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• W2466171108 abstract "•Deletion of the transcription start site increases H3K27me3 level in the gene body•Histone deacetylation mediates Ras-induced gene silencing and the H3K27me3 increase•Maximal Ras-induced accumulation of H3K27me3 requires at least 35 days•Ras-induced H3K27me3 accumulation is reversed by forced activation of transcription Trimethylated H3K27 (H3K27me3) is associated with transcriptional repression, and its abundance in chromatin is frequently altered in cancer. However, it has remained unclear how genomic regions modified by H3K27me3 are specified and formed. We previously showed that downregulation of transcription by oncogenic Ras signaling precedes upregulation of H3K27me3 level. Here, we show that lack of transcription as a result of deletion of the transcription start site of a gene is sufficient to increase H3K27me3 content in the gene body. We further found that histone deacetylation mediates Ras-induced gene silencing and subsequent H3K27me3 accumulation. The H3K27me3 level increased gradually after Ras activation, requiring at least 35 days to achieve saturation. Such maximal accumulation of H3K27me3 was reversed by forced induction of transcription with the dCas9-activator system. Thus, our results indicate that changes in H3K27me3 level, especially in the body of a subset of genes, are triggered by changes in transcriptional activity itself. Trimethylated H3K27 (H3K27me3) is associated with transcriptional repression, and its abundance in chromatin is frequently altered in cancer. However, it has remained unclear how genomic regions modified by H3K27me3 are specified and formed. We previously showed that downregulation of transcription by oncogenic Ras signaling precedes upregulation of H3K27me3 level. Here, we show that lack of transcription as a result of deletion of the transcription start site of a gene is sufficient to increase H3K27me3 content in the gene body. We further found that histone deacetylation mediates Ras-induced gene silencing and subsequent H3K27me3 accumulation. The H3K27me3 level increased gradually after Ras activation, requiring at least 35 days to achieve saturation. Such maximal accumulation of H3K27me3 was reversed by forced induction of transcription with the dCas9-activator system. Thus, our results indicate that changes in H3K27me3 level, especially in the body of a subset of genes, are triggered by changes in transcriptional activity itself. The elaborate control of histone modification is essential for various eukaryotic cellular functions including transcriptional regulation. Trimethylation at lysine-27 of histone H3 (H3K27) is mediated by Polycomb repressive complex 2 (PRC2) and associated with transcriptional repression (Di Croce and Helin, 2013Di Croce L. Helin K. Transcriptional regulation by Polycomb group proteins.Nat. Struct. Mol. Biol. 2013; 20: 1147-1155Crossref PubMed Scopus (611) Google Scholar, Simon and Kingston, 2013Simon J.A. Kingston R.E. Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put.Mol. Cell. 2013; 49: 808-824Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). The distribution of trimethylated H3K27 (H3K27me3) throughout the genome has been determined by genome-wide comprehensive analyses, such as those based on deep chromatin immunoprecipitation sequencing (ChIP-seq) (Dunham et al., 2012Dunham I. Kundaje A. Aldred S.F. Collins P.J. Davis C.A. Doyle F. Epstein C.B. Frietze S. Harrow J. Kaul R. et al.ENCODE Project ConsortiumAn integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (11030) Google Scholar, Ernst et al., 2011Ernst J. Kheradpour P. Mikkelsen T.S. Shoresh N. Ward L.D. Epstein C.B. Zhang X. Wang L. Issner R. Coyne M. et al.Mapping and analysis of chromatin state dynamics in nine human cell types.Nature. 2011; 473: 43-49Crossref PubMed Scopus (2063) Google Scholar). Such analyses have revealed an inverse relation between H3K27me3 level and transcriptional activity for various subsets of genes, including those encoding Hox proteins, cell-cycle regulators, and transcription factors. Although these observations have suggested that H3K27me3 plays a key role in transcriptional repression, they have not revealed the hierarchical order among recruitment of PRC2, deposition of H3K27me3, and transcriptional repression. Thus, it has remained unclear how genomic regions covered with H3K27me3 are specified and formed. Studies in Drosophila have identified Polycomb response elements (PREs) to which PRC2 is recruited by specific DNA-binding proteins, such as PHO (Schwartz and Pirrotta, 2007Schwartz Y.B. Pirrotta V. Polycomb silencing mechanisms and the management of genomic programmes.Nat. Rev. Genet. 2007; 8: 9-22Crossref PubMed Scopus (713) Google Scholar). PRC2 is required for maintenance of the repressed state of genes whose expression has already been attenuated by repressive transcriptional factors, including segmentation gene products. On the other hand, establishment of gene silencing by PRC2 at PREs is prevented by the Trithorax complex, which mediates the trimethylation of H3K4 and transcriptional activation (Poux et al., 2002Poux S. Horard B. Sigrist C.J. Pirrotta V. The Drosophila trithorax protein is a coactivator required to prevent re-establishment of polycomb silencing.Development. 2002; 129: 2483-2493PubMed Google Scholar). These observations suggest that transcriptional activity itself plays a role in the regulation of PRC2 at PREs. However, it remains controversial whether transcription through PREs impedes the access of PRC2 (Erokhin et al., 2015Erokhin M. Elizar’ev P. Parshikov A. Schedl P. Georgiev P. Chetverina D. Transcriptional read-through is not sufficient to induce an epigenetic switch in the silencing activity of Polycomb response elements.Proc. Natl. Acad. Sci. USA. 2015; 112: 14930-14935Crossref PubMed Scopus (27) Google Scholar, Schmitt et al., 2005Schmitt S. Prestel M. Paro R. Intergenic transcription through a polycomb group response element counteracts silencing.Genes Dev. 2005; 19: 697-708Crossref PubMed Scopus (198) Google Scholar). Furthermore, differences in PRC2 function and regulation have been detected among species, and definitive PREs corresponding to those in Drosophila have not been identified in mammalian cells to date (Bauer et al., 2016Bauer M. Trupke J. Ringrose L. The quest for mammalian Polycomb response elements: are we there yet?.Chromosoma. 2016; 125: 471-496Crossref PubMed Scopus (72) Google Scholar). Recent studies with mammalian cells have shown that CpG islands have the potential to be targeted by PRC2 (Mendenhall et al., 2010Mendenhall E.M. Koche R.P. Truong T. Zhou V.W. Issac B. Chi A.S. Ku M. Bernstein B.E. GC-rich sequence elements recruit PRC2 in mammalian ES cells.PLoS Genet. 2010; 6: e1001244Crossref PubMed Scopus (320) Google Scholar) and that PRC2 is recruited to specific regions of DNA by several mechanisms mediated by noncoding RNA (da Rocha et al., 2014da Rocha S.T. Boeva V. Escamilla-Del-Arenal M. Ancelin K. Granier C. Matias N.R. Sanulli S. Chow J. Schulz E. Picard C. et al.Jarid2 Is Implicated in the Initial Xist-Induced Targeting of PRC2 to the Inactive X Chromosome.Mol. Cell. 2014; 53: 301-316Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, Sarma et al., 2014Sarma K. Cifuentes-Rojas C. Ergun A. Del Rosario A. Jeon Y. White F. Sadreyev R. Lee J.T. ATRX directs binding of PRC2 to Xist RNA and Polycomb targets.Cell. 2014; 159: 869-883Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), transcription factors (Dietrich et al., 2012Dietrich N. Lerdrup M. Landt E. Agrawal-Singh S. Bak M. Tommerup N. Rappsilber J. Södersten E. Hansen K. REST-mediated recruitment of polycomb repressor complexes in mammalian cells.PLoS Genet. 2012; 8: e1002494Crossref PubMed Scopus (118) Google Scholar), or PRC1-dependent ubiquitylation of histone H2A (Blackledge et al., 2014Blackledge N.P. Farcas A.M. Kondo T. King H.W. McGouran J.F. Hanssen L.L. Ito S. Cooper S. Kondo K. Koseki Y. et al.Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.Cell. 2014; 157: 1445-1459Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, Kalb et al., 2014Kalb R. Latwiel S. Baymaz H.I. Jansen P.W. Müller C.W. Vermeulen M. Müller J. Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression.Nat. Struct. Mol. Biol. 2014; 21: 569-571Crossref PubMed Scopus (300) Google Scholar). On the other hand, the possibility of regulation of PRC2 recruitment by transcription has been suggested by several observations, including the finding that prevention of RNA polymerase II function with chemical inhibitors increased H3K27me3 abundance at CpG islands in embryonic stem cells (ESCs) (Riising et al., 2014Riising E.M. Comet I. Leblanc B. Wu X. Johansen J.V. Helin K. Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide.Mol. Cell. 2014; 55: 347-360Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Moreover, H3K27me3 deposition in the gene body was found to be induced when transcription was prematurely interrupted by insertion of polyadenylation site in ESCs (Kaneko et al., 2014Kaneko S. Son J. Bonasio R. Shen S.S. Reinberg D. Nascent RNA interaction keeps PRC2 activity poised and in check.Genes Dev. 2014; 28: 1983-1988Crossref PubMed Scopus (130) Google Scholar). These various observations suggest that recruitment or activation of PRC2 follows transcriptional repression. At least two mechanisms of H3K27me3 deposition in mammals have therefore been proposed: active recruitment of PRC2 by specific binding partners and passive recruitment of PRC2 after transcriptional repression (Blackledge et al., 2015Blackledge N.P. Rose N.R. Klose R.J. Targeting Polycomb systems to regulate gene expression: modifications to a complex story.Nat. Rev. Mol. Cell Biol. 2015; 16: 643-649Crossref PubMed Scopus (234) Google Scholar). Lack or mutation of PRC2 components results in developmental defects during embryogenesis or in tumorigenesis, indicating the physiological relevance of H3K27me3 (Kim and Roberts, 2016Kim K.H. Roberts C.W. Targeting EZH2 in cancer.Nat. Med. 2016; 22: 128-134Crossref PubMed Scopus (886) Google Scholar, Surface et al., 2010Surface L.E. Thornton S.R. Boyer L.A. Polycomb group proteins set the stage for early lineage commitment.Cell Stem Cell. 2010; 7: 288-298Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Furthermore, various environmental and developmental signals are known to regulate H3K27me3 levels dynamically. The small GTPase Ras transmits signals triggered by growth factor stimulation to downstream-signaling pathways, including those mediated by mitogen-activated protein kinases (MAPKs) (Karnoub and Weinberg, 2008Karnoub A.E. Weinberg R.A. Ras oncogenes: split personalities.Nat. Rev. Mol. Cell Biol. 2008; 9: 517-531Crossref PubMed Scopus (1124) Google Scholar). Activating mutations of Ras are one of the most frequent types of oncogenic mutation in human cancer. We have shown previously that oncogenic Ras signaling represses transcription of a subset of genes in association with a gradual increase in H3K27me3 level in cultured cells (Hosogane et al., 2013Hosogane M. Funayama R. Nishida Y. Nagashima T. Nakayama K. Ras-induced changes in H3K27me3 occur after those in transcriptional activity.PLoS Genet. 2013; 9: e1003698Crossref PubMed Scopus (38) Google Scholar). Although our results indicated that transcriptional repression precedes upregulation of H3K27me3 in this experimental model of abnormal epigenetic modification in cancer, it has remained unclear whether physiological changes in H3K27me3 level induced by Ras signaling are a cause or consequence of changes in transcription. We have now further examined the causal nature of the relation between H3K27 trimethylation and transcriptional silencing in response to activation of Ras signaling. With the use of the CRISPR/Cas9 system for genome editing to manipulate the state of transcription directly, we obtained evidence showing that transcription itself regulates H3K27me3 status and that the Ras-induced changes in H3K27me3 level are not completed until at least 30 days after those in transcription. Ephx1 is silenced by activation of Ras-MAPK signaling in NIH 3T3 mouse fibroblasts (Figure S1), and this silencing is accompanied by marked enrichment of H3K27me3 in the gene body (Figure 1A). We first examined whether Ras signaling or lack of transcription itself induces the change in H3K27me3 level of Ephx1. To silence gene expression without activating Ras signaling, we deleted a genomic region containing the transcription start site (TSS) of Ephx1 with the use of CRISPR/Cas9-mediated genome engineering in NIH 3T3 cells (Ran et al., 2013Ran F.A. Hsu P.D. Wright J. Agarwala V. Scott D.A. Zhang F. Genome engineering using the CRISPR-Cas9 system.Nat. Protoc. 2013; 8: 2281-2308Crossref PubMed Scopus (6409) Google Scholar, Zheng et al., 2014Zheng Q. Cai X. Tan M.H. Schaffert S. Arnold C.P. Gong X. Chen C.Z. Huang S. Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells.Biotechniques. 2014; 57: 115-124Crossref PubMed Scopus (101) Google Scholar). We therefore designed two single-guide RNAs (sgRNAs) that target a 1.5-kb genomic region around the annotated TSS of Ephx1 (Figure 1B). This region includes most of the H3K4me3 and RNA polymerase II peaks, which are indicative of the promoter region, as revealed by a public ChIP-seq database for mouse embryonic fibroblasts (MEFs). We transfected NIH 3T3 cells with plasmids encoding Streptococcus pyogenes Cas9 (SpCas9) and the two sgRNAs, and then we cloned the transfected cells for establishment of several Ephx1 TSS-deleted cell lines. Deletion of the TSS of Ephx1 was confirmed by genomic PCR analysis (Figure 1C). As expected, transcription of Ephx1 was abolished in the Ephx1 TSS-deleted cells (Figure 1D). ChIP and qPCR analysis revealed that the amount of H3K27me3 in the gene body of Ephx1 was markedly increased in these cells (Figure 1E). In contrast, H3K27me3 levels in genomic regions external to Ephx1 were similar in the Ephx1 TSS-deleted cells and parental cells, indicating that H3K27me3 was upregulated only in the gene body of Ephx1. Furthermore, consistent with the loss of Ephx1 mRNA (Figure 1D), the abundance of H3K36me3, which is an indicator of transcriptional elongation, was greatly diminished throughout the gene body of Ephx1 in the TSS-deleted cells (Figure 1F), showing that the progression of RNA polymerase II was inhibited. In contrast, the amount of acetylated H3K27 (H3K27ac), which localizes to promoter and enhancer regions, was not substantially affected by deletion of the TSS of Ephx1 (Figure 1G). We found that the amount of H3K27me3 was not increased in regions with a high H3K27ac level (regions f, h, and i) in the TSS-deleted cells (Figures 1E and 1G), consistent with previous results showing that acetylation and methylation of the same lysine residue compete with each other (Pasini et al., 2010Pasini D. Malatesta M. Jung H.R. Walfridsson J. Willer A. Olsson L. Skotte J. Wutz A. Porse B. Jensen O.N. Helin K. Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes.Nucleic Acids Res. 2010; 38: 4958-4969Crossref PubMed Scopus (253) Google Scholar). We next examined whether a decrease in H3K27me3 level associated with transcriptional activation is reversed by CRISPR/Cas9-mediated transcriptional shutoff. For this purpose, we examined Sorcs2, whose transcription is driven by Ras signaling and is accompanied by removal of H3K27me3 from the gene body (Figure 2A). To delete the TSS of Sorcs2, we introduced a pair of sgRNAs into NIH 3T3 cells expressing a constitutively active mutant of human H-Ras (RasG12V). We established several cell clones in which the TSS of Sorcs2 was homologously deleted (Figure 2B) and in which upregulation of Sorcs2 expression by RasG12V was abolished, whereas expression of the Ras-regulated genes Ephx1 and Bpil2 was largely unaltered (Figure 2C). Ras signaling reduced the amount of H3K27me3 throughout the gene body of Sorcs2 in parental cells, consistent with our previous observations (Hosogane et al., 2013Hosogane M. Funayama R. Nishida Y. Nagashima T. Nakayama K. Ras-induced changes in H3K27me3 occur after those in transcriptional activity.PLoS Genet. 2013; 9: e1003698Crossref PubMed Scopus (38) Google Scholar), but this Ras-induced epigenetic alteration was abrogated by TSS deletion (Figure 2D). The Ras-induced increase in H3K36me3 abundance also was prevented by TSS deletion (Figure 2E), indicating that transcriptional elongation along the gene body of Sorcs2 was inhibited. These observations for Sorcs2 and Ephx1 thus showed that H3K27me3 accumulation in the gene body was elicited by lack of transcription of both genes and that Ras signaling was not required for this epigenetic change. Time course analysis revealed that the PRC2 component Suz12 was gradually recruited to the gene body as well as the region around the TSS after eviction of RNA polymerase II from Ephx1 in response to activation of Ras signaling (Figure 3A). These data indicated that transcriptional repression is followed by PRC2 recruitment and that accumulation of H3K27me3 is not responsible for transcriptional repression by Ras signaling. We next searched for other molecular links between such signal activation and gene silencing. Acetylation of histone tails has been shown to enhance transcription, and we therefore explored the possibility that histone acetylation is controlled by the Ras-signaling pathway. To this end, we performed ChIP-qPCR analysis with antibodies to H3K27ac and to H3K27me3 at various times after the infection of NIH 3T3 cells with a retrovirus encoding RasG12V. We analyzed the genomic region around the TSS of Ephx1 for H3K27ac and that at the 3′ end of Ephx1 for H3K27me3. We found that the amount of H3K27ac was decreased markedly 2 days after Ras activation, at which time the amount of H3K27me3 remained unchanged (Figure 3B). Other acetylated forms of histone tails, including H3K9ac and H4ac, showed dynamics similar to those of H3K27ac in response to Ras signal activation (data not shown). These observations suggested the possibility that histone deacetylation mediates Ras-induced gene silencing, with H3K27me3 accumulating after transcriptional repression. To test this hypothesis, we treated NIH 3T3 cells expressing RasG12V with trichostatin A (TSA), an inhibitor of histone deacetylase activity, for 5 or 6 days beginning 2 days after the activation of Ras signaling (Figure 3C). Immunoblot analysis revealed that total H3K27ac and H3K27me3 levels in NIH 3T3 cells were not affected by expression of RasG12V (Figure 3D). TSA treatment increased the total amount of H3K27ac in a concentration-dependent manner without affecting that of H3K27me3, expression of Ras, or phosphorylation (activation) of the MAPKs Erk1 and Erk2 (Figure 3D). Ras-induced deacetylation of H3K27ac in the region around the TSS of Ephx1 was inhibited by TSA at even lower concentrations (Figure 3E). Moreover, transcription of Ephx1 (Figure 3F) as well as recruitment of RNA polymerase II to the region around the TSS (Figure 3G) was restored by TSA treatment to levels similar to those for parental NIH 3T3 cells. Of note, the accumulation of H3K27me3 at Ephx1 induced by Ras activation was blocked by TSA (Figure 3H), suggesting that H3K27 acetylation and transcription suppress deposition of H3K27me3. Analysis of Itgb5 yielded similar results. Ras-induced transcriptional repression and H3K27me3 deposition at Itgb5 were thus also greatly attenuated by treatment with TSA (Figure S2). Together these observations indicated that Ras activation induces deacetylation of histone tails, which is in turn responsible for gene silencing and subsequent H3K27me3 accumulation at Ephx1 and Itgb5. Our results suggested that a change in transcriptional activity gradually influences the amount of H3K27me3 in the gene body. To determine the time required for completion of the change in H3K27me3 level induced by Ras activation, we analyzed the amount of H3K27me3 at various times up to 45 days by ChIP-qPCR analysis. Transcription of Ephx1 was suppressed after ∼5 days and remained silenced for up to 45 days (Figure 4A). By contrast, the amount of H3K27me3 along the gene body of Ephx1 increased gradually and reached a maximum after ∼35 days (Figure 4B). We also observed similar time courses of transcription and H3K27me3 accumulation for Itgb5 (Figures S3A and S3B), suggesting that the change in H3K27me3 level for both genes takes at least 35 days to complete. We performed ChIP-seq analysis for H3K27me3 as well as RNA sequencing (RNA-seq) analysis with the cells in which Ras signaling had been activated for 35 days. The ChIP-seq and RNA-seq results for the genomic locus containing Ephx1 are shown together with our previous short-term time course data for H3K27me3 ChIP-seq obtained 0, 2, 4, 7, and 12 days after Ras activation (Figure 4C). In general, H3K27me3 did not manifest focal enrichment, but rather accumulated over several tens of kilobases of the genomic region. Visual inspection revealed that the amount of H3K27me3 in the gene body of Ephx1 was much higher at 35 days than at the other time points, confirming that H3K27me3 is deposited progressively over time. To quantitate the H3K27me3 level, we used a peak caller that is optimized for detection of broad peaks. As exemplified by the Ephx1 locus in Figure 4C, broad peaks of H3K27me3 were identified at 0 and 35 days after Ras activation. This analysis revealed that 18.7% of the entire genome was covered with such broad peaks, in both control and RasG12V-expressing cells, and that 72.5% of the entire genome was devoid of such broad peaks in both types of cells (Figure 4D). Furthermore, 2.9% or 5.9% of the entire genome was not covered with H3K27me3 at day 0 but was covered at 35 days after Ras activation or vice versa. We then calculated the normalized H3K27me3 signal within each defined H3K27me3 domain for each time point. The mean signals for both the common regions covered with H3K27me3 broad peaks and the common uncovered regions remained constant from 0 to 35 days after Ras activation (Figure 4E). In contrast, the signals for the regions covered in only control or RasG12V-expressing cells continued to change from 0 to 35 days (Figure 4E). Of note, the normalized H3K27me3 signal in the region containing Ephx1 also showed a gradual increase (Figure 4E), with the level at 35 days being much higher than that at 12 days and similar to that for common covered regions. We obtained similar, although less pronounced, results for the genomic region containing Itgb5 (Figure S3C). On the basis of these data, we concluded that changes in H3K27me3 content, especially around the Ephx1 locus, were not completed until at least 35 days after Ras activation. To determine whether transcriptional activation is sufficient for removal of H3K27me3 broad peaks, we activated Ephx1 transcription with the use of the dCas9-activator system based on a catalytically inactive mutant of Cas9 (dCas9) (Konermann et al., 2015Konermann S. Brigham M.D. Trevino A.E. Joung J. Abudayyeh O.O. Barcena C. Hsu P.D. Habib N. Gootenberg J.S. Nishimasu H. et al.Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.Nature. 2015; 517: 583-588Crossref PubMed Scopus (1661) Google Scholar). Various proteins fused to dCas9 can be recruited to specific genomic regions in the presence of sgRNAs. We first introduced RasG12V into NIH 3T3 cells and cultured the cells for at least 30 days in order to allow completion of H3K27me3 peak formation around Ephx1. We then sequentially introduced three components into the cells to activate Ephx1 transcription (Figure 5A): a fusion protein consisting of dCas9 and the transcriptional activator Vp64 (dCas9-Vp64), a fusion protein containing the bacteriophage coat protein MS2 and the transactivation domains of p65 and HSF1 (MS2-p65HSF1), and a fusion RNA that includes the sgRNA and an RNA sequence with an MS2-binding site (sgRNA-MS2RNA) and mediates the recruitment of dCas9-Vp64 and MS2-p65HSF1 to the promoter region of Ephx1 (Figure 5B). Immunoblot analysis showed that dCas9-Vp64 was efficiently expressed without affecting the amount of Ras protein or the extent of downstream-signaling activity, as indicated by the phosphorylation level of Erk1/2 (Figure 5C). Transcription of Ephx1 was suppressed by RasG12V, and this suppression was reversed by targeting of the Ephx1 promoter by the specific sgRNA, but it was not affected by a control sgRNA (Figure 5D). Importantly, the increase in H3K27me3 level induced by Ras signaling also was reversed by the specific sgRNA, but not by the control sgRNA (Figure 5E). Such an effect on H3K27me3 level was not observed in genomic regions external to the gene body of Ephx1 (Figure 5E), possibly suggesting the importance of RNA polymerase II for erasure of H3K27me3. Collectively, these results showed that transcription itself erased broad peaks of H3K27me3 that had accumulated in response to prolonged activation of Ras signaling. Given that CpG islands are thought to be potential targets of PRC2 in mammalian cells (Mendenhall et al., 2010Mendenhall E.M. Koche R.P. Truong T. Zhou V.W. Issac B. Chi A.S. Ku M. Bernstein B.E. GC-rich sequence elements recruit PRC2 in mammalian ES cells.PLoS Genet. 2010; 6: e1001244Crossref PubMed Scopus (320) Google Scholar), we examined whether our results for gene bodies also might extend to H3K27me3 at CpG-rich promoters. We and others have shown that Ras signaling generates a focal H3K27me3 peak at the CpG-rich promoter of Smad6 (Hosogane et al., 2013Hosogane M. Funayama R. Nishida Y. Nagashima T. Nakayama K. Ras-induced changes in H3K27me3 occur after those in transcriptional activity.PLoS Genet. 2013; 9: e1003698Crossref PubMed Scopus (38) Google Scholar, Kaneda et al., 2011Kaneda A. Fujita T. Anai M. Yamamoto S. Nagae G. Morikawa M. Tsuji S. Oshima M. Miyazono K. Aburatani H. Activation of Bmp2-Smad1 signal and its regulation by coordinated alteration of H3K27 trimethylation in Ras-induced senescence.PLoS Genet. 2011; 7: e1002359Crossref PubMed Scopus (49) Google Scholar). In contrast to the other genes studied here, Ras induced the accumulation of H3K27me3 predominantly in the promoter region and, to a lesser extent, in the gene body of Smad6 (Figure 6A; Figure S4A). We established several NIH 3T3 cell clones in which the TSS of Smad6 was homologously deleted (Figure 6B). The expression level of Smad6 in these clones was even lower than that apparent after Ras-induced gene silencing in parental cells (Figure 6C). Although Ras signaling increased the level of H3K27me3 around the CpG island in parental cells, transcriptional inhibition by TSS deletion did not trigger the focal accumulation of H3K27me3 in this region (Figure 6E). Of note, we observed an increase in H3K27me3 abundance in the gene body in response to either Ras activation or TSS deletion at sites at which the amount of H3K36me3 decreased (Figures 6D and 6E). These observations thus suggested that H3K27me3 accumulation around the CpG island of Smad6 was not induced by abrogation of transcription alone, with Ras signaling or a DNA element within the deleted region also being required. We also activated Smad6 transcription with the use of the dCas9-activator system. Suppression of Smad6 transcription by RasG12V was reversed by targeting of the gene promoter with two different specific sgRNAs (Figure S4B). Consistent with this result, the amount of H3K4me3 around the CpG island was reduced in RasG12V-expressing cells, but it was recovered in cells also expressing specific sgRNAs (Figure S4C). Importantly, the increase in H3K27me3 level induced by Ras signaling also was reversed by the specific sgRNAs, both around the CpG island as well as in the gene body of Smad6 (Figure S4D). Collectively, these results suggested that transcriptional activation is sufficient for removal of a focal peak of H3K27me3 around a CpG island. Although Ras signaling affects transcription and H3K27me3 level, the relation between and sequence of these events have remained unclear. We have now shown that Ras-induced changes in H3K27me3 level in the gene body are dependent on transcriptional changes. We further found that histone deacetylation is required for Ras-induced changes in both transcription and H3K27me3 level. In addition, we found that the accumulation of H3K27me3 in the gene body of Ephx1 induced by constitutive Ras signaling required at least 35 days to reach a plateau, and we learned that even this maximal level of H3K27me3 deposition was erased by forced activation of transcription. The focal accumulation of H3K27me3 around the CpG island of Smad6 was not induced by TSS deletion, however, although such focal deposition of H3K27me3 was erased by forced activation of transcription. Collectively, our results thus show that transcription itself regulates H3K27me3 level in the body of at least a subset of genes in cells with activated Ras signaling. Chemical inhibition of RNA polymerase II with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or triptolide recently was shown to increase H3K27me3 abundance at CpG islands in mouse ESCs (Riising et al., 2014Riising E.M. Comet I. Leblanc B. Wu X. Johansen J.V. Helin K. Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide.Mol. Cell. 2014; 55: 347-360Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Thi" @default.
• W2466171108 created "2016-07-22" @default.
• W2466171108 creator A5013878863 @default.
• W2466171108 creator A5045423153 @default.
• W2466171108 creator A5060059918 @default.
• W2466171108 creator A5074539682 @default.
• W2466171108 date "2016-07-01" @default.
• W2466171108 modified "2023-10-13" @default.
• W2466171108 title "Lack of Transcription Triggers H3K27me3 Accumulation in the Gene Body" @default.
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