Ash1 counteracts Polycomb repression independent of histone H3 lysine 36 methylation
2019; Springer Nature; Volume: 20; Issue: 4 Linguagem: Inglês
10.15252/embr.201846762
ISSN1469-3178
AutoresEshagh Dorafshan, Tatyana G. Kahn, A.G. Glotov, Mikhail Savitsky, Matthias Walther, Günter Reuter, Yuri B. Schwartz,
Tópico(s)RNA modifications and cancer
ResumoArticle4 March 2019free access Source DataTransparent process Ash1 counteracts Polycomb repression independent of histone H3 lysine 36 methylation Eshagh Dorafshan Department of Molecular Biology, Umeå University, Umeå, Sweden Search for more papers by this author Tatyana G Kahn Department of Molecular Biology, Umeå University, Umeå, Sweden Search for more papers by this author Alexander Glotov Department of Molecular Biology, Umeå University, Umeå, Sweden Search for more papers by this author Mikhail Savitsky Department of Molecular Biology, Umeå University, Umeå, Sweden Search for more papers by this author Matthias Walther Institute of Developmental Genetics, Martin-Luther University of Halle-Wittenberg, Halle, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Gunter Reuter Institute of Developmental Genetics, Martin-Luther University of Halle-Wittenberg, Halle, Germany Search for more papers by this author Yuri B Schwartz Corresponding Author [email protected] orcid.org/0000-0003-4790-3920 Department of Molecular Biology, Umeå University, Umeå, Sweden Search for more papers by this author Eshagh Dorafshan Department of Molecular Biology, Umeå University, Umeå, Sweden Search for more papers by this author Tatyana G Kahn Department of Molecular Biology, Umeå University, Umeå, Sweden Search for more papers by this author Alexander Glotov Department of Molecular Biology, Umeå University, Umeå, Sweden Search for more papers by this author Mikhail Savitsky Department of Molecular Biology, Umeå University, Umeå, Sweden Search for more papers by this author Matthias Walther Institute of Developmental Genetics, Martin-Luther University of Halle-Wittenberg, Halle, Germany Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Gunter Reuter Institute of Developmental Genetics, Martin-Luther University of Halle-Wittenberg, Halle, Germany Search for more papers by this author Yuri B Schwartz Corresponding Author [email protected] orcid.org/0000-0003-4790-3920 Department of Molecular Biology, Umeå University, Umeå, Sweden Search for more papers by this author Author Information Eshagh Dorafshan1, Tatyana G Kahn1, Alexander Glotov1, Mikhail Savitsky1, Matthias Walther2,3, Gunter Reuter2 and Yuri B Schwartz *,1 1Department of Molecular Biology, Umeå University, Umeå, Sweden 2Institute of Developmental Genetics, Martin-Luther University of Halle-Wittenberg, Halle, Germany 3Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany *Corresponding author. Tel: +46 90 785 6784; E-mail: [email protected] EMBO Rep (2019)20:e46762https://doi.org/10.15252/embr.201846762 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Polycomb repression is critical for metazoan development. Equally important but less studied is the Trithorax system, which safeguards Polycomb target genes from the repression in cells where they have to remain active. It was proposed that the Trithorax system acts via methylation of histone H3 at lysine 4 and lysine 36 (H3K36), thereby inhibiting histone methyltransferase activity of the Polycomb complexes. Here we test this hypothesis by asking whether the Trithorax group protein Ash1 requires H3K36 methylation to counteract Polycomb repression. We show that Ash1 is the only Drosophila H3K36-specific methyltransferase necessary to prevent excessive Polycomb repression of homeotic genes. Unexpectedly, our experiments reveal no correlation between the extent of H3K36 methylation and the resistance to Polycomb repression. Furthermore, we find that complete substitution of the zygotic histone H3 with a variant in which lysine 36 is replaced by arginine does not cause excessive repression of homeotic genes. Our results suggest that the model, where the Trithorax group proteins methylate histone H3 to inhibit the histone methyltransferase activity of the Polycomb complexes, needs revision. Synopsis This study shows that H3K36 methylation is not required for Polycomb repression. It indicates that the model that Trithorax group proteins methylate histone H3 to inhibit the histone methyltransferase activity of Polycomb complexes needs to be revised. Ash1 is the only Drosophila H3K36-specific methyltransferase necessary to prevent excessive Polycomb repression of homeotic genes. The SET domain of Ash1 is required to counteract Polycomb repression. Substitution of the zygotic histone H3 with a variant in which lysine 36 is replaced by arginine does not cause excessive repression of homeotic genes. Introduction Polycomb repression is essential to maintain cell type specific gene expression programmes in a wide range of multicellular animals including Drosophila, mice and humans 1-3. It is potent and once established tends to repress target genes for many cell generations. Polycomb proteins, the building blocks of the repressive system, are ubiquitous but the set of genes they repress varies between different cell types. For example, homeotic selector (HOX) genes of the bithorax complex are repressed by Polycomb in the anterior half of the fly body but remain transcriptionally active in the posterior half 4, 5. The genetic evidence indicates that Trithorax (Trx) and Absent, small or homeotic discs 1 (Ash1) proteins are critical to safeguard Drosophila Polycomb target genes from erroneous repression in cells where they have to remain active 6-8. Importantly, neither Trx nor Ash1 are responsible for transcriptional activation of the Polycomb target genes, which is done by appropriate enhancers and associated transcription factors. Instead, the two, in some way, specifically antagonize Polycomb repression 9, 10. Polycomb proteins act as multisubunit complexes that affect chromatin organization in multiple ways, which includes posttranslational modification of histone proteins 2, 11, 12. Of those, tri-methylation of lysine 27 of histone H3 (H3K27me3) by Polycomb Repressive Complex 2 (PRC2) is essential for repression 13. In vitro experiments indicate that the catalytic activity of PRC2 is inhibited by prior methylation of histone H3 tail at lysine 4 (K4) and lysine 36 (K36) 14-16. Trx and Ash1 both have SET domains that can methylate H3K4 and H3K36, respectively 16-20. From this, it was proposed that Trx and Ash1 counteract Polycomb repression by inhibiting PRC2 catalytic activity via H3K4 and H3K36 methylation 14-16. The model provides a simple mechanistic explanation of the antagonism between Trx/Ash1 and Polycomb repression. However, several observations do not easily fit to the model. First, there are other Drosophila histone methyltransferases, Set1 and Trr 21-23 that can methylate H3K4 and two histone methyltransferases NSD and Set2 24, 25 that can methylate H3K36. Why these are not redundant with Trx and Ash1 is unclear. Second, methylated H3K4 or H3K36 have to be present on the same H3 tail to inhibit PRC2 activity 15. Therefore, nucleosomes have to be extensively methylated by Trx and Ash1 to block Polycomb repression efficiently. However, recent quantitative mass spectrometry study indicates that in Drosophila cells only a very small fraction of histone H3 is methylated at K36 (H3K36me1 = 2.5% of total, H3K36me2 = 0.5% of total and H3K36me3 = 1.5% of total) 26. Since these modifications are widely spread over the Drosophila genome 27, their density at any given site is expected to be very low. Third, transgenic experiments of Hödl and Basler 28 indicate that flies in which all zygotic histone H3 molecules carry arginine or alanine instead of lysine 4 (K4) have no ectopic repression of HOX or other developmental genes. The latter cannot be ascribed to the redundancy with Ash1-mediated H3K36 methylation as individual loss-of-function mutations in trx and ash1 both cause stochastic loss of HOX gene expression 6-9. Finally, chromatin immunoprecipitation studies in Drosophila embryos and cultured cells indicate that, when Polycomb-regulated genes are transcriptionally active, they often lose PRC2 binding 30, 31, 29. The loss of methyltransferase would automatically cause the loss of H3K27me3 leaving no need to invoke special mechanisms to inhibit catalytic activity of PRC2. HOX genes specify anterior–posterior axis of multicellular animals. In Drosophila, the HOX genes are grouped in two clusters: the Antennapedia complex that encompasses genes responsible for the identity of the segments that form the head and the anterior thorax 32 and the bithorax complex, which groups genes that specify the third thoracic and all abdominal segments 4. Both clusters are classical targets of Polycomb/Trithorax regulation and changes in their gene expression patterns proved to be one of the best readouts to detect defects in the Polycomb or Trithorax functions 6, 34-36, 33. For example, in mutants lacking any of the core Polycomb proteins, the expression of the HOX genes is not confined to appropriate segments, which leads to transformations of multiple segments towards the more posterior neighbours 6, 34, 35, 33. On the other hand, impaired trx or ash1 function causes stochastic loss of HOX gene expression and partial transformation of corresponding segments towards the anterior fate 6, 36. Here we investigated whether Ash1 counteracts Polycomb repression by methylating K36 on histone H3. Unlike the Trx protein, which binds Polycomb target genes both when they are repressed and when they are transcriptionally active 31, 37, Ash1 is a hallmark of the de-repressed state 31, 38, 39. Using a combination of genetic and biochemical approaches, we, for the first time, showed that Ash1 is the only Drosophila H3K36-specific methyltransferase required to prevent excessive Polycomb repression of the HOX genes. Surprisingly, our experiments demonstrated that complete substitution of the zygotic histone H3 with a variant in which lysine 36 is replaced by arginine does not lead to ectopic repression of homeotic genes. Altogether, our results suggest that the model, where the Trithorax group proteins methylate histone H3 to inhibit the histone methyltransferase activity of PRC2, may need to be reconsidered. Results If Ash1 counteracts Polycomb repression by methylating H3K36, other H3K36-specific histone methyltransferases may also contribute to the process. To address this question, we examined stochastic loss of the homeotic gene expression in flies with mutations in the ash1, NSD and Set2 genes. As previously reported 7, 8, 40, 41, flies with combinations of mutant ash1 alleles lose expression of the bithorax complex genes and display transformations of thoracic and abdominal segments. The ash122 allele is a point mutation that converts Gln 129 into a stop codon (Fig 1A, 8). The truncated open reading frame of ash122 encodes for a short non-functional polypeptide that lacks all conserved domains. The ash121 allele is a substitution of Glu 1365 to Lys within the Associated With SET (AWS) domain (Fig 1A, 8). The ash122/ash121 heterozygotes develop to pharate adult stage and about 12% survive as adults. The mutant adult flies show haltere to wing and third leg towards second leg transformations, characteristic of partial loss of the Ubx gene expression (Fig 1B and C). In addition, they show transformations of the 5th and 6th abdominal segments towards more anterior fate (Fig 1B and C), which reflect partial loss of the Abd-B gene expression. The ash1Df(3L)Exel9011 allele (hereafter referred to ash19011) is a large deletion that removes the entire ash1 and several other genes 42. Nearly all ash122/ash19011 animals die at early pupal stage before the adult cuticle is formed. The single ash122/ash19011 male with the adult cuticle developed enough to examine its morphology showed clear posterior to anterior transformations of the third thoracic and abdominal segments. Figure 1. Ash1 is the only H3K36-specific methyltransferase critical to counteract Polycomb repression of the Drosophila HOX genes The schematic of the Drosophila Ash1 protein organization. Ash1 is 2,226 amino acid long and contains eight domains (indicated by coloured rectangles). The SET domain together with the AWS (Associated With SET) and the post-SET domains are necessary and sufficient for Ash1 histone methyltransferase (HMTase) activity. The functions of the BAH (Bromo Adjacent Homology), PHD (Plant homeodomain) and AT-hook domains are unknown. The positions of ash122 and ash121 point mutations are indicated by arrows. Segmental expression of the Drosophila bithorax complex genes. The three genes of the complex, Ubx, abd-A and Abd-B, are shown as coloured rectangles. The expression of Ubx gives identity to the third thoracic (T3) and the first abdominal (A1) segments, the expression of abd-A defines the second, third and fourth abdominal segments (A2–A4), and the expression of Abd-B gives identity to the rest of the abdominal segments (illustrated with corresponding colour code). Adult phenotypes of the ash1 and NSD mutants. In ash122/ash121 mutants (designated as ash1−), the loss of Abd-B expression results in partial transformation of abdominal segments 6 and 5 towards segments 5 and 4, which is visible from the partial loss of pigmentation on tergites 5 and 6 (t5 and t6) and appearance of bristles on sternite 6 (s6, marked with black arrows). The loss of Ubx expression causes transformation of the third thoracic to the second thoracic segment visible as partial haltere (H) to wing and third leg (3L) to second leg (2L) transformations. The former is evident from the change in the haltere shape and the appearance of multiple bristles (black arrowheads). The latter is indicated by the apical and pre-apical bristles (red arrows) on the tibia of the third leg of ash1 mutants. These are normally present on 2L but absent on 3L (compare to wild-type). Also note the appearance of additional hypopleural bristles on the third thoracic segment of the ash1− flies (red arrowheads), which indicate its transformation towards the second thoracic segment. Phenotype of the NSDds46/NSDds46 (NSD−) flies is indistinguishable from wild-type and the phenotype of the double ash122,NSDds46/ash122,NSDds46 (ash1−,NSD−) flies is no more severe than that of the single ash122/ash121 (ash1−) mutants. Ubx expression in the haltere imaginal discs. The expression was assayed by immunostaining with antibodies against Ubx (red) and acetylated H3K18 (green, positive control). While ash122/ash121 (ash1−) larvae show stochastic clonal loss of the Ubx immunostaining in haltere discs (yellow dashed lines), Set2− larvae have uniform expression of Ubx throughout the haltere disc, resembling that in the wild-type larvae. Scale bars indicate 100 μm. Download figure Download PowerPoint No loss-of-function alleles for the NSD gene have been described to date. Therefore, we used CRISPR/Cas9-mediated mutagenesis 43 to replace the entire NSD open reading frame with a DsRed transgene driven by a synthetic eye-specific 3xP3 promoter (Fig EV1). Flies homozygous for resulted NSDds46 allele are viable, fertile and show no homeotic transformations (Fig 1C). Both Ash1 and NSD were said to di-methylate H3K36 24, 44. Nevertheless, the NSDds46 allele does not reduce the viability or enhance the homeotic transformations of ash1 mutants (Fig 1C). These observations indicate that NSD is not required to counteract Polycomb repression of the homeotic genes. Click here to expand this figure. Figure EV1. Generation of the NSD loss-of-function allele The structure of the Drosophila NSD locus. Red arrows indicate locations of the gRNA sites used for CRISPR/Cas9-mediated replacement of the NSD Open Reading Frame (ORF) with DsRed. The homology regions HA-1 and HA-2, used for the replacement, are shown with bold lines. The half-arrows represent the primers used for genotyping of the mutant allele. The dashed red line in NSD_RT_1f primer, used for RT–qPCR, indicates the intronic region that is excluded from the primer. The NSDds46 allele. After Homology-directed Repair (HDR), the insertion of the DsRed cassette generates the loss-of-function allele NSDds46, where DsRed substitutes most of the NSD ORF. The DsRed expression is controlled by the 3xP3 promoter. In addition to DsRed, the replacement cassette contains two loxP sites to remove DsRed via Cre-mediated recombination and an attP docking site to insert variants of the NSD ORF. The half-arrows represent the primers used to genotype the mutant allele. Genotyping of the NSDds46 allele by PCR. The replacement of the NSD ORF by the DsRed cassette was confirmed by PCR with four different primers pairs. Three primer pairs (top row of images) yield the product only if the replacement has happened. The expected sizes of the PCR products are 469, 427 and 186 bp for DonR_revA and NSD_back, DonR_revA and gen_16_NSD, and gene_1_NSD and DonR_forB primer pairs, respectively. The PCR with Mes-4-RI-2.1 and Mes-4-RI-2.2 primer pair amplifies the 830 bp product only from the wild-type allele. PCR with His3.3B_fwd and His3.3B_rev primer pair, amplifying the 495 bp DNA fragment from the His3.3B gene, was used as a positive control. The NSDds46 allele produces no messenger RNA. This was confirmed by RT–PCR with NSD_Rt_1f and NSD_Rt_1r primer pair, which yields the 513 bp product only when the intact NSD mRNA is produced. RT–PCR with rp49for and rp49rev primer pair that amplifies 132 bp fragment from the cDNA of constitutively expressed RpL32 (a.k.a rp49) gene was used as positive control. Download figure Download PowerPoint The reported mutation of the Drosophila Set2 gene (Set21) removes the N-terminal half of its open reading frame, which includes the catalytic SET domain 25. Most of the Set21 homozygous flies die during metamorphosis. The lethality can be complemented by a transgenic copy of the Set2 genomic region, which indicates that the mutant chromosome does not contain second site lethal mutations 25. In our hands, from 300 Set21 homozygous larvae picked at the first instar and grown separately from their heterozygous siblings, 221 formed pupal cases but only five males and three females formed some chitin structures, including one male with fully developed adult cuticle (Appendix Fig S1). None of the cuticle structures showed signs of homeotic transformations suggesting that the Set2 protein is not essential to counteract Polycomb repression of the HOX genes. To test this further, we compared the haltere imaginal discs from the wild-type, ash122/ash121 and Set21 third instar larvae stained with antibodies against the Ubx protein. Consistent with previous reports, the ash122/ash121 discs showed clonal patches of cells lacking Ubx immunostaining 9, 40. In contrast, the discs from the wild-type and the Set21 larvae displayed uniform Ubx staining (Fig 1D), supporting the conclusion that Set2 is not required to counteract Polycomb repression of the HOX genes. To summarize, our observations suggest that, from the three Drosophila H3K36-specific histone methyltransferases, only Ash1 is critical to prevent the unscheduled Polycomb repression of the homeotic genes. Ash1 SET domain is required to counteract Polycomb repression If Ash1 is the only H3K36-specific methyltransferase critical to counteract Polycomb repression, something in its mode of action must differ from that of NSD and Set2. Ash1 may be specifically targeted to Polycomb-regulated genes, it may methylate some non-histone substrates, or it could have functions unrelated to methyltransferase activity. The latter option seems unlikely considering recent reports that Ash1 interacts with Mrg15 and that this interaction enhances the catalytic activity of Ash1 and helps to antagonize Polycomb repression 40, 41. To investigate this option further, we generated transgenic fly strain that expressed the full-length Ash1 (Ash1-FL) protein as well as strains that expressed the Ash1 variants lacking either the SET domain (Ash1∆SET) or the PHD domain (Ash1∆PHD), all under control of the Ubi-p63E promoter 45. The flies carrying either of the transgenic constructs along with a copy of the endogenous wild-type ash1 are fully viable, fertile and display no homeotic transformations. This indicates that the deletion of the SET or PHD domains, or potential overexpression of the transgenic Ash1 proteins, does not have adverse dominant effects. When introduced into the ash122/ash19011 background, one or two copies of the transgene expressing full-length Ash1 restore the viability and yield flies with no homeotic transformations (Fig 2A, Appendix Fig S2). Somewhat surprisingly, the ash122/ash19011 strain supplemented with two copies of the ash1∆SET or the ash1∆PHD transgene also yields viable adult flies (Appendix Fig S2). These flies, however, have low fitness and no stable stocks could be established. Importantly, all ash122/ash19011 flies expressing the Ash1∆SET or Ash1∆PHD proteins show homeotic transformations indicating that the expression of HOX genes is still stochastically lost (Fig 2A). Since transgenic Ash1-FL, Ash1∆SET or Ash1∆PHD are expressed at comparable levels (Fig 2B and C), this argues that the SET and the PHD domains of Ash1 are required to counteract Polycomb repression of the HOX genes. Figure 2. Ash1 SET domain is required to counteract Polycomb repression Adult phenotypes of the ash122/ash19011 flies supplemented with transgenic constructs expressing either the full-length Ash1 (Ash1FL) or the truncated variants lacking the PHD (Ash1ΔPHD) or the SET (Ash1ΔSET) domains. Note extra hypopleural bristles (red arrows), the third leg (L3) to second leg (L2), haltere (H) to wing, t5–t4 and t6–t5 transformations in the Ash1ΔPHD and the Ash1ΔSET but not in the Ash1FL flies. The latter are evident from the partial loss of pigmentation in t6 and t5, or the appearance of small bristles (trichomas) on t6 of the Ash1ΔPHD and the Ash1∆SET flies in the area that is normally naked (Ash1FL, yellow dashed line). The transformed L3 acquire apical and pre-apical bristles on the tibia (black triangles) while halteres change shape and acquire rows of bristles (black arrows). Twofold dilutions of total nuclear protein extracts from the third instar larvae of the ash122/ash19011 mutants supplemented with various transgenic constructs and wild-type flies were analysed by Western blot with anti-Ash1 antibodies. Arrow indicates the position of Ash1 protein. Note that transgenic proteins are expressed at comparable level. Coomassie staining of SDS–PAGE separated protein extracts from (B) was used to control the loading. Source data are available online for this figure. Source Data for Figure 2 [embr201846762-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Western blot analyses showed no major difference in the overall levels of H3K36me1, H3K36me2 and H3K36me3 between the wild-type and ash122/ash19011 third instar larvae (Fig 3A and C). However, we noted mild (~ 2-fold) reduction of H3K36me1, also visible in the ash122/ash19011, NSDds46 double mutants but not in the Set21 mutants (Fig 3A). Consistent with previous reports, the Set21 mutant larvae displayed tenfold reduction in H3K36me3 25 as well as slight loss of H3K36me2 (Fig 3B and C). Neither mutant showed increase in the level of bulk di- or tri-methylated H3K27 (Fig EV2). Altogether, these results suggest that Ash1 is not solely responsible for any of the H3K36 methylation states and that aside from H3K36me3, which is produced predominantly by Set2, all three methylases contribute to the H3K36me1 and H3K36me2 pools. Figure 3. Western blot analysis of the bulk H3K36 methylation in larval tissues of various mutants A–C. Twofold serial dilutions of the total protein extracts from the wild-type, ash122/ash19011 (ash1−), ash122,NSDds46/ash19011,NSDds46 (ash1−, NSD−) and Set21 (Set2−) larval brains, imaginal discs and salivary glands were analysed by Western blot with antibodies against H3K36me1 (A), H3K36me2 (B) and H3K36me3 (C). Note the strong (> 10-fold) reduction of H3K36me3 signal in the Set2− extract and the slight (˜ 2-fold) reduction of H3K36me1 signal in the ash1− and ash1−, NSD− extracts. The protein extracts from the wild-type, double ash1−, NSD− and single NSD− and Set2− mutants (right panels) were analysed together on the same membrane; however, the images of the H3K36me1 and H3K36me3 Western blots were modified to splice out the marker lane between the ash1−, NSD− and the Set2− extracts. Western blots with constitutively expressed BEAF-32 protein were used as loading controls. Source data are available online for this figure. Source Data for Figure 3 [embr201846762-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. H3K27 methylation in the Drosophila larvae lacking different H3K36-specific histone methyltransferasesWestern blot analysis of the ash1− (ash122/ash19011), ash1−,NSD− (ash122,NSDds46/ash19011,NSDds46), Set2− (Set21/ Set21) and NSD− (NSDds46/NSDds46) mutants shows no change in global levels of di- and tri-methyl H3K27 compared to wild-type (Oregon R). Twofold serial dilutions of total protein extracts from larval brains, imaginal discs and salivary glands were analysed by Western blot with corresponding antibodies. The protein extracts from the wild-type, double ash1−, NSD− and single NSD− and Set2− mutants (right panels) were analysed together on the same membrane; however, the images were modified to splice out the marker lane between the ash1−, NSD− and the Set2− extracts. Western blots with constitutively expressed BEAF-32 protein were used as loading controls. Source data are available online for this figure. Download figure Download PowerPoint Although its contribution to the bulk H3K36 methylation is limited, Ash1 may be critical for the appropriate level of H3K36 methylation at de-repressed Polycomb target genes. To address this question, we assayed the presence of Ash1, H3K36me1 and H3K36me2 at five genomic sites using Chromatin Immunoprecipitation coupled with quantitative PCR (ChIP-qPCR). Studies in cultured Drosophila cells suggested two modes of Ash1 binding to the genome. The first mode produces numerous weak binding sites biased towards long 5′ introns of transcriptionally active genes 38, 41. The second mode results in few dozens of strongly bound regions many of which represent Polycomb-regulated genes captured in transcriptionally active state 31, 38, 41. Here we selected three developmental genes no ocelli (noc), homothorax (hth) and extra macrochaetae (emc), which displayed the strong Ash1 binding in multiple cultured cell lines 31, 38. One of the genes, hth, was previously shown to bind Polycomb proteins when transcriptionally inactive 31. We also included the two well-known Polycomb target genes Su(z)2 and Ubx. Both were shown to bind Ash1 when transcriptionally active 31, 39 and Ubx is one of the genes whose erroneous repression causes some of the homeotic transformations seen in the ash1 mutants. Finally, we assayed an intergenic region on chromosome 3R and the constitutively active TBP-associated factor 4 (Taf4) gene. Neither of them is known to bind Ash1 31, 38 and, therefore, can serve as negative controls. Chromatin immunoprecipitation (ChIP) and quantitative PCR analysis (ChIP-qPCR) indicates that, following the Ash1 depletion in the ash122/ash19011 third instar larvae, H3K36me1 is mildly (~ 2-fold) reduced at some of the selected genes (Figs 4A and EV3), while the H3K36me2 levels are not measurably affected (Fig 4B). The detected reduction of H3K36me1 is small. Nevertheless, the partial loss of H3K36me1 is completely reversed by the re-introduction of the transgenes expressing the wild-type and the PHD-deficient but not the SET domain-deficient Ash1 proteins (Fig 4A). This suggests that the partial loss of H3K36me1 from the Ash1-bound genes is caused by the loss of Ash1 catalytic activity. Importantly, while the Ash1∆SET and Ash1∆PHD proteins are expressed (Fig 2B) and bind the chromatin with comparable efficiency (Fig 4C) and the Ash1∆PHD protein restores the wild-type levels of H3K36me1 (Fig 4A), the transgenic ash1∆PHD, ash122/ash19011 flies still have homeotic transformations (Fig 2A). Altogether, our observations indicate that Ash1 mono-methylates a measurable fraction of the nuclear H3K36 and that Ash1 SET domain is required to counteract Polycomb repression of the HOX genes. Yet, there seems to be no correlation between the levels of methylated H3K36 (bulk or at specific loci) and the extent of the erroneous Polycomb repression. Figure 4. ChIP and quantitative PCR (ChIP-qPCR) analysis of H3K36 methylation and Ash1 binding A–C. Chromatin from the wild-type (dark blue bars), ash122/ash19011 (ash1−, red bars) and transgenic ash122/ash19011 (Ash1FL, light blue bars; Ash1ΔPHD, green bars; Ash1ΔSET, orange bars) larvae was subjected to immunoprecipitation with the antibodies against H3K36me1 (A), H3K36me2 (B) and Ash1 (C). Histograms show the mean of the two independent experiments (n = 2) with dots indicating individual experimental results. An intergenic region on chromosome 3R (intergenic) and the constitutively active TBP-associated factor 4 (Taf4) gene serve as controls. The loss of Ash1 ChIP signal in the ash1− larvae indicates that the selected genes are the genuine Ash1 binding sites. Download figure Download PowerPoint Click
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