FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2258223596> ?p ?o ?g. }

• W2258223596 endingPage "1168" @default.
• W2258223596 startingPage "1156" @default.
• W2258223596 abstract "•MacroH2A1.2 is enriched at prospective muscle-specific enhancers•Activation of muscle-specific enhancers requires macroH2A1.2•MacroH2A1.2 is required for the activation of the myogenic regulatory network•Recruitment of Pbx1 at muscle regulatory regions is contingent on macroH2A1.2 Histone variants complement and integrate histone post-translational modifications in regulating transcription. The histone variant macroH2A1 (mH2A1) is almost three times the size of its canonical H2A counterpart, due to the presence of an ∼25 kDa evolutionarily conserved non-histone macro domain. Strikingly, mH2A1 can mediate both gene repression and activation. However, the molecular determinants conferring these alternative functions remain elusive. Here, we report that mH2A1.2 is required for the activation of the myogenic gene regulatory network and muscle cell differentiation. H3K27 acetylation at prospective enhancers is exquisitely sensitive to mH2A1.2, indicating a role of mH2A1.2 in imparting enhancer activation. Both H3K27 acetylation and recruitment of the transcription factor Pbx1 at prospective enhancers are regulated by mH2A1.2. Overall, our findings indicate a role of mH2A1.2 in marking regulatory regions for activation. Histone variants complement and integrate histone post-translational modifications in regulating transcription. The histone variant macroH2A1 (mH2A1) is almost three times the size of its canonical H2A counterpart, due to the presence of an ∼25 kDa evolutionarily conserved non-histone macro domain. Strikingly, mH2A1 can mediate both gene repression and activation. However, the molecular determinants conferring these alternative functions remain elusive. Here, we report that mH2A1.2 is required for the activation of the myogenic gene regulatory network and muscle cell differentiation. H3K27 acetylation at prospective enhancers is exquisitely sensitive to mH2A1.2, indicating a role of mH2A1.2 in imparting enhancer activation. Both H3K27 acetylation and recruitment of the transcription factor Pbx1 at prospective enhancers are regulated by mH2A1.2. Overall, our findings indicate a role of mH2A1.2 in marking regulatory regions for activation. Histone post-translational modifications shape the epigenome and regulate transcription (Jenuwein and Allis, 2001Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7632) Google Scholar, Kundaje et al., 2015Kundaje A. Meuleman W. Ernst J. Bilenky M. Yen A. Heravi-Moussavi A. Kheradpour P. Zhang Z. Wang J. Ziller M.J. et al.Roadmap Epigenomics ConsortiumIntegrative analysis of 111 reference human epigenomes.Nature. 2015; 518: 317-330Crossref PubMed Scopus (3578) Google Scholar). The nucleosome incorporation of histone variants provides an additional regulatory layer that influences the formation of chromatin states associated with either transcriptional repression or activation (Jin and Felsenfeld, 2007Jin C. Felsenfeld G. Nucleosome stability mediated by histone variants H3.3 and H2A.Z.Genes Dev. 2007; 21: 1519-1529Crossref PubMed Scopus (401) Google Scholar, Jin et al., 2009Jin C. Zang C. Wei G. Cui K. Peng W. Zhao K. Felsenfeld G. H3.3/H2A.Z double variant-containing nucleosomes mark ‘nucleosome-free regions’ of active promoters and other regulatory regions.Nat. Genet. 2009; 41: 941-945Crossref PubMed Scopus (581) Google Scholar, Barski et al., 2007Barski A. Cuddapah S. Cui K. Roh T.Y. Schones D.E. Wang Z. Wei G. Chepelev I. Zhao K. High-resolution profiling of histone methylations in the human genome.Cell. 2007; 129: 823-837Abstract Full Text Full Text PDF PubMed Scopus (5056) Google Scholar, Maze et al., 2014Maze I. Noh K.M. Soshnev A.A. Allis C.D. Every amino acid matters: essential contributions of histone variants to mammalian development and disease.Nat. Rev. Genet. 2014; 15: 259-271Crossref PubMed Scopus (230) Google Scholar). Localized replacement of canonical histones by histone variants modifies the chromatin structure to attract or repel transcription factors, chromatin writers, readers, and erasers (Skene and Henikoff, 2013Skene P.J. Henikoff S. Histone variants in pluripotency and disease.Development. 2013; 140: 2513-2524Crossref PubMed Scopus (111) Google Scholar). Among the different histone variants, the two isoforms macroH2A1.1 and -1.2 are characterized by the presence of an evolutionarily conserved, ∼25-kDa carboxyl-terminal globular region called the macro domain (Pehrson and Fried, 1992Pehrson J.R. Fried V.A. MacroH2A, a core histone containing a large nonhistone region.Science. 1992; 257: 1398-1400Crossref PubMed Scopus (278) Google Scholar), serving as surface for interaction with metabolites and histone modifiers (Ladurner, 2003Ladurner A.G. Inactivating chromosomes: a macro domain that minimizes transcription.Mol. Cell. 2003; 12: 1-3Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, Kustatscher et al., 2005Kustatscher G. Hothorn M. Pugieux C. Scheffzek K. Ladurner A.G. Splicing regulates NAD metabolite binding to histone macroH2A.Nat. Struct. Mol. Biol. 2005; 12: 624-625Crossref PubMed Scopus (245) Google Scholar, Chakravarthy et al., 2005Chakravarthy S. Gundimella S.K. Caron C. Perche P.Y. Pehrson J.R. Khochbin S. Luger K. Structural characterization of the histone variant macroH2A.Mol. Cell. Biol. 2005; 25: 7616-7624Crossref PubMed Scopus (136) Google Scholar, Gamble and Kraus, 2010Gamble M.J. Kraus W.L. Multiple facets of the unique histone variant macroH2A: from genomics to cell biology.Cell Cycle. 2010; 9: 2568-2574Crossref PubMed Scopus (69) Google Scholar, Hussey et al., 2014Hussey K.M. Chen H. Yang C. Park E. Hah N. Erdjument-Bromage H. Tempst P. Gamble M.J. Kraus W.L. The histone variant MacroH2A1 regulates target gene expression in part by recruiting the transcriptional coregulator PELP1.Mol. Cell. Biol. 2014; 34: 2437-2449Crossref PubMed Scopus (16) Google Scholar). A role for mH2A1 in mediating gene repression was initially suggested by observations linking it to female X chromosome inactivation (Costanzi and Pehrson, 1998Costanzi C. Pehrson J.R. Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals.Nature. 1998; 393: 599-601Crossref PubMed Scopus (478) Google Scholar, Csankovszki et al., 2001Csankovszki G. Nagy A. Jaenisch R. Synergism of Xist RNA, DNA methylation, and histone hypoacetylation in maintaining X chromosome inactivation.J. Cell Biol. 2001; 153: 773-784Crossref PubMed Scopus (373) Google Scholar). More recently, mH2A1 has been shown to contrast reprogrammed pluripotency (Gaspar-Maia et al., 2013Gaspar-Maia A. Qadeer Z.A. Hasson D. Ratnakumar K. Leu N.A. Leroy G. Liu S. Costanzi C. Valle-Garcia D. Schaniel C. et al.MacroH2A histone variants act as a barrier upon reprogramming towards pluripotency.Nat. Commun. 2013; 4: 1565Crossref PubMed Scopus (146) Google Scholar, Barrero et al., 2013Barrero M.J. Sese B. Kuebler B. Bilic J. Boue S. Martí M. Izpisua Belmonte J.C. Macrohistone variants preserve cell identity by preventing the gain of H3K4me2 during reprogramming to pluripotency.Cell Rep. 2013; 3: 1005-1011Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, Pasque et al., 2011Pasque V. Gillich A. Garrett N. Gurdon J.B. Histone variant macroH2A confers resistance to nuclear reprogramming.EMBO J. 2011; 30: 2373-2387Crossref PubMed Scopus (115) Google Scholar), to repress expression of the HoxA cluster (Buschbeck et al., 2009Buschbeck M. Uribesalgo I. Wibowo I. Rué P. Martin D. Gutierrez A. Morey L. Guigó R. López-Schier H. Di Croce L. The histone variant macroH2A is an epigenetic regulator of key developmental genes.Nat. Struct. Mol. Biol. 2009; 16: 1074-1079Crossref PubMed Scopus (148) Google Scholar) and of the α-globin locus in erythroleukemic cells (Ratnakumar et al., 2012Ratnakumar K. Duarte L.F. LeRoy G. Hasson D. Smeets D. Vardabasso C. Bönisch C. Zeng T. Xiang B. Zhang D.Y. et al.ATRX-mediated chromatin association of histone variant macroH2A1 regulates α-globin expression.Genes Dev. 2012; 26: 433-438Crossref PubMed Scopus (94) Google Scholar), and to suppress melanoma progression through regulation of cyclin-dependent protein kinase (CDK)8 (Kapoor et al., 2010Kapoor A. Goldberg M.S. Cumberland L.K. Ratnakumar K. Segura M.F. Emanuel P.O. Menendez S. Vardabasso C. Leroy G. Vidal C.I. et al.The histone variant macroH2A suppresses melanoma progression through regulation of CDK8.Nature. 2010; 468: 1105-1109Crossref PubMed Scopus (288) Google Scholar). However, there is evidence to suggest that mH2A1 has a multifaceted function in controlling gene transcription (Gamble et al., 2010Gamble M.J. Frizzell K.M. Yang C. Krishnakumar R. Kraus W.L. The histone variant macroH2A1 marks repressed autosomal chromatin, but protects a subset of its target genes from silencing.Genes Dev. 2010; 24: 21-32Crossref PubMed Scopus (128) Google Scholar). Reducing mH2A1 levels not only does not result in generalized de-repression of mH2A1-bound genes but is, in fact, associated with failure to activate up to 75% of its targets (Gamble et al., 2010Gamble M.J. Frizzell K.M. Yang C. Krishnakumar R. Kraus W.L. The histone variant macroH2A1 marks repressed autosomal chromatin, but protects a subset of its target genes from silencing.Genes Dev. 2010; 24: 21-32Crossref PubMed Scopus (128) Google Scholar). Moreover, while inhibiting p300-dependent histone acetylation in vitro (Doyen et al., 2006Doyen C.M. An W. Angelov D. Bondarenko V. Mietton F. Studitsky V.M. Hamiche A. Roeder R.G. Bouvet P. Dimitrov S. Mechanism of polymerase II transcription repression by the histone variant macroH2A.Mol. Cell. Biol. 2006; 26: 1156-1164Crossref PubMed Scopus (110) Google Scholar), mH2A1 has been recently reported to cooperate with PARP-1 to regulate transcription by promoting CBP (CREB-binding protein)-mediated acetylation of histone H2B at lysines 12 and 120, with opposing effects on transcription (Chen et al., 2014Chen H. Ruiz P.D. Novikov L. Casill A.D. Park J.W. Gamble M.J. MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation.Nat. Struct. Mol. Biol. 2014; 21: 981-989Crossref PubMed Scopus (82) Google Scholar). These and other observations (Creppe et al., 2012Creppe C. Janich P. Cantariño N. Noguera M. Valero V. Musulén E. Douet J. Posavec M. Martín-Caballero J. Sumoy L. et al.MacroH2A1 regulates the balance between self-renewal and differentiation commitment in embryonic and adult stem cells.Mol. Cell. Biol. 2012; 32: 1442-1452Crossref PubMed Scopus (77) Google Scholar, Podrini et al., 2014Podrini C. Koffas A. Chokshi S. Vinciguerra M. Lelliott C.J. White J.K. Adissu H.A. Williams R. Greco A. MacroH2A1 isoforms are associated with epigenetic markers for activation of lipogenic genes in fat-induced steatosis.FASEB J. 2014; 29: 1676-1687Crossref PubMed Scopus (30) Google Scholar) indicate that mH2A1 may exert a dual function in regulating gene expression. Here, we report that mH2A1.2 is involved in imparting enhancer competency in skeletal muscle cells. In agreement with previous findings, mH2A1.2 was localized to the H3K27me3 promoter regions of repressed genes. However, mH2A1.2-occupied and -repressed targets were not reactivated upon mH2A1.2 knockdown. Instead, activation of muscle enhancers was dependent on mH2A1.2, as its reduction brought about decreased H3K27 acetylation. Reducing mH2A1.2 impaired expression of the master developmental regulator Myogenin, resulting in defective activation of the myogenic gene regulatory network and muscle cell differentiation. Notably, mH2A1.2 mediated chromatin engagement of Pbx1, a homeodomain transcription factor priming MyoD gene targets for activation (Berkes et al., 2004Berkes C.A. Bergstrom D.A. Penn B.H. Seaver K.J. Knoepfler P.S. Tapscott S.J. Pbx marks genes for activation by MyoD indicating a role for a homeodomain protein in establishing myogenic potential.Mol. Cell. 2004; 14: 465-477Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, Maves et al., 2007Maves L. Waskiewicz A.J. Paul B. Cao Y. Tyler A. Moens C.B. Tapscott S.J. Pbx homeodomain proteins direct Myod activity to promote fast-muscle differentiation.Development. 2007; 134: 3371-3382Crossref PubMed Scopus (106) Google Scholar). In aggregate, these findings assign a role to mH2A1.2 in conferring enhancer marking and activation via regulation of transcription factors’ recruitment and H3K27 acetylation. To investigate the role of the histone variant mH2A1 in transcriptional regulation of cell differentiation, we used the mouse skeletal muscle C2C12 cell line, as a model system. C2C12 cells recapitulate muscle differentiation in culture, as they can be kept in an undifferentiated state as myoblasts (MBs) and induced to differentiate to form multinucleated myotubes (MTs) (Yaffe and Saxel, 1977Yaffe D. Saxel O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.Nature. 1977; 270: 725-727Crossref PubMed Scopus (1559) Google Scholar). Both alternatively spliced mH2A1.1 and 1.2 isoforms (Rasmussen et al., 1999Rasmussen T.P. Huang T. Mastrangelo M.A. Loring J. Panning B. Jaenisch R. Messenger RNAs encoding mouse histone macroH2A1 isoforms are expressed at similar levels in male and female cells and result from alternative splicing.Nucleic Acids Res. 1999; 27: 3685-3689Crossref PubMed Scopus (81) Google Scholar, Costanzi and Pehrson, 2001Costanzi C. Pehrson J.R. MACROH2A2, a new member of the MARCOH2A core histone family.J. Biol. Chem. 2001; 276: 21776-21784Crossref PubMed Scopus (123) Google Scholar) were expressed in C2C12 cells (Figure S1A). Since RNA sequencing (RNA-seq) analysis indicated that the mH2A1.2 isoform was the most represented in MBs and expressed at levels similar to those of mH2A1.1 in MTs (Figure S1B), we chose to focus our study on the mH2A1.2 isoform. Analysis of chromatin immunoprecipitation sequencing (ChIP-seq) data generated from two experiments with two different mH2A1.2 antibodies (see Experimental Procedures) identified ∼77,000 overlapping enriched genomic regions in MBs and ∼36,600 in MTs, respectively (Figures S1C and S1D; Table S1). Peak calling with either the MACS2 (Feng et al., 2012Feng J. Liu T. Qin B. Zhang Y. Liu X.S. Identifying ChIP-seq enrichment using MACS.Nat. Protoc. 2012; 7: 1728-1740Crossref PubMed Scopus (863) Google Scholar) or the SICER (Zang et al., 2009Zang C. Schones D.E. Zeng C. Cui K. Zhao K. Peng W. A clustering approach for identification of enriched domains from histone modification ChIP-Seq data.Bioinformatics. 2009; 25: 1952-1958Crossref PubMed Scopus (729) Google Scholar) algorithm identified largely overlapping mH2A1.2-enriched regions (Figure S1E). Examples of mH2A1.2-occupied regions, as called by the MACS2 algorithm, are illustrated in Figure S1F. A global reduction of the mH2A1.2 signal was observed after mH2A1.2 knockdown, indicating that the majority of peaks correspond to the mH2A1.2 isoform (Figures S1G and S1H). Genome-wide distribution of mH2A1.2 was similar in MBs and MTs (Figure 1A). Genome-wide maps of mH2A1.2, intersected with those of active and repressive epigenetic marks in MBs, revealed that the majority of mH2A1.2 peaks was localized at active regions (Figure 1B). Specifically, 32% of mH2A1.2 peaks occurred at H3K4me1+/H3K27ac+ regions (active enhancers), 21% occurred at H3K4me1+ regions, 19% overlapped with H3K4me3+/H3K27ac+ (active promoters), and 25% of mH2A1.2 peaks were located at regions not occupied by any of the aforementioned epigenetic marks considered (mH2A1.2 only). In contrast, only 3% of mH2A1.2 peaks co-localized with the repressive mark H3K27me3 (Figure 1B). Furthermore, among mH2A1.2-bound promoters, only 8% were H3K27me3+, while 67% of these promoters were occupied by both H3K4me3 and H3K27ac (Figure S2A). In MTs, the percentage of mH2A1.2+/H3K27me3+ regions increased to 18% (Figure 1B), and a Gene Ontology (GO) analysis of the newly acquired mH2A1.2+/H3K27me3+ TSS (transcription start site) identified terms related to, among others, “neuron differentiation,” “ pattern specification process,” and “embryonic morphogenesis” (Table S1). Reduction of mH2A1.2 peaks at active enhancers (32% in MB versus 7% in MT; Figure 1B) occurred at MT-specific enhancers (i.e., enhancers active in MT; discussed later) (Figure S2B) and coincided with increased mH2A1.2 occupancy at H3K4me1+ and otherwise non-epigenetically defined genomic regions (64%; Figure 1B). mH2A1.2 occupancy was also reduced, but more modestly, at constitutive enhancers (i.e., enhancers active in both MB and MT; discussed later) in MTs (Figure S2C). Examples of expressed genes occupied by mH2A1.2 are shown in Figure 1C. Developmental regulators of other cell lineages, such as Neurog2 and Wnt1, which are transcriptionally silent in C2C12 cells (Mousavi et al., 2012Mousavi K. Zare H. Wang A.H. Sartorelli V. Polycomb protein Ezh1 promotes RNA polymerase II elongation.Mol. Cell. 2012; 45: 255-262Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), are among mH2A1.2-bound genes with H3K27me3 (Figure 1D). We assigned MB-mH2A1.2+ active enhancers or MB-mH2A1.2+ regions acquiring either H3K4me1+ or H3K4me1+/H3K27ac+ in MTs to genes by proximity (Whyte et al., 2013Whyte W.A. Orlando D.A. Hnisz D. Abraham B.J. Lin C.Y. Kagey M.H. Rahl P.B. Lee T.I. Young R.A. Master transcription factors and mediator establish super-enhancers at key cell identity genes.Cell. 2013; 153: 307-319Abstract Full Text Full Text PDF PubMed Scopus (2289) Google Scholar, Mousavi et al., 2013Mousavi K. Zare H. Dell’orso S. Grontved L. Gutierrez-Cruz G. Derfoul A. Hager G.L. Sartorelli V. eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci.Mol. Cell. 2013; 51: 606-617Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar) and queried gene expression changes occurring during the transition from MB to MT. Enhancers residing within 100 kb, 50 kb, or 20 kb from the closest promoter were considered. While the number of enhancer-assigned genes increased with increasing genomic intervals (Figure S2D), GO analyses for 100-kb and 50-kb intervals captured essentially all the terms returned by the analysis conducted for the 20-kb interval, including “muscle cell differentiation” and “muscle and muscle system process” (Figure S2E; Table S1). Therefore, for further analysis, we considered a proximity measure of 20 kb to assign genes to identified enhancers. Genomic regions that became active enhancers in MT displayed a clear association with upregulated genes (Figure 1E). Similarly, a smaller set comprising genes assigned to mH2A1.2+ regions and occupied by H3K4me1 and H3K27me3 marks in MBs was also enriched for upregulated genes in MTs (Table S2). Overall, these results indicate that mH2A1.2 preferentially occupies transcriptionally active genomic regions in MB or regions programmed to be activated in MT. We addressed the function of mH2A1.2 during muscle cell differentiation by transfecting C2C12 cells with either control or two different mH2A1.2 small interfering RNAs (siRNAs; mH2A1.2 interference, mH2A1.2i) (Figure 2A; Figure S3A) and inducing them to differentiate to form MTs. For further analysis, we chose to use mH2A1.2i_2 siRNAs, as they were the most effective (Figure S3A). mH2A1.2 siRNA specifically reduced mH2A1.2 but not the closely related mH2A1.1 isoform (Figure S3B). MB growth was not affected by mH2A1.2i (Figure S3C). However, Myogenin, a myogenic transcription factor required for muscle differentiation (Tapscott, 2005Tapscott S.J. The circuitry of a master switch: Myod and the regulation of skeletal muscle gene transcription.Development. 2005; 132: 2685-2695Crossref PubMed Scopus (552) Google Scholar), was reduced (Figures 2A–2C; Figures S3A and S3D), and formation of muscle-specific myosin-heavy-chain (MHC)-positive, multinucleated MTs was compromised by mH2A1.2i (Figure 2D). The expression of the muscle-specific gene troponin T type 1 (Tnnt1) was also greatly reduced (Figure S3E). To complement knockdown experiments, exogenous FLAG-tagged mH2A1.2 was expressed in C2C12 cells and found to increase Myogenin expression (Figures 2E and 2F). To define the global impact of reducing mH2A1.2 on the transcriptome, RNA-seq experiments were performed in control and mH2A1.2i C2C12 cells. When mH2A1.2i C2C12 MBs were induced to differentiate, a profound effect on transcriptional dynamics was observed. As indicated in the scatterplot representing changes in gene expression (Figure 3A), genes physiologically upregulated during cell differentiation failed to be properly activated in mH2A1.2i cells, while genes downregulated during differentiation remained transcribed. In control cells, expression of 2,392 genes was increased during the transition from MBs to MTs (Figure 3B; Table S3). Compared to control MTs, 1,786 gene transcripts were reduced by mH2A1.2i. Out of these 1,786 transcripts, 1,440 (80.5%) corresponded to transcripts increased during the differentiation of MBs to MTs (Figure 3B). GO analysis of the transcripts that failed to be appropriately upregulated in mH2A1.2i cells returned terms related to “muscle cell development” and “muscle cell differentiation” (Figure 3C). GO terms for the transcripts that remained elevated in mH2A1.2i cells were related to “cell cycle,” “and “DNA replication” (Figure 3D). Myogenin and its downstream targets muscle creatine kinase (Ckm) and troponin T type 2 (Tnnt2) were not properly activated in mH2A1.2i cells (Figure 3E). Conversely, transcripts of the Inhibitor of DNA Binding 3 (Id3), a member of the Id family of helix-loop-helix proteins counteracting muscle differentiation (Benezra et al., 1990Benezra R. Davis R.L. Lockshon D. Turner D.L. Weintraub H. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins.Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1801) Google Scholar), cyclin D1 (Ccnd1), and the cell-cycle regulator Mcm5, which are physiologically downregulated upon C2C12 differentiation, remained abnormally elevated in mH2A1i cells (Figure 3F). To validate these findings, we used a different mH2A1.2 siRNA (mH2A1.2i_1) (Figure S3A). In mH2A1.2i_1-transfected cells, transcripts of Myogenin, muscle-specific MHC 3 (Myh3), cardiac actin (Actc1), and creatine kinase (Ckm) were reduced, while those of cyclin D (Ccnd1) remained elevated (Figure 3G). These findings indicate that mH2A1.2 is required to activate muscle gene expression during cell differentiation. We used the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) (Buenrostro et al., 2013Buenrostro J.D. Giresi P.G. Zaba L.C. Chang H.Y. Greenleaf W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.Nat. Methods. 2013; 10: 1213-1218Crossref PubMed Scopus (3186) Google Scholar) to define chromatin accessibility in C2C12 MBs and MTs. In ATAC-seq, tagging of nucleosome-free genomic regions is mediated by transposase-mediated delivery of sequencing adapters. Tagged regions correlate with DNase I hypersentitive sites (open chromatin), which are generally found within genomic regulatory functions. Using two independent replicates, ∼47,300 and ∼17,200 transposase-accessible or open chromatin regions were reproducibly identified in MBs and MTs, respectively (Figure 4A). More than 84% of these genomic regions (14,448/17,200) were open in both MBs and MTs (Figure 4B). The remaining ATAC-seq MT sites (∼2,650) were closed in MBs and open in MTs, and almost all of them (∼2,500) were located outside the promoter regions (Figures 4A and 4B). We refer to these two groups as constitutive (open in both MBs and MTs) and MT-specific enhancers (present only in MTs), respectively. mH2A1.2 occupied both enhancer groups in MBs (Figure 4B). While constitutive enhancers were similarly acetylated at H3K27 in both MBs and MTs (Figure 4C; compare MB-WT, black line, and MT-WT, blue line), MT-specific enhancers acquired H3K27ac only in MTs (Figure 4D; compare MB-WT, black line, and MT-WT, blue line). To determine whether mH2A1.2 regulates the activity of constitutive and MT-specific enhancers, we assigned genes to these two groups of enhancers (based on proximity distance ±20 kb) and evaluated how mH2A1.2i affected expression of the enhancer-assigned genes. Using gene set enrichment analysis (GSEA), we found that genes assigned to constitutive enhancers were positively correlated with genes whose expression was reduced by mH2A1.2i in MBs (Figure 4E), whereas genes whose expression was diminished by mH2A1.2i in MTs correlated with genes assigned to MT-specific enhancers (Figure 4F). Since H3K27 acetylation is a defining step associated with enhancer activation (Creyghton et al., 2010Creyghton M.P. Cheng A.W. Welstead G.G. Kooistra T. Carey B.W. Steine E.J. Hanna J. Lodato M.A. Frampton G.M. Sharp P.A. et al.Histone H3K27ac separates active from poised enhancers and predicts developmental state.Proc. Natl. Acad. Sci. USA. 2010; 107: 21931-21936Crossref PubMed Scopus (2493) Google Scholar, Heintzman et al., 2009Heintzman N.D. Hon G.C. Hawkins R.D. Kheradpour P. Stark A. Harp L.F. Ye Z. Lee L.K. Stuart R.K. Ching C.W. et al.Histone modifications at human enhancers reflect global cell-type-specific gene expression.Nature. 2009; 459: 108-112Crossref PubMed Scopus (1779) Google Scholar, Rada-Iglesias et al., 2011Rada-Iglesias A. Bajpai R. Swigut T. Brugmann S.A. Flynn R.A. Wysocka J. A unique chromatin signature uncovers early developmental enhancers in humans.Nature. 2011; 470: 279-283Crossref PubMed Scopus (1523) Google Scholar, Zentner et al., 2011Zentner G.E. Tesar P.J. Scacheri P.C. Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions.Genome Res. 2011; 21: 1273-1283Crossref PubMed Scopus (387) Google Scholar, Bonn et al., 2012Bonn S. Zinzen R.P. Girardot C. Gustafson E.H. Perez-Gonzalez A. Delhomme N. Ghavi-Helm Y. Wilczyński B. Riddell A. Furlong E.E. Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development.Nat. Genet. 2012; 44: 148-156Crossref PubMed Scopus (345) Google Scholar), we evaluated whether mH2A1.2 was involved in conferring H3K27 acetylation by performing H3K27ac ChIP-seq on mH2A1.2i. H3K27 acetylation at constitutive enhancers was slightly reduced (Figure 4C; compare MT-WT, blue line, with MT-mH2A1.2i, red line). A most profound effect of mH2A1.2i on H3K27 acetylation was observed at MT-specific enhancers. At these enhancers, mH2A1i.2 reduced H3K27ac to background levels observed in MBs, where the chromatin of MT-specific enhancers is closed (Figure 4D; compare MT-WT, blue line, with MT-mH2A1.2i, red line). Consistent with a more limited reduction of H3K27ac at constitutive enhancers (Figure 4C), the transcription of genes assigned to constitutive enhancers was less affected than that of genes controlled by MT-specific enhancers (Figure 4E; enrichment score [ES] < 0.30; Figure 4F, ES > 0.45) in mH2A1.2i cells. Next, we analyzed H3K27ac at promoter regions. mH2A1.2i did not modify H3K27ac at constitutive promoters but reduced it at MT-specific promoters (Figures S4A and S4B). These findings are consistent with the impaired acquisition of MT-specific enhancer competency upon mH2A1.2i and consequent failure to induce promoter activation (H3K27ac) and gene transcription. To establish whether a direct link exists between reduced H3K27 acetylation and mH2A1.2i, we attempted rescue experiments by overexpressing mH2A1.2 in mH2A1.2i cells. mH2A1.2 overexpression partially restored H3K27ac at both the Myogenin and Myh3 loci in mH2A1.2i cells (Figure 4G). In summary, these results indicate that, during muscle cell differentiation, mH2A1.2 is involved in conferring enhancer activation by regulating H3K27 acetylation. The presence of mH2A1.2 in MBs at both TSSs and enhancers destined to become activated in MTs (MT-specific enhancers), as well as its requirement for their activation, prompted us to investigate a potential link between mH2A1.2 and the transcription factor Pbx1. The TALE (three-amino-acid loop extension) homeodomain-containing transcription factor Pbx1 is required to assist MyoD-dependent activation of Myogenin (Berkes et al., 2004Berkes C.A. Bergstrom D.A. Penn B.H. Seaver K.J. Knoepfler P.S. Tapscott S.J. Pbx marks genes for activation by MyoD indicating a role for a homeodomain protein in establishing myogenic potential.Mol. Cell. 2004; 14: 465-477Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, de la Serna et al., 2005de la Serna I.L. Ohkawa Y. Berkes C.A. Bergstrom D.A. Dacwag C.S. Tapscott S.J. Imbalzano A.N. MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex.Mol. Cell. Biol. 2005; 25: 3997-4009Crossref PubMed Scopus (222) Google Scholar). Pbx1 is constitutively bound to the Myogenin gene in fibroblasts prior to MyoD-mediated conversion to muscle and, by directly interacting with two specific domains, ensures productive and stable MyoD recruitment at the Myogenin promoter (Berkes et al., 2004Berkes C.A. Bergstrom D.A. Penn B.H. Seaver K.J. Knoepfler P.S. Tapscott S.J. Pbx marks genes for activation by MyoD indicating a role for a homeodomain protein in establishing myogenic potential.Mol. Cell. 2004; 14: 465-477Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). More recently, the Pbx1/MyoD interaction has been shown to regulate expression of a large cohort of MyoD-dependent genes (Fong et al., 2015Fong A.P. Yao Z. Zhong J.W. Johnson N.M. Farr 3rd, G.H. Maves L. Tapscott S.J. Conversion of MyoD to a neurogenic factor: binding site specificity determines lineage.Cell Rep. 2015; 10: 1937-1946Abstract Fu" @default.
• W2258223596 created "2016-06-24" @default.
• W2258223596 creator A5007756720 @default.
• W2258223596 creator A5024927053 @default.
• W2258223596 creator A5035350667 @default.
• W2258223596 creator A5057320865 @default.
• W2258223596 creator A5061857523 @default.
• W2258223596 creator A5063928673 @default.
• W2258223596 creator A5069959515 @default.
• W2258223596 creator A5082564918 @default.
• W2258223596 creator A5088934837 @default.
• W2258223596 creator A5090051911 @default.
• W2258223596 date "2016-02-01" @default.
• W2258223596 modified "2023-10-15" @default.
• W2258223596 title "The Histone Variant MacroH2A1.2 Is Necessary for the Activation of Muscle Enhancers and Recruitment of the Transcription Factor Pbx1" @default.
• W2258223596 cites W1522147759 @default.
• W2258223596 cites W1755602580 @default.
• W2258223596 cites W1965514686 @default.
• W2258223596 cites W1967835379 @default.
• W2258223596 cites W1970461933 @default.
• W2258223596 cites W1971451591 @default.
• W2258223596 cites W1971578941 @default.
• W2258223596 cites W1973022314 @default.
• W2258223596 cites W1978125302 @default.
• W2258223596 cites W1978607217 @default.
• W2258223596 cites W1978690835 @default.
• W2258223596 cites W1981030303 @default.
• W2258223596 cites W1985061074 @default.
• W2258223596 cites W1992296270 @default.
• W2258223596 cites W1993113643 @default.
• W2258223596 cites W1997053831 @default.
• W2258223596 cites W2002670570 @default.
• W2258223596 cites W2004863894 @default.
• W2258223596 cites W2005144583 @default.
• W2258223596 cites W2007639313 @default.
• W2258223596 cites W2013660990 @default.
• W2258223596 cites W2014677321 @default.
• W2258223596 cites W2015416439 @default.
• W2258223596 cites W2018863876 @default.
• W2258223596 cites W2019005509 @default.
• W2258223596 cites W2020956187 @default.
• W2258223596 cites W2025525406 @default.
• W2258223596 cites W2037231961 @default.
• W2258223596 cites W2038208040 @default.
• W2258223596 cites W2038356000 @default.
• W2258223596 cites W2044524973 @default.
• W2258223596 cites W2055322019 @default.
• W2258223596 cites W2057586248 @default.
• W2258223596 cites W2074998492 @default.
• W2258223596 cites W2076154138 @default.
• W2258223596 cites W2080482028 @default.
• W2258223596 cites W2082161548 @default.
• W2258223596 cites W2083405476 @default.
• W2258223596 cites W2083441653 @default.
• W2258223596 cites W2087476954 @default.
• W2258223596 cites W2087573417 @default.
• W2258223596 cites W2093146349 @default.
• W2258223596 cites W2097065948 @default.
• W2258223596 cites W2100189018 @default.
• W2258223596 cites W2102619694 @default.
• W2258223596 cites W2105892090 @default.
• W2258223596 cites W2117757143 @default.
• W2258223596 cites W2122097157 @default.
• W2258223596 cites W2122813253 @default.
• W2258223596 cites W2123106337 @default.
• W2258223596 cites W2123564569 @default.
• W2258223596 cites W2124985265 @default.
• W2258223596 cites W2126806930 @default.
• W2258223596 cites W2128940002 @default.
• W2258223596 cites W2130410032 @default.
• W2258223596 cites W2133465414 @default.
• W2258223596 cites W2135396849 @default.
• W2258223596 cites W2139811812 @default.
• W2258223596 cites W2143658931 @default.
• W2258223596 cites W2144091055 @default.
• W2258223596 cites W2145683850 @default.
• W2258223596 cites W2146648681 @default.
• W2258223596 cites W2154746182 @default.
• W2258223596 cites W2157569563 @default.
• W2258223596 cites W2158217645 @default.
• W2258223596 cites W2158901355 @default.
• W2258223596 cites W2159511823 @default.
• W2258223596 cites W2166820820 @default.
• W2258223596 cites W2167776710 @default.
• W2258223596 cites W2169567788 @default.
• W2258223596 cites W2171808845 @default.
• W2258223596 cites W2739174192 @default.
• W2258223596 doi "https://doi.org/10.1016/j.celrep.2015.12.103" @default.
• W2258223596 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4749450" @default.
• W2258223596 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/26832413" @default.
• W2258223596 hasPublicationYear "2016" @default.
• W2258223596 type Work @default.
• W2258223596 sameAs 2258223596 @default.
• W2258223596 citedByCount "48" @default.
• W2258223596 countsByYear W22582235962016 @default.
• W2258223596 countsByYear W22582235962017 @default.
• W2258223596 countsByYear W22582235962018 @default.
• W2258223596 countsByYear W22582235962019 @default.

• next