear extraction from 3XFLAG-tagged cells lines , 0 . 5–1 billion cells were collected by centrifugation at 500 g for 5 min . Cells were washed with 10 ml of PBS , resuspended in 10 ml of ice-cold NBT-10 buffer [15 mM HEPES pH 7 . 5 , 15 mM NaCl , 60 mM KCl , 0 . 5 mM EGTA pH 8 , 10% Sucrose , 0 . 15% Triton-X-100 , 1 mM PMSF , 0 . 1 mM DTT , 1X cOmplete EDTA-free Protease Inhibitor ( Roche , Cat . No 5056489001 ) ] and rotated for 10 min at 4°C . Lysed cells were gently overlaid on 20 ml of ice-cold NB-1 . 2 buffer ( 15 mM HEPES pH 7 . 5 , 15 mM NaCl , 60 mM KCl , 0 . 5 mM EGTA pH 8 , 1 . 2 M Sucrose , 1 mM PMSF , 0 . 1 mM DTT , 1X cOmplete EDTA-free Protease Inhibitor ) and spun at 4000 g for 15 min . Pelleted nuclei were washed once with 10 ml of ice-cold NB-10 buffer ( 15 mM HEPES pH 7 . 5 , 15 mM NaCl , 60 mM KCl , 0 . 5 mM EGTA pH 8 , 10% Sucrose , 1 mM PMSF , 0 . 1 mM DTT , 1X cOmplete EDTA-free Protease Inhibitor ) and resuspended in ice-cold Protein-RIPA buffer ( 50 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 1 mM EDTA , 0 . 5% IGEPAL CA-630 , 0 . 5% Na-Deoxycholate , 0 . 1% SDS , 1 mM PMSF , 0 . 1 mM DTT , 1X cOmplete EDTA-free Protease Inhibitor ) . Nuclei were sonicated in 15 mL Falcons tubes using Diagenode Bioruptor ( 20 cycles , 30 s ON/30 s OFF ) . Extract was spun at 16000 g for 15 min at 4°C in a table-top centrifuge . Soluble extract was collected and total protein concentration determined using Protein Assay Dye Reagent Concentrate ( BIO-RAD , Cat No 5000006 ) with BSA as standard . 2 mg aliquots were flash-frozen . For FLAG-immunoprecipitation , 2 mg of nuclear protein were thawed , spun at 160 , 000 g for 15 min at 4°C to remove aggregates . Extracts were diluted 1:1 with Benzonase dilution buffer ( 50 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 4 mM MgCl2 , 0 . 5% NP-40 , 1 mM PMSF , 0 . 1 mM DTT , 1X cOmplete EDTA-free Protease Inhibitor ) . 60 µL ( 50% slurry ) of Protein-RIPA-equilibrated FLAG-m2 beads ( Sigma-Aldrich , Cat . No A2220 ) were added together with 1 µL of Benzonase ( Merck-Millipore , Cat . No 1 . 01654 . 0001 ) . After 3 hr of incubation at 4°C on a rotating wheel , the beads were washed 3 times with ice-cold Protein-RIPA buffer and thrice with ice-cold TBS ( 5 mM Tris-HCl pH 7 . 5 , 150 mM NaCl ) ( 5 min rotation each , 4°C ) . For western blots , beads were then resuspended in 50 µL of 5X Laemmli Sample buffer ( 250 mM Tris-HCl pH 6 . 8 , 10% w/v SDS , 50% v/v glycerol , 0 . 1% w/v bromophenol blue , 10% β-mercaptoethanol ) and boiled for 5 min at 95°C . For mass-spectrometry , beads were incubated with 50 µL of elution buffer ( 2 M urea , 50 mM Tris-HCl , pH 7 . 5 , 2 mM DTT and 10 µg ml–1 trypsin ) for 30 min at 37°C . The eluate was removed and beads were incubated in 50 µL of alkylation buffer ( 2 M urea , 50 mM Tris-HCl , pH 7 . 5 and 10 mM chloroacetamide ) at 37°C for 5 min . Combined eluates were further incubated overnight at room temperature . Tryptic-peptide mixtures were acidified with 1% Trifluoroacetic acid ( TFA ) and desalted with Stage Tips containing three layers of SDB-RPS ( Polystyrene-divinylbenzene copolymer partially modified with sulfonic acid ) material . To this end , samples were mixed 1:1 with 1% TFA in isopropanol and loaded onto the stagetip . After two washes with 100 µL 1%TFA in Isopropanol and two washes with 100 µl 0 . 2%TFA in water , samples were eluted with 80 µl of 2% ( v/v ) ammonium hydroxide , 80% ( v/v ) acetonitrile ( ACN ) and dried on a centrifugal evaporator . Samples were dissolved in 10 µL Buffer A* ( 2% ACN/0 . 1% TFA ) for mass spectrometry . Peptides were separated on 50‐cm columns packed in house with ReproSil‐Pur C18‐AQ 1 . 9 μm resin ( Dr Maisch ) . Liquid chromatography was performed on an EASY‐nLC 1200 ultra‐high‐pressure system coupled through a nanoelectrospray source to a Q-Exactive HF-X mass spectrometer ( Thermo Fisher ) . Peptides were loaded in buffer A ( 0 . 1% formic acid ) and separated by application of a non-linear gradient of 5–30% buffer B ( 0 . 1% formic acid , 80% ACN ) at a flow rate of 300 nl min–1 over 70 min . Data acquisition switched between a full scan and 10 data‐dependent MS/MS scans . Full scans were acquired with target values of 3 × 106 charges in the 300–1 , 650 m/z range . The resolution for full-scan MS spectra was set to 60 , 000 with a maximum injection time of 20 ms . The 10 most abundant ions were sequentially isolated with an ion target value of 1 × 105 and an isolation window of 1 . 4 m/z . Fragmentation of precursor ions was performed by higher energy C-trap dissociation with a normalized collision energy of 27 eV . Resolution for HCD spectra was set to 15 , 000 with a maximum ion-injection time of 60 ms . Multiple sequencing of peptides was minimized by excluding the selected peptide candidates for 30 s . In total , 3 technical replicates ( parallel immunoprecipitations ) for each of the 3 biological replicates ( 1 clone = 1 replicate , extract prepared on different days ) were analyzed . Raw mass spectrometry data were analyzed with MaxQuant ( version 1 . 5 . 6 . 7 ) ( Cox and Mann , 2008 ) and Perseus ( version 1 . 5 . 4 . 2 ) software packages . Peak lists were searched against the Drosophila melanogaster UniProt FASTA database combined with 262 common contaminants by the integrated Andromeda search engine ( Cox et al . , 2011 ) . The false discovery rate ( FDR ) was set to 1% for both peptides ( minimum length of 7 amino acids ) and proteins . ‘Match between runs’ ( MBR ) with a maximum time difference of 0 . 7 min was enabled . Relative protein amounts were determined with the MaxLFQ algorithm ( Cox et al . , 2014 ) , with a minimum ratio count of two . Missing values were imputed from a normal distribution , by applying a width of 0 . 2 and a downshift of 1 . 8 standard deviations . Imputed LFQ values of the technical replicates for each biological replicate were averaged . Differential enrichment analysis was performed in R using the limma package as previously described ( Kammers et al . , 2015; Ritchie et al . , 2015 ) . Adjusted p-values ( FDR ) were calculated using the p . adjust function ( method = ‘fdr’ ) ( Source code 1 ) . Primers for dsRNA templates were either obtained from the TRiP website ( https://fgr . hms . harvard . edu/fly-in-vivo-rnai ) , designed using SnapDragon ( https://www . flyrnai . org/snapdragon ) or designed using E-RNAi ( https://www . dkfz . de/signaling/e-rnai3/ ) ( Horn and Boutros , 2010; Perkins et al . , 2015 ) , except one primer pair for Tip60 RNAi which was obtained from Kusch et al . ( 2004 ) ( Supplementary file 4 ) . Templates for in vitro transcription were generated by PCR-amplification using Q5 Polymerase ( New England Biolabs , Cat No . M0491S ) . dsRNAs were generated by in vitro transcription using MEGAScript T7 kit ( Invitrogen , Cat . No AMB 13345 ) , followed by incubation at 85°C for 5 min and slow cool-down to room temperature . D . melanogaster Kc167 cell were collected by spinning at 500 g for 5 min . Cells were washed once with PBS and resuspended in Schneider’s Drosophila Medium without serum and Penicillin-Streptomycin at a concentration of 1 . 5 million/ml ( for RNAi in 12-well and 6-well plates ) or 3 million/ml ( for RNAi in T-75 flasks ) . 0 . 75 million ( 12-well ) , 1 . 5 million ( 6-well ) or 15 million ( T-75 flasks ) cells were plated and 5 µg ( 12-well ) , 10 ug ( 6-well ) or 50 µg ( T-75 flasks ) of dsRNA was added . Cells were incubated for 1 hr with gentle rocking and 3 volumes of Schneider’s Drosophila Medium ( supplemented with 10% FBS and 1% Penicillin-Streptomycin solution ) was added . After 6 days cells were collected and analyzed . Two million of Kc167 cells were pelleted at 500 g for 5 min . Cells were resuspended in 1 mL of PBS and 1 million of D . virilis 79f7Dv3 cells were added . Cells were pelleted at 500 g for 5 min and total RNA was extracted using RNeasy Mini Kit ( QIAgen , Cat No . 74104 ) , including DNAse digestion step ( QIAgen , Cat No . 79254 ) . mRNA was purified using Poly ( A ) RNA selection kit ( Lexogen , Cat . No M039100 ) . Both total RNA and mRNA quality was verified on a 2100 Bioanalyzer ( Agilent Technologies , Cat . No G2939BA ) . Libraries for sequencing were prepared using NEBnext Ultra II directional RNA library prep kit for Illumina ( New England Biolabs , Cat . No E7760L ) . Libraries were sequenced on an Illumina HiSeq 1500 instrument at the Laboratory of Functional Genomic Analysis ( LAFUGA , Gene Center Munich , LMU ) . For the analysis , 50 bp single reads were mapped to the D . melanogaster ( release 6 ) or independently to the D . virilis ( release 1 ) genome using STAR ( version 2 . 5 . 3a ) with the GTF annotation dmel-all-r6 . 17 . gtf or dvir-all-r1 . 07 . gtf , respectively . Multi-mapping reads were filtered out by the parameter --outFilterMultimapNmax 1 . Genic reads were counted with the parameter --quantMode GeneCounts . Read count tables were imported to R and low count genes were removed ( at least 1 read per gene in 6 of the samples ) . Normalization factors ( sizeFactors ) were calculated for D . melanogaster or D . virilis count tables independently using DESeq2 package ( version 1 . 24 ) . Normalization factors derived from D . virilis were applied to D . melanogaster counts . Statistical analysis was carried out using DESeq2 by providing replicate information as batch covariate . Estimated log2 fold-change and adjusted p-values were obtained by the results function ( DESeq2 ) and adjusted p-value threshold was set 0 . 01 . Batch effect was corrected by the ComBat function from the sva package ( version 3 . 32 ) on the log2-transformed normalized read counts . Batch adjusted counts were plotted relative to control or used for principal component analysis ( PCA ) . Plots were generated using R graphics . Scripts are available on GitHub ( https://github . com/tschauer/Domino_RNAseq_2020 ) . cDNA was synthesized from 1 µg of total RNA ( extracted as previously described but omitting the addition of D . virilis spike-in cells ) using Superscript III First Strand Synthesis System ( Invitrogen , Cat . No 18080–051 , random hexamer priming ) and following standard protocol . cDNA was diluted 1:100 , qPCR reaction was assembled using Fast SYBR Green Mastermix ( Applied Biosystem , Cat . No 4385612 ) and ran on a Lightcycler 480 II ( Roche ) instrument . Primer efficiencies were calculated via serial dilutions . Sequences as available in Supplementary file 4 . For nuclear fractionation , 5–10 million cells were pelleted at 500 g for 5 min and washed with PBS . Cell pellets were either used directly or flash-frozen for later processing . Pellets were suspended in 300 µL of ice-cold NBT-10 buffer and rotated for 10 min at 4°C . Lysed cells were gently overlaid on 500 µL of ice-cold NB-1 . 2 buffer and spun at 5000 g for 20 min . Pelleted nuclei were washed once with 500 µL of ice-cold NB-10 buffer . Nuclei were resuspended in 60 µL of 1:1 mix of Protein-RIPA buffer and 5X Laemmli Sample buffer . Samples were boiled for 5 min at 95°C and ran on pre-cast 8% or 14% polyacrylamide gels ( Serva , Cat . No 43260 . 01 and 43269 . 01 ) under denaturing conditions in 1X Running Buffer ( 25 mM Tris , 192 mM glycine , 0 . 1% SDS ) . Gels were wet-transferred onto nitrocellulose membranes ( GE Healthcare Life Sciences , Cat . No 10600002 ) in ice-cold 1X Transfer Buffer ( 25 mM Tris , 192 mM glycine ) with 20% methanol ( for histones ) or with 10% methanol + 0 . 1% SDS ( for DOM proteins ) at 400 mA for 45–60 min . Membranes were blocked with 5% BSA in TBS buffer for 1 hr and incubated with primary antibodies in TBST buffer ( TBS + 0 . 1% Tween-20 ) + 5% non-fat milk at 4°C overnight . Membrane were washed 3 times ( 5 min each ) with TBST buffer and incubated with secondary antibodies in TBST buffer for 1 hr at room temperature . Membrane were washed 3 times with TBST buffer , 2 times with TBS buffer ( 5 min each ) , and dried before imaging . Images were taken on a LI-COR Odyssey or a LI-COR Odyssey CLx machine ( LI-COR Biosciences ) . 70 to 130 million D . melanogaster Kc167 cells , resuspended in 20 ml of complete Schneider’s Drosophila Medium , were crosslinked by adding 1:10 of the volume of Fixing Solution ( 100 mM NaCl , 50 mM Hepes pH 8 , 1 mM EDTA , 0 . 5 mM EGTA , 10% methanol-free formaldehyde ) and rotated at room temperature for 8 min . 1 . 17 ml of freshly-prepared 2 . 5 M glycine was added to stop the fixation ( final conc . 125 mM ) . Cells were pelleted at 500 g for 10 min ( 4°C ) and resuspended in 10 mL of ice-cold PBS . 3 . 5 million of fixed D . virilis cells ( fixed as described for D . melanogaster cells ) were added for every 70 million D . melanogaster cells . Cells were pelleted at 526 g for 10 min ( 4°C ) and resuspended in 1 ml of PBS + 0 . 5% Triton-X-100 + 1X cOmplete EDTA-free Protease Inhibitor for every 70 million D . melanogaster cells and rotated at 4°C for 15 min to release nuclei . Nuclei were pelleted at 2000 g for 10 min and washed once with 10 ml of ice-cold PBS . Nuclei were suspended in 1 ml of RIPA buffer ( 10 mM Tris-HCl pH 8 , 140 mM NaCl , 1 mM EDTA , 0 . 1% Na-deoxycholate , 1% Triton-X-100 , 0 . 1% SDS , 1 mM PMSF , 1X cOmplete EDTA-free Protease Inhibitor ) + 2 mM CaCl2 for every 70 million D . melanogaster cells , divided into 1 ml aliquots and flash-frozen in liquid N2 . 1 mL of fixed nuclei was quickly thawed and 1 µL of MNase ( to 0 . 6 units ) ( Sigma-Aldrich , Cat . No N5386 ) added . Chromatin was digested for 35 min at 37°C . MNase digestion was stopped by transferring the samples on ice and adding 22 µL of 0 . 5 M EGTA . Samples were mildly sonicated using a Covaris S220 instrument with the following settings: 50 W peak power , 20% duty factor , 200 cycles/burst , 8 min total time . Insoluble chromatin was removed by centrifugation at 16 , 000 g for 30 min at 4°C . Soluble chromatin was pre-cleared by incubation with 10 µL of 50% RIPA-equilibrated Protein A + G sepharose bead slurry ( GE Healthcare , Cat . No 17-5280-11 and 17-0618-06 ) for every 100 µL of chromatin for 1 hr at 4°C . 100 µL of pre-cleared chromatin were set aside ( input ) and kept overnight at 4°C , while each primary antibody was added to 300 µL of chromatin and incubated overnight at 4°C . 40 µL of 50% RIPA-equilibrated Protein A + G sepharose bead slurry was added for each immunoprecipitation and rotated 3 hr at 4°C . Beads were washed 5 times with 1 ml of RIPA ( 5 min rotation at 4°C , pelleted at 3000 g for 1 min between washes ) and resuspended in 100 µL of TE ( 10 mM Tris pH 8 , 1 mM EDTA ) . 0 . 5 µL of RNAseA ( Sigma-Aldrich , Cat . No . R4875 ) was added to both input samples and resuspended beads , followed by incubation at 37°C for 30 min . After addition of 6 µL of 10% SDS , protease digestion ( 250 ng/ µL Proteinase K , Genaxxon , Cat . no . M3036 . 0100 ) and crosslink reversal were performed simultaneously at 68°C for 2 hr . DNA was purified using 1 . 8X Agencourt AMPure XP beads ( Beckman Coulter , Cat No A63880 ) following the standard protocol and eluted in 30 µL of 5 mM Tris-HCl pH 8 . Libraries for sequencing were prepared using NEBNext Ultra II DNA library prep kit for Illumina ( New England Biolabs , E7465 ) . Libraries were sequenced on an Illumina HiSeq 1500 instrument at the Laboratory of Functional Genomic Analysis ( LAFUGA , Gene Center Munich , LMU ) . 50 bp single-end reads were mapped to the D . melanogaster ( release 6 ) or independently to the D . virilis ( release 1 ) genome using bowtie2 ( Langmead and Salzberg , 2012 ) using standard parameters . Tag directories and input-normalized coverage files were generated using Homer ( Heinz et al . , 2010 ) with the parameters -fragLength 150 and -totalReads was set to the number of reads mapped to D . virilis genome . Input-normalized , scaled D . melanogaster coverage files were visualized using the Integrative Genomics Viewer ( Robinson et al . , 2011 ) . Scripts for D . virilis scaling and input normalization are available on GitHub ( https://github . com/tschauer/Domino_ChIPseq_2020 ) . Composite plots were generated using tsTools ( https://github . com/musikutiv/tsTools ) and base R graphics . Annotations are derived from TxDb . Dmelanogaster . UCSC . dm6 . ensGene_3 . 4 . 4 ( http://bioconductor . org/packages/release/data/annotation/html/TxDb . Dmelanogaster . UCSC . dm6 . ensGene . html ) . Heatmaps were generated using pheatmap ( https://cran . r-project . org/web/packages/pheatmap/index . html ) . Violin-boxplots were generated using ggplot2 ( https://cran . r-project . org/web/packages/ggplot2/index . html ) . DOM-A and DOM-B cDNAs were cloned into pENTR3c vector ( Thermo Fischer Scientific , Cat . No A10464 ) by In-Fusion Cloning ( Takara Bio , Cat . No 638909 ) using LD35056 , LD03212 , LD32234 plasmids Drosophila Genomic Resource Center ) as templates for PCR . RNAi-resistant DOM-A and DOM-B were generated by substituting around 500 bp of the original cDNA sequence with manually mutagenized , synthesized DNA constructs ( gBlock , Integrated DNA Technology ) , by restriction cloning . DOM-A and DOM-B K945G mutants were generated by site-directed mutagenesis ( New England Biolabs , Cat . No E0554S ) . For transfection in Drosophila cells , the constructs in pENTR3c were recombined in pHWG ( expression driven by hsp70 promoter , C-terminal GFP tag;DrosophilaGenomic Resource Center ) by Gateway cloning ( Thermo Fischer Scientific ) . 1–2 million Kc167 cells in 2 ml complete Schneider’sDrosophilaMedium were seeded in each well of a 6-well plate . After 4 hr , cells were transfected with 500 ng of complementation plasmid ( described before ) + 25 ng of pCoBlast ( Thermo Fischer , Cat . No K5150-01 ) using Effectene Transfection Reagent ( QIAgen , Cat . No 301425 ) . 48 hr after transfection , 2 ml of the cells were collected , transferred into T-25 flasks and diluted with 4 ml of complete Schneider’sDrosophilaMedium + Blasticidin at a final concentration of 50 ng/ul . 7–8 days after selection the cells were collected and treated with dsRNA as described before . For immunofluorescence , 0 . 2–0 . 4 million cells in 200 µL of complete Schneider’s Drosophila Medium were seeded onto a round 12 mm coverslips ( Paul Marienfeld GmbH and Co . , Cat No . 0117520 ) placed separately inside wells of 12-well plates . Cell were allowed to attach for 2–4 hr and the coverslips were gently rinsed with 500 µL of PBS . Cells were fixed in 500 µL of ice-cold PBS + 2% formaldehyde for 7 . 5 min . After removal of fixative , cells were permeabilized by adding 500 µL of ice-cold PBS + 0 . 25% Triton-X-100 + 1% formaldehyde and incubating for 7 . 5 min . Coverslips were washed two times with 1 ml of PBS and blocked with PBS + 3% BSA for 1 hr at room temperature . Coverslip were transferred onto a piece of parafilm , placed into a wet chamber and 40 µL of primary antibody solution was gently added onto the coverslip . After overnight incubation at 4°C , coverslips were transferred back to 12-well plates and washed twice with 1 ml of PBS . Coverslip were transferred again onto a piece of parafilm , placed into a wet chamber and 40 µL of secondary antibody was gently added onto the coverslip . After 1 hr incubation at room temperature , coverslips were transferred back to 12-well plates and washed twice with 1 ml of PBS . Cells were incubated with 1 ml of 0 . 2 µg/ml DAPI ( Sigma-Aldrich , Cat . No 10236276001 ) for 5 min at room temperature . Coverslips were washed with PBS and with deionized water , mounted on slides with 8 µL of Vectashield ( Vector Laboratories , Cat . No H-1000 ) and sealed with nail polish . Images were taken on a Leica SP5 confocal microscope . Images were processed and analyzed using Fiji ( Source code 2 ) and data plotted using R-Studio . p-values were calculated using linear regression ( lm function in R ) . Kc167 cells were treated with dsRNAs in 6-well plates as described before . Cells were counted , pelleted and snap-frozen in liquid N2 . For histone acid extraction , pellets from 4 to 12 million cells were resuspended in 500 µL of ice-cold 0 . 2M H2SO4 and histone were extracted by rotating overnight at 4°C . Cell debris were removed by centrifugation at 16 , 000 g for 10 min at 4°C . Histone were precipitated by adding trichloroacetic acid to reach 26% final concentration . Tubes were mixed and incubated at 4°C for 2 hr and spun at 16 , 000 g for 45 min . Pellets were washed twice with ice-cold 100% acetone ( 5 min rotation at 4°C , 10 min of 16 , 000 g spin at 4°C between washes ) , dried for 30 min at room temperature and resuspended in 10 µL of 2 . 5x Laemmli sample buffer for every initial cell million and boiled at 95°C for 5 min . Samples were stored at −20°C until further use . The histones corresponding to 10 million cells were separated onto 4–20% pre-cast polyacrylamide gels ( Serva , Cat . No 43277 . 01 ) . Gels were briefly stained with Coomassie ( Serva , Cat . No 17524 . 01 ) and stored in water at 4°C . For targeted mass-spectrometry analysis , histones were excised , washed once with water and de-stained twice by incubating 30 min at 37°C with 200 µL of 50% acetonitrile ( ACN ) in 50 mM NH4HCO3 . Gel pieces were then washed twice with 200 µL water and twice with 200 µL of 100% ACN to dehydrate them , followed by 5 min of speed-vac to remove residual ACN . Histones were in-gel acylated by adding 10 µL of deuterated acetic anhydride ( Sigma-Aldrich , Cat . No 175641 ) and 20 µL of 100 mM NH4HCO3 . After 1 min , 70 µL of 1 M NH4HCO3 were slowly added to the reaction . Samples were incubated at 37°C for 45 min with vigorous shaking . Samples were washed 5 times with 200 µL of 100 mM NH4HCO3 , 5 times with 200 µL of water and twice with 200 µL of 100% ACN , followed by 3 min of speed-vac . Gel pieces were rehydrated in 20 µL of trypsin solution ( 25 ng/ µL trypsin in 100 mM NH4HCO3 ) ( Promega , Cat . No V5111 ) and incubated at 4°C for 20 min . After the addition of 100 µL of 50 mM NH4HCO3 , histones were in-gel digested overnight at 37°C . Peptides were sequentially extracted by incubating 10 min at room temperature twice with 60 µL of 50% ACN 0 . 25% trifluoroacetic acid ( TFA ) and twice 40 µL of 100% ACN . The total volume ( around 250 µL ) was evaporated in a centrifugal evaporator and the dried peptides were stored at −20°C until resuspension in 100 µL of 0 . 1% TFA . Peptides were loaded in a C18 Stagetip ( pre-washed with ACN and conditioned with 0 . 1% TFA ) , washed 3 times with 20 µL of 0 . 1% TFA and peptides were eluted 3 times with 20 µL of 80% ACN 0 . 25% TFA . Eluted peptides were evaporated in a centrifugal evaporator , resuspended in 15 µl of 0 . 1% TFA and stored at −20°C . Desalted peptides were injected in an RSLCnano system ( Thermo Fisher Scientific ) and separated in a 15 cm analytical column ( 75 µm ID home-packed with ReproSil-Pur C18-AQ 2 . 4 μm from Dr . Maisch ) with a 50 min gradient from 4% to 40% acetonitrile in 0 . 1% formic acid at 300 nl/min flowrate . The effluent from the HPLC was electrosprayed into Q Exactive HF mass spectrometer ( Thermo Fisher Scientific ) . The MS instrument was programmed to target several ions as described before ( Feller et al . , 2015 ) except for the MS3 fragmentation . Survey full scan MS spectra ( from m/z 270–730 ) were acquired with resolution R = 60 , 000 at m/z 400 ( AGC target of 3 × 106 ) . Targeted ions were isolated with an isolation window of 0 . 7 m/z to a target value of 2 × 105 and fragmented at 27% normalized collision energy . Typical mass spectrometric conditions were: spray voltage , 1 . 5 kV; no sheath and auxiliary gas flow; heated capillary temperature , 250°C . Peak integration was performed using Skyline ( https://skyline . ms/project/home/software/Skyline/begin . view ) . Quantified data was further analyzed in R according to the formulas described in Feller et al . ( 2015 ) ( Supplementary file 5; Source code 3 and Source code 4 ) . DOM-A and DOM-B polyclonal antibody were generated by expression of C-terminal specific polypeptides . For DOM-A , residues 2963 to 3188 were expressed as C-terminal Glutathione-S-transferase ( GST ) fusion in E . coli , purified using Glutathione Sepharose resin ( GE Healthcare , Cat . No 17075605 ) and eluted with glutathione . For DOM-B , residues 2395 to 2497 were expressed as C-terminal Maltose Binding Protein ( MBP ) fusion in E . coli , absorbed to amylose resin ( New England Biolabs , Cat . No E8121S ) and eluted with maltose . Antibody production in rabbit was done by Eurogentec ( https://secure . eurogentec . com/eu-home . html ) . Both antibodies were validated by RNAi and western blot . For the monoclonal antibody against TIP60 , N-terminal 6xHis-tagged TIP60 ( full length ) was expressed in E . coli , purified over a Ni-NTA column and eluted with imidazole . Monoclonal antibodies were developed by Dr . Elizabeth Kremmer ( BioSysM , LMU Munich ) . Antibodies were validated by RNAi and western blot . Sources of other antibodies were: DOM-A monoclonal and H2A . V polyclonal ( Börner and Becker , 2016 ) . Histone H4 rabbit polyclonal antibody: Abcam ( Cat . No ab10158 ) . Mouse anti- FLAG monoclonal antibody: Sigma-Aldrich ( Cat . No F3165 ) . Anti H4K12ac rabbit polyclonal antibody: Merck-Millipore ( Cat . No 07–595 ) . Anti-GFP mouse monoclonal antibody: Roche ( Cat . No 11814460001 ) . Anti-lamin mouse monoclonal antibody: kind gift of Dr . Harald Saumweber . Next Generation sequencing data are available at the Gene Expression Omnibus under accession number GSE145738 . Targeted proteomics data are available at ProteomeXchange under accession number PXD017729 . Scripts for D . virilis scaling and input normalization for ChIP-seq are available on GitHub ( https://github . com/tschauer/Domino_ChIPseq_2020; Schauer , 2020a; copy archived at https://github . com/elifesciences-publications/Domino_ChIPseq_2020 ) . Scripts for RNA-seq analysis are available on GitHub ( https://github . com/tschauer/Domino_RNAseq_2020; Schauer , 2020b; copy archived at https://github . com/elifesciences-publications/Domino_RNAseq_2020 ) . Immunofluorescence images used for quantification of the complementation assays are available on Dryad ( https://doi . org/10 . 5061/dryad . 1rn8pk0qt ) .
Histone acetylation and deposition of H2A . Z variant are integral aspects of active transcription . In Drosophila , the single DOMINO chromatin regulator complex is thought to combine both activities via an unknown mechanism . Here we show that alternative isoforms of the DOMINO nucleosome remodeling ATPase , DOM-A and DOM-B , directly specify two distinct multi-subunit complexes . Both complexes are necessary for transcriptional regulation but through different mechanisms . The DOM-B complex incorporates H2A . V ( the fly ortholog of H2A . Z ) genome-wide in an ATP-dependent manner , like the yeast SWR1 complex . The DOM-A complex , instead , functions as an ATP-independent histone acetyltransferase complex similar to the yeast NuA4 , targeting lysine 12 of histone H4 . Our work provides an instructive example of how different evolutionary strategies lead to similar functional separation . In yeast and humans , nucleosome remodeling and histone acetyltransferase complexes originate from gene duplication and paralog specification . Drosophila generates the same diversity by alternative splicing of a single gene .
Cells contain a large number of proteins that control the activity of genes in response to various signals and changes in their environment . Often these proteins work together in groups called complexes . In the fruit fly Drosophila melanogaster , one of these complexes is called DOMINO . The DOMINO complex alters gene activity by interacting with other proteins called histones which influence how the genes are packaged and accessed within the cell . DOMINO works in two separate ways . First , it can replace certain histones with other variants that regulate genes differently . Second , it can modify histones by adding a chemical marker to them , which alters how they interact with genes . It was not clear how DOMINO can do both of these things and how that is controlled; but it was known that cells can make two different forms of the central component of the complex , called DOM-A and DOM-B , which are both encoded by the same gene . Scacchetti et al . have now studied fruit flies to understand the activities of these forms . This revealed that they do have different roles and that gene activity in cells changes if either one is lost . The two forms operate as part complexes with different compositions and only DOM-A includes the TIP60 enzyme that is needed to modify histones . As such , it seems that DOM-B primarily replaces histones with variant forms , while DOM-A modifies existing histones . This means that each form has a unique role associated with each of the two known behaviors of this complex . The presence of two different DOMINO complexes is common to flies and , probably , other insects . Yet , in other living things , such as mammals and yeast , their two roles are carried out by protein complexes originating from two distinct genes . This illustrates a concept called convergent evolution , where different organisms find different solutions for the same problem . As such , these findings provide an insight into the challenges encountered through evolution and the diverse solutions that have developed . They will also help us to understand the ways in which protein activities can adapt to different needs over evolutionary time .