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• W2118612858 abstract "Control of gene expression during development requires the concerted action of sequence-specific transcriptional regulators and epigenetic modifiers, which are spatially coordinated within the nucleus through mechanisms that are poorly understood. Here we show that transcriptional repression by the Msx1 homeoprotein in myoblast cells requires the recruitment of Polycomb to target genes located at the nuclear periphery. Target genes repressed by Msx1 display an Msx1-dependent enrichment of Polycomb-directed trimethylation of lysine 27 on histone H3 (H3K27me3). Association of Msx1 with the Polycomb complex is required for repression and regulation of myoblast differentiation. Furthermore, Msx1 promotes a dynamic spatial redistribution of the H3K27me3 repressive mark to the nuclear periphery in myoblast cells and the developing limb in vivo. Our findings illustrate a hitherto unappreciated spatial coordination of transcription factors with the Polycomb complex for appropriate regulation of gene expression programs during development. Control of gene expression during development requires the concerted action of sequence-specific transcriptional regulators and epigenetic modifiers, which are spatially coordinated within the nucleus through mechanisms that are poorly understood. Here we show that transcriptional repression by the Msx1 homeoprotein in myoblast cells requires the recruitment of Polycomb to target genes located at the nuclear periphery. Target genes repressed by Msx1 display an Msx1-dependent enrichment of Polycomb-directed trimethylation of lysine 27 on histone H3 (H3K27me3). Association of Msx1 with the Polycomb complex is required for repression and regulation of myoblast differentiation. Furthermore, Msx1 promotes a dynamic spatial redistribution of the H3K27me3 repressive mark to the nuclear periphery in myoblast cells and the developing limb in vivo. Our findings illustrate a hitherto unappreciated spatial coordination of transcription factors with the Polycomb complex for appropriate regulation of gene expression programs during development. Msx1 homeoprotein located at the nuclear periphery with its repressed target genes Repressed target genes of Msx1 are enriched for the Polycomb H3K27me3 chromatin mark Msx1 interaction with Polycomb required for repression and myoblast differentiation Msx1 redistributes the Polycomb H3K27me3 mark to the periphery in the developing limb Appropriate spatial and temporal control of gene expression during development involves a coordinated network of chromatin-modifying complexes and sequence-specific DNA binding proteins, which operate within the dynamic spatial organization of the nucleus. Indeed, the nucleus is comprised of distinct functional and morphological compartments, such as those dedicated to transcription (Lanctôt et al., 2007Lanctôt C. Cheutin T. Cremer M. Cavalli G. Cremer T. Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions.Nat. Rev. Genet. 2007; 8: 104-115Crossref PubMed Scopus (632) Google Scholar, Misteli, 2007Misteli T. Beyond the sequence: cellular organization of genome function.Cell. 2007; 128: 787-800Abstract Full Text Full Text PDF PubMed Scopus (897) Google Scholar), and wherein chromosomes tend to be organized with gene-rich regions near the interior and gene-poor regions near the periphery (Fraser and Bickmore, 2007Fraser P. Bickmore W. Nuclear organization of the genome and the potential for gene regulation.Nature. 2007; 447: 413-417Crossref PubMed Scopus (577) Google Scholar, Towbin et al., 2009Towbin B.D. Meister P. Gasser S.M. The nuclear envelope—a scaffold for silencing?.Curr. Opin. Genet. Dev. 2009; 19: 180-186Crossref PubMed Scopus (120) Google Scholar, Zhao et al., 2009Zhao R. Bodnar M.S. Spector D.L. Nuclear neighborhoods and gene expression.Curr. Opin. Genet. Dev. 2009; 19: 172-179Crossref PubMed Scopus (131) Google Scholar). Among chromatin regulators, the polycomb repressive complexes, PRC1 and PRC2, act sequentially and coordinately to repress gene expression by covalent modification of chromatin (Schuettengruber and Cavalli, 2009Schuettengruber B. Cavalli G. Recruitment of polycomb group complexes and their role in the dynamic regulation of cell fate choice.Development. 2009; 136: 3531-3542Crossref PubMed Scopus (323) Google Scholar). The PRC2 complex, which includes the enzymatic component, Ezh2, as well as Suz12 and EED, imparts a repressive trimethyl mark at lysine 27 of histone H3 (H3K27me3) (Cao et al., 2002Cao R. Wang L. Wang H. Xia L. Erdjument-Bromage H. Tempst P. Jones R.S. Zhang Y. Role of histone H3 lysine 27 methylation in Polycomb-group silencing.Science. 2002; 298: 1039-1043Crossref PubMed Scopus (2842) Google Scholar, Kuzmichev et al., 2002Kuzmichev A. Nishioka K. Erdjument-Bromage H. Tempst P. Reinberg D. Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein.Genes Dev. 2002; 16: 2893-2905Crossref PubMed Scopus (1271) Google Scholar, 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 (714) Google Scholar), associated with lineage commitment in embryonic stem cells and differentiation during development (Boyer et al., 2006Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. Wernig M. Tajonar A. Ray M.K. et al.Polycomb complexes repress developmental regulators in murine embryonic stem cells.Nature. 2006; 441: 349-353Crossref PubMed Scopus (2042) Google Scholar, Bracken et al., 2006Bracken A.P. Dietrich N. Pasini D. Hansen K.H. Helin K. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions.Genes Dev. 2006; 20: 1123-1136Crossref PubMed Scopus (1011) Google Scholar). PRC2 complexes are highly dynamic, tending to be active during developmental stages when cells are proliferating but not yet differentiated, and associated with genes that are repressed but poised for expression when differentiation ensues (Bracken et al., 2006Bracken A.P. Dietrich N. Pasini D. Hansen K.H. Helin K. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions.Genes Dev. 2006; 20: 1123-1136Crossref PubMed Scopus (1011) Google Scholar, Ezhkova et al., 2009Ezhkova E. Pasolli H.A. Parker J.S. Stokes N. Su I.H. Hannon G. Tarakhovsky A. Fuchs E. Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells.Cell. 2009; 136: 1122-1135Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). This is exemplified in the myogenic lineage, where Ezh2 is expressed in myoblast cells but downregulated in myotubes, while its forced expression inhibits the formation of myotubes (Caretti et al., 2004Caretti G. Di Padova M. Micales B. Lyons G.E. Sartorelli V. The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation.Genes Dev. 2004; 18: 2627-2638Crossref PubMed Scopus (510) Google Scholar). Although mammalian Polycomb response elements (PRE) have recently been identified (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, Sing et al., 2009Sing A. Pannell D. Karaiskakis A. Sturgeon K. Djabali M. Ellis J. Lipshitz H.D. Cordes S.P. A vertebrate Polycomb response element governs segmentation of the posterior hindbrain.Cell. 2009; 138: 885-897Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, Woo et al., 2010Woo C.J. Kharchenko P.V. Daheron L. Park P.J. Kingston R.E. A region of the human HOXD cluster that confers polycomb-group responsiveness.Cell. 2010; 140: 99-110Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), how PRC2 complexes are recruited to genomic targets in dynamic spatial and temporal contexts has not been fully elucidated. Homeoproteins comprise one of the major classes of transcriptional regulators that control differentiation during development. Among these, Msx1 is expressed in diverse spatial and temporal domains during development, where a unifying feature is its restricted expression to proliferating cells that are poised to differentiate and its downregulation prior to differentiation (Bendall and Abate-Shen, 2000Bendall A.J. Abate-Shen C. Roles for Msx and Dlx homeoproteins in vertebrate development.Gene. 2000; 247: 17-31Crossref PubMed Scopus (227) Google Scholar, Davidson, 1995Davidson D. The function and evolution of Msx genes: pointers and paradoxes.Trends Genet. 1995; 11: 405-411Abstract Full Text PDF PubMed Scopus (309) Google Scholar). In the developing limb, for example, where Msx1, together with Msx2, is required for proper limb formation (Lallemand et al., 2005Lallemand Y. Nicola M.A. Ramos C. Bach A. Cloment C.S. Robert B. Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development.Development. 2005; 132: 3003-3014Crossref PubMed Scopus (115) Google Scholar), Msx1 is expressed in a zone of undifferentiated mesenchymal cells destined to form structural elements of the limb, but not in the adjacent cells differentiating to form these structures (Bendall et al., 1999Bendall A.J. Ding J. Hu G. Shen M.M. Abate-Shen C. Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors.Development. 1999; 126: 4965-4976Crossref PubMed Google Scholar, Catron et al., 1996Catron K.M. Wang H. Hu G. Shen M.M. Abate-Shen C. Comparison of MSX-1 and MSX-2 suggests a molecular basis for functional redundancy.Mech. Dev. 1996; 55: 185-199Crossref PubMed Scopus (113) Google Scholar, Davidson, 1995Davidson D. The function and evolution of Msx genes: pointers and paradoxes.Trends Genet. 1995; 11: 405-411Abstract Full Text PDF PubMed Scopus (309) Google Scholar, Davidson et al., 1991Davidson D.R. Crawley A. Hill R.E. Tickle C. Position-dependent expression of two related homeobox genes in developing vertebrate limbs.Nature. 1991; 352: 429-431Crossref PubMed Scopus (165) Google Scholar). Similarly, in the myogenic lineage Msx1 is expressed in myogenic precursors during development as well as in adult satellite cells (i.e., stem cells), but not in differentiated myotubes (Bendall et al., 1999Bendall A.J. Ding J. Hu G. Shen M.M. Abate-Shen C. Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors.Development. 1999; 126: 4965-4976Crossref PubMed Google Scholar, Cornelison et al., 2000Cornelison D.D. Olwin B.B. Rudnicki M.A. Wold B.J. MyoD(-/-) satellite cells in single-fiber culture are differentiation defective and MRF4 deficient.Dev. Biol. 2000; 224: 122-137Crossref PubMed Scopus (211) Google Scholar, Houzelstein et al., 1999Houzelstein D. Auda-Boucher G. Chéraud Y. Rouaud T. Blanc I. Tajbakhsh S. Buckingham M.E. Fontaine-Pérus J. Robert B. The homeobox gene Msx1 is expressed in a subset of somites, and in muscle progenitor cells migrating into the forelimb.Development. 1999; 126: 2689-2701PubMed Google Scholar). Furthermore, forced expression of Msx1 in myoblast cells inhibits their differentiation (Hu et al., 2001Hu G. Lee H. Price S.M. Shen M.M. Abate-Shen C. Msx homeobox genes inhibit differentiation through upregulation of cyclin D1.Development. 2001; 128: 2373-2384Crossref PubMed Google Scholar, Woloshin et al., 1995Woloshin P. Song K. Degnin C. Killary A.M. Goldhamer D.J. Sassoon D. Thayer M.J. MSX1 inhibits myoD expression in fibroblast x 10T1/2 cell hybrids.Cell. 1995; 82: 611-620Abstract Full Text PDF PubMed Scopus (131) Google Scholar), whereas its forced expression in myotubes results in their dedifferentiation (Odelberg et al., 2000Odelberg S.J. Kollhoff A. Keating M.T. Dedifferentiation of mammalian myotubes induced by msx1.Cell. 2000; 103: 1099-1109Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). The inhibitory consequences of Msx1 for differentiation are mediated, in part, by its actions as a transcriptional repressor. For instance, Msx1 represses the expression of MyoD, a principal regulator of myogenic differentiation, by binding to the core enhancer region (CER) (Bendall et al., 1999Bendall A.J. Ding J. Hu G. Shen M.M. Abate-Shen C. Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors.Development. 1999; 126: 4965-4976Crossref PubMed Google Scholar, Lee et al., 2004Lee H. Habas R. Abate-Shen C. MSX1 cooperates with histone H1b for inhibition of transcription and myogenesis.Science. 2004; 304: 1675-1678Crossref PubMed Scopus (197) Google Scholar, Lee et al., 2006Lee H. Quinn J.C. Prasanth K.V. Swiss V.A. Economides K.D. Camacho M.M. Spector D.L. Abate-Shen C. PIAS1 confers DNA-binding specificity on the Msx1 homeoprotein.Genes Dev. 2006; 20: 784-794Crossref PubMed Scopus (84) Google Scholar, Woloshin et al., 1995Woloshin P. Song K. Degnin C. Killary A.M. Goldhamer D.J. Sassoon D. Thayer M.J. MSX1 inhibits myoD expression in fibroblast x 10T1/2 cell hybrids.Cell. 1995; 82: 611-620Abstract Full Text PDF PubMed Scopus (131) Google Scholar), which regulates the timing of MyoD expression in vivo (Goldhamer et al., 1995Goldhamer D.J. Brunk B.P. Faerman A. King A. Shani M. Emerson Jr., C.P. Embryonic activation of the myoD gene is regulated by a highly conserved distal control element.Development. 1995; 121: 637-649Crossref PubMed Google Scholar). Notably, this interaction occurs at the nuclear periphery, and this subnuclear localization is required for repression by Msx1 (Lee et al., 2006Lee H. Quinn J.C. Prasanth K.V. Swiss V.A. Economides K.D. Camacho M.M. Spector D.L. Abate-Shen C. PIAS1 confers DNA-binding specificity on the Msx1 homeoprotein.Genes Dev. 2006; 20: 784-794Crossref PubMed Scopus (84) Google Scholar). In the current study, we investigate the consequences of Msx1 localization to the nuclear periphery for its function in transcriptional repression in myoblast cells and the murine embryonic limb. We identify Msx1 target genes and find that the repressed targets are preferentially located at the nuclear periphery in myoblast cells, where Msx1 is also located. We further show that their repression requires association of Msx1 with the PRC2 complex, resulting in an Msx1-dependent enrichment of H3K27me3 on Msx1 genomic binding sites as well as a striking redistribution of this repressive mark to the nuclear periphery. Our findings highlight a hitherto unappreciated role of spatial context of chromatin marks as a key factor in regulating gene expression during development. We have shown previously that transcriptional repression by Msx1 in C2C12 myoblast cells requires its localization to the nuclear periphery (Lee et al., 2006Lee H. Quinn J.C. Prasanth K.V. Swiss V.A. Economides K.D. Camacho M.M. Spector D.L. Abate-Shen C. PIAS1 confers DNA-binding specificity on the Msx1 homeoprotein.Genes Dev. 2006; 20: 784-794Crossref PubMed Scopus (84) Google Scholar). We now find that this striking localization of Msx1 to the periphery is distinctive, as it is not shared by other classes of homeoproteins (Figure 1A and see Figure S1A available online); notably, the C-terminal region of Msx1 that is required for localization to the periphery (Lee et al., 2006Lee H. Quinn J.C. Prasanth K.V. Swiss V.A. Economides K.D. Camacho M.M. Spector D.L. Abate-Shen C. PIAS1 confers DNA-binding specificity on the Msx1 homeoprotein.Genes Dev. 2006; 20: 784-794Crossref PubMed Scopus (84) Google Scholar and below) is not conserved with other homeoproteins (Bendall and Abate-Shen, 2000Bendall A.J. Abate-Shen C. Roles for Msx and Dlx homeoproteins in vertebrate development.Gene. 2000; 247: 17-31Crossref PubMed Scopus (227) Google Scholar). Furthermore, this subnuclear localization by Msx1 also occurs in primary myoblasts in culture, as well as the developing limb of mouse embryos in vivo, but not in all other tissues where Msx1 is expressed during development, such as the neural tube (Figures 1B and 1C). Thus, localization to the nuclear periphery is a distinctive feature of Msx1 that occurs in specific biological contexts. To further investigate the significance of this subnuclear localization for transcriptional regulation, we first identified Msx1 target genes and then assessed their subnuclear localization in myoblast cells. Target genes were identified using a combination of gene expression profiling and chromatin-immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) to identify genes that are both differentially expressed and bound by Msx1 (Figure 2A ). Because Msx1 is virtually undetectable in most cultured cells, including myoblasts (J.W. and C.A.-S., unpublished data), we performed these analyses in C2C12 cells expressing exogenous Msx1 (Hu et al., 2001Hu G. Lee H. Price S.M. Shen M.M. Abate-Shen C. Msx homeobox genes inhibit differentiation through upregulation of cyclin D1.Development. 2001; 128: 2373-2384Crossref PubMed Google Scholar, Lee et al., 2004Lee H. Habas R. Abate-Shen C. MSX1 cooperates with histone H1b for inhibition of transcription and myogenesis.Science. 2004; 304: 1675-1678Crossref PubMed Scopus (197) Google Scholar, Lee et al., 2006Lee H. Quinn J.C. Prasanth K.V. Swiss V.A. Economides K.D. Camacho M.M. Spector D.L. Abate-Shen C. PIAS1 confers DNA-binding specificity on the Msx1 homeoprotein.Genes Dev. 2006; 20: 784-794Crossref PubMed Scopus (84) Google Scholar), and then validated our findings for endogenous Msx1 in the developing limb, comparing Msx1 mutant with wild-type embryos (Figure 2A). We performed Affymetrix gene expression profiling using RNA from C2C12 cells expressing tamoxifen-inducible Msx1 (Hu et al., 2001Hu G. Lee H. Price S.M. Shen M.M. Abate-Shen C. Msx homeobox genes inhibit differentiation through upregulation of cyclin D1.Development. 2001; 128: 2373-2384Crossref PubMed Google Scholar), focusing on genes whose expression was differentially regulated shortly after (i.e., within 6 hr) induction to enrich for direct target genes (Table S1). We compared these with genes bound by Msx1 within 10 kb of the transcription start site (TSS), as determined by ChIP-Seq analyses of genomic DNA from C2C12 cells expressing a constitutively-active epitope-tagged Msx1 (Lee et al., 2006Lee H. Quinn J.C. Prasanth K.V. Swiss V.A. Economides K.D. Camacho M.M. Spector D.L. Abate-Shen C. PIAS1 confers DNA-binding specificity on the Msx1 homeoprotein.Genes Dev. 2006; 20: 784-794Crossref PubMed Scopus (84) Google Scholar) (Figure S2A and Table S2). Comparison of differentially expressed genes (i.e., from the gene expression profiling data) with those bound by Msx1 (i.e., from the ChIP-Seq analyses) revealed 79 upregulated and 87 downregulated target genes (total of 166) (Figure 2B and Table S3). Although many more genes were bound by Msx1 (i.e., 8606 genes) than were differentially expressed (i.e., 221 genes), more than 75% of the differentially expressed genes were also bound by Msx1 (p = 9.8 × 10−10), and particularly the downregulated genes had a bias for Msx1 binding at or near the TSS (p = 0.01) (Figures S2B and S2C and Table S3). Interestingly, Msx1 genomic binding sites were enriched for its consensus DNA binding site (i.e., TAATT) (Figure S2D), while de novo motif discovery analyses revealed the prevalence of additional sequence motifs, including an AP1 binding site (see Supplemental Experimental Procedures and Figure S2E). Thus, recognition of target sequences by Msx1 in vivo may involve both direct and indirect interactions. Consistent with the known functions of Msx1 as a negative regulator of differentiation (Bendall and Abate-Shen, 2000Bendall A.J. Abate-Shen C. Roles for Msx and Dlx homeoproteins in vertebrate development.Gene. 2000; 247: 17-31Crossref PubMed Scopus (227) Google Scholar), functional annotation of target genes revealed that those upregulated were enriched for genes involved in controlling the cell cycle, cellular proliferation, and/or development, whereas those downregulated tended to be involved in differentiation, bone remodeling, and/or myogenesis, as well as development (Figure S2F). We validated the differential expression of selected target genes, which were chosen on the basis of their potential relevance for myogenesis and/or differentiation, by real-time PCR using RNA from C2C12 cells expressing or lacking exogenous Msx1 (Figure 2C and Table S4). The differential expression of target genes was further validated in developing limbs of Msx1 mutant versus wild-type embryos, using real-time PCR as well as in situ hybridization, which showed that genes downregulated in C2C12 cells expressing exogenous Msx1 were upregulated in the mutant limb lacking Msx1 and vice versa (Figures 2C and 2D, Table S4, and Figure S2G). Notably, our finding that target genes identified in C2C12 cells are also regulated by Msx1 in murine embryos indicates that they are indeed bona fide targets in vivo. We next investigated the subnuclear localization of Msx1 target genes in myoblast cells using fluorescence in situ hybridization (FISH) analyses. We found that the genes bound and downregulated by Msx1 exhibited strong (i.e., Myc, Met, MyoD, and Myf5 > 75%) or moderate (i.e., Six1, Angpt1, Snai2, and Ezh1 > 65%) association with the nuclear periphery in both C2C12 and primary myoblast cells (Figures 2E and 2F and Figures S1B and S1C). Importantly, this was only the case for the repressed targets (i.e., the downregulated genes), because the genes that were bound and upregulated by Msx1, as well as other genes that were neither bound nor regulated by Msx1 were not preferentially associated with the nuclear periphery (Figures 2E and 2F and Figures S1B and S1C). As we had observed previously (Lee et al., 2006Lee H. Quinn J.C. Prasanth K.V. Swiss V.A. Economides K.D. Camacho M.M. Spector D.L. Abate-Shen C. PIAS1 confers DNA-binding specificity on the Msx1 homeoprotein.Genes Dev. 2006; 20: 784-794Crossref PubMed Scopus (84) Google Scholar), the localization of target genes at the nuclear periphery was not dependent on expression of Msx1 (Figures 2E and 2F and Figures S1B and S1C). Because target genes that are repressed, but not activated, were localized to the nuclear periphery, our subsequent analyses to investigate the consequences of Msx1 localization to the nuclear periphery for transcriptional control in myoblast cells was focused primarily on the repressed target genes. Consistent with the known functions of Msx1 as a transcriptional repressor and negative regulator of muscle cell differentiation (Hu et al., 2001Hu G. Lee H. Price S.M. Shen M.M. Abate-Shen C. Msx homeobox genes inhibit differentiation through upregulation of cyclin D1.Development. 2001; 128: 2373-2384Crossref PubMed Google Scholar, Lee et al., 2004Lee H. Habas R. Abate-Shen C. MSX1 cooperates with histone H1b for inhibition of transcription and myogenesis.Science. 2004; 304: 1675-1678Crossref PubMed Scopus (197) Google Scholar, Lee et al., 2006Lee H. Quinn J.C. Prasanth K.V. Swiss V.A. Economides K.D. Camacho M.M. Spector D.L. Abate-Shen C. PIAS1 confers DNA-binding specificity on the Msx1 homeoprotein.Genes Dev. 2006; 20: 784-794Crossref PubMed Scopus (84) Google Scholar, Odelberg et al., 2000Odelberg S.J. Kollhoff A. Keating M.T. Dedifferentiation of mammalian myotubes induced by msx1.Cell. 2000; 103: 1099-1109Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, Woloshin et al., 1995Woloshin P. Song K. Degnin C. Killary A.M. Goldhamer D.J. Sassoon D. Thayer M.J. MSX1 inhibits myoD expression in fibroblast x 10T1/2 cell hybrids.Cell. 1995; 82: 611-620Abstract Full Text PDF PubMed Scopus (131) Google Scholar), these repressed target genes include known regulators of muscle cell differentiation, such as Six1, Snai2, and Myf5 (Figures 2B–2D and Table S4). Inspection of the ChIP-Seq binding data for these targets, as well that of a previously known target, MyoD, revealed Msx1 binding to multiple sites at these genes (Figure 3A and Table S5). Chromatin immunoprecipitation (ChIP) analyses of endogenous Msx1 in embryonic limb as well as exogenous Msx1 in C2C12 cells confirmed binding to these genomic sites, but not to other sites where Msx1 was not bound in the ChIP-Seq analyses (i.e., negative control sites) (Figure 3B and Figure S2H). Notably, these Msx1 genomic binding sites include regulatory regions such as the CER of MyoD, as well as the −58 kb distal regulatory element of Myf5, which are known homeoprotein-binding elements that control expression of these respective myogenic regulators in the developing limb (Buchberger et al., 2007Buchberger A. Freitag D. Arnold H.H. A homeo-paired domain-binding motif directs Myf5 expression in progenitor cells of limb muscle.Development. 2007; 134: 1171-1180Crossref PubMed Scopus (37) Google Scholar, Goldhamer et al., 1995Goldhamer D.J. Brunk B.P. Faerman A. King A. Shani M. Emerson Jr., C.P. Embryonic activation of the myoD gene is regulated by a highly conserved distal control element.Development. 1995; 121: 637-649Crossref PubMed Google Scholar, Hadchouel et al., 2003Hadchouel J. Carvajal J.J. Daubas P. Bajard L. Chang T. Rocancourt D. Cox D. Summerbell D. Tajbakhsh S. Rigby P.W. Buckingham M. Analysis of a key regulatory region upstream of the Myf5 gene reveals multiple phases of myogenesis, orchestrated at each site by a combination of elements dispersed throughout the locus.Development. 2003; 130: 3415-3426Crossref PubMed Scopus (84) Google Scholar). In the course of these analyses, we noticed a significant overlap between target genes repressed by Msx1 and genes previously identified as Polycomb targets in mouse embryonic stem (ES) cells (34/87 genes; p = 8.6 × 10−6), as well as genes enriched for H3K27me3 in mouse ES cells (27/87 genes; p = 2 × 10−4) (Table S6) (Boyer et al., 2006Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. Wernig M. Tajonar A. Ray M.K. et al.Polycomb complexes repress developmental regulators in murine embryonic stem cells.Nature. 2006; 441: 349-353Crossref PubMed Scopus (2042) Google Scholar, Mikkelsen et al., 2007Mikkelsen T.S. Ku M. Jaffe D.B. Issac B. Lieberman E. Giannoukos G. Alvarez P. Brockman W. Kim T.K. Koche R.P. et al.Genome-wide maps of chromatin state in pluripotent and lineage-committed cells.Nature. 2007; 448: 553-560Crossref PubMed Scopus (3241) Google Scholar). Considering these observations, as well as our previous findings showing that Msx1 genomic binding at the CER is correlated with increased levels of repressive chromatin marks at this site (Lee et al., 2004Lee H. Habas R. Abate-Shen C. MSX1 cooperates with histone H1b for inhibition of transcription and myogenesis.Science. 2004; 304: 1675-1678Crossref PubMed Scopus (197) Google Scholar), we asked whether repressive chromatin marks were enriched on Msx1 target genes. We used chromatin immunoprecipitation coupled with DNA microarray hybridization (ChIP-Chip) to examine the relationship between Msx1 genomic binding and levels of the Polycomb mark, H3K27me3, as well as another repressive mark, H3K9me2, and a mark of active chromatin, H3K4me3 (Figure 3C and Figure S3A). These studies were done using a high-density array containing several hundred developmental regulatory genes, including Msx1 target genes (Supplemental Experimental Procedures) to evaluate the levels of these histone marks relative to Msx1 genomic binding in C2C12 cells expressing or lacking Msx1. These ChIP-Chip binding data revealed an Msx1-dependent overall enrichment of H3K27me3 on repressed target genes (MyoD, net change +34.7; Myf5, net change +23.2; Six1, net change +22.7), but no such enrichment of the H3K9me2 repressive mark or the H3K4me3 activator mark on these repressed genes (Figure 3C and Figure S3A). More generally, the overall abundance of H3K27me3 on repressed Msx1-bound genes versus comparable genes not bound by Msx1 (N = 15/group) revealed a significant enrichment of H3K27me3 in Msx1-expressing cells (median net change +28.6; p = 1.8 × 10−6), which was not the case for H3K9me2 or H3K4me3 (Figure 3D). Interestingly, not only did we observe an increase in the H3K27me3 mark on repressed genes bound by Msx1, we observed a decrease in H3K27me3 levels on genes not bound by Msx1 (median net change −14; p = 1.8 × 10−6). In particular, Dkk1 and En2, which are neither bound nor regulated by Msx1, had reduced levels of H3K27me3 in Msx1-expressing cells (net change, Dkk1 −78.8; En2 −85.8) (Figure S3B). To rule out any potential bias due to selection of genes for these analyses, we examined the genomic loci for two Hox clusters, each spanning >100 kb, and found that the HoxC cluster, which is strongly bound by Msx1, was significantly enriched for H3K27me3 in Msx1-expressing cells (net change +383), whereas the HoxD cluster, which was virtually devoid of Msx1 binding, had reduced levels of H3K27me3 in Msx1-expressing cells (net change −174) (Figure S3C). Taken together, these findings suggest that Msx1 promotes the redistribution of the H3K27me3 mark from genomic regions where Msx1 is not bound to those where it is bound. Notably, enrichment of the H3K27me3 mark was also observed on genes that were activated, rather than repressed by Msx1 (median net change +24.9; p = 8.2 × 10−5) yet, unlike the repressed genes, the activated genes, which are not localized to the nuclear periphery, were also enriched for the H3K4me3 mark (median net change +6.3; p = 0.04) (Figure 3D and Figure S3A). Therefore, although Msx1 binding may promote enrichment of the H3K27me3 mark on target genes, whether the outcome is repression or activation may be influenced by the status of other histone marks, as well as the localization of targets within the nuclear compartment. Further comparison of the ChIP-Seq binding data" @default.
• W2118612858 created "2016-06-24" @default.
• W2118612858 creator A5014280273 @default.
• W2118612858 creator A5015258958 @default.
• W2118612858 creator A5030633309 @default.
• W2118612858 creator A5033587895 @default.
• W2118612858 creator A5057814473 @default.
• W2118612858 creator A5061402622 @default.
• W2118612858 creator A5083124918 @default.
• W2118612858 creator A5086675366 @default.
• W2118612858 date "2011-09-01" @default.
• W2118612858 modified "2023-10-10" @default.
• W2118612858 title "The Msx1 Homeoprotein Recruits Polycomb to the Nuclear Periphery during Development" @default.
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