The Msx1 Homeoprotein Recruits G9a Methyltransferase to Repressed Target in Myoblast Cells

Jingqiang Wang, Cory Abate-Shen* Departments of Urology and Pathology & Cell Biology and Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York, United States of America

Abstract Although the significance of lysine modifications of core histones for regulating expression is widely appreciated, the mechanisms by which these modifications are incorporated at specific regulatory elements during cellular differentiation remains largely unknown. In our previous studies, we have shown that in developing myoblasts the Msx1 homeoprotein represses by influencing the modification status of chromatin at its target genes. We now show that genomic binding by Msx1 promotes enrichment of the H3K9me2 mark on repressed target genes via recruitment of G9a histone methyltransferase, the enzyme responsible for catalyzing this histone mark. Interaction of Msx1 with G9a is mediated via the homeodomain and is required for transcriptional repression and regulation of cellular differentiation, as well as enrichment of the H3K9me2 mark in proximity to Msx1 binding sites on repressed target genes in myoblast cells as well as the developing limb. We propose that regulation of chromatin status by Msx1 recruitment of G9a and other histone modifying enzymes to regulatory regions of target genes represents an important means of regulating the gene expression during development.

Citation: Wang J, Abate-Shen C (2012) The Msx1 Homeoprotein Recruits G9a Methyltransferase to Repressed Target Genes in Myoblast Cells. PLoS ONE 7(5): e37647. doi:10.1371/journal.pone.0037647 Editor: Charalampos Babis Spilianakis, University of Crete, Greece Received December 23, 2011; Accepted April 27, 2012; Published May 22, 2012 Copyright: ß 2012 Wang, Abate-Shen. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by The T.J. Martell Foundation for Leukemia, Cancer and AIDS Research, http://www.tjmartellfoundation.org (C.A.-S.) and by grant HD029446, http://www.nichd.nih. gov (C.A.-S.). Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction nent inhibition of gene expression), and is mediated by another SET domain , Suv39H1 [10,13–15]. Cellular differentiation during development involves the coor- The homeoprotein family comprises one of the major classes of dinated change in expression of many thousands of genes in sequence-specific DNA binding , which regulate gene appropriate spatial and temporal contexts. A principal mechanism expression and cellular differentiation during development. by which this occurs is through modification of the core histones Among its members is the Msx1 homeoprotein, which is expressed (H3, H4, H2A, and H2B) that comprise nucleosomes, which are in diverse spatial and temporal domains during development but the fundamental units of chromatin. There are at least 8 distinct restricted to cells that have not yet begun to differentiate [16,17]. types of histone modifications, of which the most critical for In the myogenic lineage, for example, Msx1 is expressed in transcriptional repression is lysine methylation, the enzymatic myogenic precursors during development as well as in adult transfer of one or more methyl groups from the donor S- myogenic satellite cells (i.e., stem cells), but not in differentiated Adenosylmethionine (SAM) onto the e-nitrogen of lysine [1,2]. myotubes [18–20]. Forced expression of Msx1 in myoblast cells Genome-wide mapping studies using ChIP-Chip and ChIP-Seq inhibits their differentiation [21,22], which is mediated by the have shown that di-methylation of histone H3 at lysine 9 actions of Msx1 as a transcriptional repressor. In particular, Msx1 (H3K9me2) is widely found at repressed genes during develop- represses MyoD, which is a principal regulator of myogenic ment and in embryonic stem cells [3,4] while perturbation of the differentiation, by binding to a key regulatory element, the Core H3K9me2 mark results in a profound change in the repression Enhancer Region (CER) [20,22–25] that regulates the timing of status [5,6]. The relevant histone lysine methyltransferase enzyme MyoD expression in vivo [26]. is G9a, a member of the highly conserved SET domain family, Previously, we have shown that regulation of myoblast which, as part of a complex containing GLP, is responsible for differentiation and repression of MyoD expression by Msx1 is catalyzing the H3K9me2 mark [7–11]. Thus, mutant mice lacking correlated with increased repressor marks at the CER of MyoD G9a are seriously impaired in the H3K9me2 mark and display [25], which include increased tri-methylation of H3K27 embryonic lethality reflecting the consequences of global pertur- (H3K27me3) [23]. We now show that in myoblasts as well as bation of gene repression [12]. Notably, the H3K9me2 mark, the developing limb genomic binding by the Msx1 homeoprotein which is associated with transcriptional repression (regulated promotes enrichment of the H3K9me2 mark on repressed target inhibition of gene expression), is distinct from tri-methylation of genes via recruitment of the G9a histone methyltransferase, the H3K9, which is associated with transcriptional silencing (perma- enzyme responsible for catalyzing this histone mark [7–11]. Interaction of Msx1 with G9a is mediated via the homeodomain

PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e37647 Msx1 Recruits G9a to Target Genes and is required for transcriptional repression and regulation of In particular, we examined the status of H3K9me2 as a cellular differentiation, as well as enrichment of the H3K9me2 consequence of Msx1 expression at several sites on MyoD as well mark in proximity to Msx1 binding sites on repressed target genes. as several other myogenic regulators that are repressed by Msx1 Based on our findings on the recruitment of the H3K9me2 mark, [23], namely Myf5, Angpt1, , Six1, and Snai2 [28–34] (Figure 2A in conjunction with our recently published findings regarding the and 2B). Interestingly, we found that binding by Msx1 resulted in role of Msx1 in recruiting H3K27me3 to target genes [23], we increased levels of H3K9me2 on certain but not all of these describe four distinct categories of Msx1 target genes that are genomic binding sites. For example, Msx1 binding was associated distinguished by differential recruitment of the relevant histone with increased H3K9me2 on the CER regions of MyoD (MyoD-4; methyltransferases. Our findings suggest that an important means P = 4.461025) and the 258 kb site of Myf5 (Myf5-2; of regulating gene expression during development involves the P = 2.461027), both of which are known homeoprotein regulatory differential recruitment of histone modifying enzymes to regula- elements that control expression of these respective genes in vivo tory regions of target genes to influence chromatin status. [26,33,34]. In contrast, H3K9me2 was not increased at the Msx1 binding sites of other target genes, such as Snai2 (Figure 2B). Results Interestingly, for the target gene, Six1, where Msx1 binds to two sites (Six1–3 and Six1–6), only one of the sites (Six1–6) was Msx1 is Associated with H3K9me2 and Binds to G9a enriched for H3K9me2 (Figure 2B); notably, we have found that Following from our previous study showing that repression of the other site (Six1–3) displays an Msx1-dependent enrichment for MyoD by Msx1 is correlated with increased repressor marks at a an alternative methyl mark, H3K27me3 [23]. Furthermore, the key regulatory element, the CER [25] and associated with Msx1-dependent enrichment of H3K9me2 was well correlated increased tri-methylation of H3K27 (H3K27me3) [23], we looked with recruitment of G9a binding to these sites (Figure 2C). In more generally at how Msx1 may influence the modification status particular, ChIP-qPCR analyses revealed that G9a binding was of core histones on target genes in myoblast cells. We found that significantly enriched at relevant Msx1 binding sites, including the Msx1 associates specifically with H3K9me2 but not H3K9me3 in CER (MyoD-4; P=0.001) and the 258 kb site of Myf5 (Myf5-2; co-immunoprecipitation assays using proteins immunopurified P=5.861025), but not on its binding site on Snai2, which is also from C2C12 cells (Figure 1A). Notably, the H3K9me2 mark, not enriched for H3K9me2 (Figure 2C, compare with Figure 2B). which is associated with repression, is distinct from tri-methylation In the case of Six1, G9a was bound at only at the Msx1 site that of H3K9, which is associated with transcriptional silencing [10,13– was enriched for H3K9me2 (Six1–6; Figure 2B). 15]. To investigate whether these findings were also relevant for Considering that Msx1 is associated with H3K9me2, we next endogenous Msx1 in vivo, we performed ChIP analysis using limb asked whether Msx1 interacts with G9a, which is the enzyme that from wild-type or Msx mutant embryos. We found that the levels is responsible for this methyl mark. We found that both exogenous of H3K9me2 were significantly reduced in the Msx mutant versus Msx1 expressed in C2C12 myoblast cells and endogenous Msx1 wild-type limb at the MyoD CER (MyoD-4; P = 1.761025) and expressed in the developing limb interacted strongly with G9a, the 258 kb region of Myf5 (Myf5-2; P = 4.961025) but not at regardless of whether co-immunoprecipitation assays were done Snai2-1 (Figure 3). Notably, similar to our findings in the myoblast using antibodies to pull down Msx1 or G9a (Figure 1B and 1C). cells, levels of H3K9me2 on the Six1 gene were significantly Msx1 also associated with GLP (Figure 1C), which forms a reduced in the Msx mutant versus wild-type limb at the Six1–6, complex with G9a, but it did not interact with Suv39H1 but not at Six1–3 (Figure 3). Taken together, these data suggest (Figure 1C), which is responsible for tri-methylation of H3K9 that, for the endogenous protein in vivo as well as the exogenous and associated with gene silencing rather than repression [14]. protein in myoblast cells, Msx1 recruits G9a to selected genomic Msx1 has multiple functional domains that mediate interactions targets where it promotes enrichment of the H3K9me2 repressive with protein partners, DNA binding, transcriptional repression mark in the vicinity of its binding. and/or sub-nuclear location [23–25]. Analyses of truncated Msx1 proteins lacking these various functional domains revealed that the Association of Msx1 with G9a is Required for Regulation homeodomain of Msx1 is the primary domain required for its of Myoblast Differentiation interaction with G9a (Figure 1D and 1E). In particular, a We next investigated the consequences of Msx1 association with truncated Msx1 protein lacking the homeodomain [Msx1(1– G9a for regulation of myogenic differentiation by evaluating the 172)] did not interact with G9a, while various other truncated consequences of G9a knock-down in C2C12 myoblast cells. We Msx1 proteins that contained the homeodomain but lacked for used two independent G9a siRNAs and verified their efficacy and example domains required for repression Msx1(139–303); specificity in C2C12 cells using q-PCR to evaluate G9a mRNA Msx1(1–239); and Msx1(1–271)] interacted with G9a, albeit with levels and Western blotting to detect levels of G9a protein or levels varying degrees of efficacy (Figure 1D and 1E). Notably the of histone marks (Figure 4A-C); for most assays, only one siRNA is homeodomain is required for DNA binding by Msx1 but also shown. The consequences of G9a knock-down for regulation of mediates interactions of Msx1 with other protein partners differentiation by Msx1 were evaluated by the appearance of [16,20,23,27]. Taken together, these findings indicate that Msx1 myotubes and by Western blot detection of markers of terminal associates with G9a histone methyltransferase via the homeodo- muscle differentiation, namely myosin heavy chain (MHC) and main, although the important caveat to these studies in the (Figure 4D and 4E). Without exogenous Msx1 possible influence of these altered domains on the structure of the expression, C2C12 cells (Vector) were differentiated by 3 days protein overall. after induction regardless of whether they expressed the control or G9a siRNA as evident by myotubes formation (Figure 4D, top Msx1 Genomic Binding Associates with Enrichment of panel) and expression of MHC and Myogenin (Figure 4E, left the H3K9me2 Repressive Mark panel). However, as we have shown previously [21,24,25], Msx1 Having established that Msx1 interacts with G9a, we next asked completely abrogates differentiation of C2C12 cells, as evident by whether genomic binding by Msx1 is associated with increased the absence of myotubes (Figure 4D, left bottom panel) and lack of levels of H3K9me2 on its repressed target genes in myoblast cells. expression of MHC and Myogenin (Figure 4E, right panel). In

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Figure 1. Msx1 binds to G9a/GLP via the homeodomain and the C-terminal region. (A) Co-immunoprecipitation assays were done using C2C12 cell protein extracts expressing Flag-Msx1 and immunoprecipitated with anti-Flag followed by immunoblotting for the indicated histone marks. (B) 293T cells were transfected with the indicated expression plasmids. Proteins were immunoprecipitated with Flag antibodies and immunoblotted with anti-Flag or Anti-Myc, as indicated. (C) (Left) C2C12 cell protein extracts expressing Flag-Msx1 were immunoprecipitated with anti-Flag following by immunoblotting with antibodies to detect the indicated proteins. (Right) Limb extracts (11.5 dpc) expressing endogenous Msx1 were immunoprecipitated with anti-Msx1 antibody (4F11) following by immunoblotting with antibodies to detect the indicated proteins. (D) Truncated Msx1 proteins expressed in 293T cells were immunopurified with anti-Flag followed by immunoblotting to detect G9a. Shown is the quantitative analyses of G9a, normalize to G9a input and Msx1 input. In each immunoprecipitation assays, 1 mg of protein was used and input was 1% of the total protein. (E) Schematic representation of Msx1 and truncated derivatives showing a summary of data. doi:10.1371/journal.pone.0037647.g001 contrast, depletion of G9a reverted these inhibitory effects of Msx1 C2C12 myoblast cells. Depletion of G9a significantly reduced on myoblast differentiation, as evident from the appearance of Msx1 binding to genomic sites of repressed genes in myoblast cells myotubes (Figure 4D, right bottom panel) and expression of MHC in ChIP assays, as exemplified for the CER (MyoD-4; and Myogenin (Figure 4E, right panel). P = 1.161027) and the 258 kb element of Myf5 (Myf5-2; We quantified the differentiation status of the C2C12 cells at 2 P = 3.861024) (Figure 5A). Interestingly however, analyses of days after induction of differentiation using an antibody for MHC Msx1 binding in vitro in gel shift assays showed that G9a knock- to detect the MHC+ cells and determine the myogenic index [35]. down did not inhibit binding by Msx1 to these genomic target In vector cells, depletion of G9a resulted in a higher myogenic sequences, namely the MyoD CER (MyoD-4) or Myf5-1 differentiation compared with those expressing the control siRNA (Figure 5B). Notably, we have previously shown a distinction (Figure 4F and 4G), consistent with a role G9a in myogenic between binding of Msx1 in vitro and its ability to bind with target differentiation as reported recently [35]. However, in Msx1- sequences in vivo, which is dependent on interactions with its expressing cells, depletion of G9a restored the myogenic differen- protein partners in vivo [23,24]. Thus, while G9a may not directly tiation nearly the levels without Msx1 (Figure 4F and 4G). These affect the affinity of Msx1 for its binding sites (Figure 5B), findings demonstrate that G9a is essential for Msx1 to inhibit considering that depletion of G9a impaired in Msx1 ability to myogenic differentiation in these myoblast cells. interact with genomic binding sites in vivo (Figure 5A), G9a may affect the ability of Msx1 to access authentic target sites in Association of Msx1 with G9a is Required for myoblast cells. Transcriptional Repression Notably, the diminished binding of Msx1 to repressed genes in We next investigated the consequences of Msx1 association with myoblast cells as a consequence of G9a knock-down was G9a for transcriptional repression following knock-down of G9a in accompanied by a partial abrogation of repression of its target

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Figure 2. Msx1 genomic binding associates with enrichment of the H3K9me2 repressive mark in myoblast cells. (A) Diagram of six Msx1 repressed target genes [23] showing the positions of Msx1 binding sites and known regulatory regions as well as their overlap; also shown is a negative control site. DRR: Distal Regulatory Region. (B) ChIP-qPCR analyses showing the relative levels of H3K9me2 at Msx1 genomic binding sites in C2C12 cells. ChIP data are expressed as relative enrichment of H3K9me2 normalized to input in C2C12 cells expressing or lacking Msx1. (Inset) ChIP

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data expressed as fold enrichment of H3K9me2 in C2C12 cells expressing Msx1 versus the control cells lacking Msx1 (and normalized to input). (C) ChIP-qPCR assays were done using C2C12 cells expressing Flag-Msx1 or a control vector to evaluate binding of G9a to the indicated Msx1 target sequences. ChIP data are expressed as relative enrichment of G9a binding normalized to input in C2C12 cells expressing or lacking Msx1. (Inset) ChIP data are expressed as fold enrichment of G9a binding in C2C12 cells expressing Msx1 versus the control cells lacking Msx1 (and normalized to input). In B and C, the * indicate the following: ***P,0.0001, **P,0.001, *P,0.01. doi:10.1371/journal.pone.0037647.g002 genes, including MyoD, Angpt1, Myc and Six1 following knock-down affected the H3K27me3 mark on target genes. Since depletion of of G9a in Msx1-expressing but not the cells without Msx1 (Vector) G9a did not reduce the total level of H3K27me3 (Figure 4C), we (Figure 5C and 5D). The notable exception was Myf5, which was asked whether Msx1 association with G9a is required for the increased in expression at the protein and mRNA levels in the Msx1-dependent redistribution of H3K27me3 on its genomic vector cells (lacking exogenous Msx1) following G9a knock-down bound sites. In the presence of control siRNA, Msx1 promotes (Figure 5C and 5D). Although depletion of G9a in C2C12 cells enrichment of the H3K27me3 repressive mark on its genomic does not affect MyoD mRNA and protein levels (see Figure 5C sites, such as MyoD-2, MyoD-4, Myf5-1 and Myf5-2 [23] (Figure 7A). and 5D), it may inhibit its transcriptional activity, in turn leading In contrast, genes that were neither bound nor regulated by Msx1, to up-regulation of Myf5 (Figure 5C and 5D, top panel), which is such as Dkk1, En2, and Irx1 had reduced levels of H3K27me3 in consistent with a recent study showing that G9a interacts with Msx1-expressing C2C12 cells with control siRNA [23] (Figure 7B). MyoD to constrain its transcriptional activity [35]. Finally, However in Msx1 expressing C2C12 cells, knock-down of G9a following G9a knock-down in Msx1 expressing C2C12 cells, we dramatically reduced the H3K27me3 mark on Msx1 genomic sites observed a significant reduction in the H3K9me2 mark, but not (Figure 7A), while it resulted in reversed the H3K27me3 reduction the H3K9me3 mark, on Msx1 target genomic binding sites such as at Dkk1, En2, and Irx1 associated with exogenous Msx1 expression CER (MyoD-4; P = 7.861025) and the 258 kb region of Myf5 (Figure 7B). Furthermore, since the association of Msx1 with the (Myf5-2, P = 4.261024), as well as on the genes that are regulated nuclear periphery is associated with the redistribution of the by G9a but not Msx1 such as MageA2, Major satellite, and Wfdc15a, H3K27me3 mark [23], we asked whether G9a affects the sub- but not on a gene (GAPDH) that is not regulated by either Msx1 or nuclear location of Msx1. Strikingly, we found that depletion of G9a (Figure 6A–H) [6,12]. Taken together, these findings G9a dramatically interrupted Msx1 nuclear periphery localization demonstrate that the interaction of Msx1 with G9a in myoblast (Figure 7C). These findings suggest that Msx1 association with cells is essential for Msx1 to bind and repress target genes in cells G9a contributes to the Msx1-dependent redistribution of and for enrichment of the H3K9me2 repressive mark at specific H3K27me3 genomic bound sites by affecting the nuclear genomic binding sites. periphery localization of Msx1.

The G9a associated with Redistribution of H3K27me3 by Discussion Msx1 to Genomic Sites The impact of Msx1 on cellular differentiation during develop- Finally, since we have shown previously that repression by Msx1 ment is dependent on its ability to repress the expression of is correlated with increased tri-methylation of H3K27 regulatory genes in specific cellular contexts, such as occurs for (H3K27me3) [23], we asked whether Msx1 association with G9a MyoD in cells of the myogenic lineage [22,24,25]. Our current

Figure 3. Msx1 genomic binding associated with enrichment of the H3K9me2 repressive mark in the developing limb. ChIP-qPCR analyses show relative levels of H3K9me2 at Msx1 genomic binding sites in Msx1; Msx2 conditional mutant versus wild-type limb (13.5 dpc). ChIP data are expressed as relative enrichment of the H3K9me2 mark normalized to input. (inset) ChIP data expressed as fold enrichment in wild-type embryonic limb versus Msx1; Msx2 conditional mutant embryonic limb (and normalized to input). The * indicate the following: ***P,0.0001, **P,0.001, *P,0.01. doi:10.1371/journal.pone.0037647.g003

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Figure 4. Association of Msx1 with G9a is required for regulation of myoblast differentiation. (A) Quantitative PCR to determine the level of G9a mRNA following knock-down by siRNA, mRNAs were prepared 48 hours after transfection with the indicated siRNA. (B) Western blot assay for efficiency of G9a knock-down, C2C12 cells were transfected with the indicated siRNA, extracts prepared 72 hours after transfection and analyzed by western blotting for the indicated proteins. (C) Western blot assay for histone marks. C2C12 cells were transfected with the indicated siRNA, extracts prepared 72 hours after transfection and analyzed by western blotting for the indicated histone marks. (D) Differentiation assay of C2C12 cells expressing (+) or lacking (-) Msx1 along with the control or G9a siRNA. Micrographs of C2C12 cells show the absence of myotubes in Msx1-expressing cells but not in cells also expressing the G9a siRNA on Day 3 after differentiation. (E) Western blots analyses of markers of terminal muscle differentiation, MHC and Myogenin on Day 3 after differentiation. (F) Immunofluorescence assays done on Day 2 after differentiation as detected using antibody for MHC and the nuclear marker TOPRO3. The scale bars represent 50 mm. (G) Quantitative analyses of myogenic index on Day 2 after differentiation. The percentage of MHC+ cells in vector cells with control siRNA was given a value of 100%. In A and G, the * indicate the following: ***P,0.0001, **P,0.001, *P,0.01. doi:10.1371/journal.pone.0037647.g004 findings suggest that these activities of Msx1 are mediated by its histone methyltransferase, Ezh2, which leads to enrichment of its ability to directly influence the methylation status of nucleosomes respective histone mark, namely H3K27me3, on certain target on selected regulatory elements to which it is bound in myoblast genes (Figure 8; Table S1) [23]. In fact, we can identify 4 scenarios cells (Figure 8). Our findings further indicate that this reflects, in in which Msx1 differentially recruits histone methyltransferases to part, recruitment of the G9a histone methyltransferase to these target genes to regulate their expression. In particular, in the first regulatory sites, which in turn promotes methylation of core case Msx1 recruits both H3K9me2 and H3K27me3 repressive histones in the vicinity of Msx1 binding (Figure 8; Table S1). marks at the same binding site, as is the case for target genes, such Notably, recruitment of the G9a histone methyltransferase by as, MyoD, Myf5, Myc, and Angpt1 [18,26,28,31,33,34]. In the Msx1 is mediated via the homeodomain, which is the defining second scenario, Msx1 recruits both H3K9me2 and H3K27me3 feature of this family of sequence specific developmental regulators repressive marks to target genes, but at the different sites. For [16,20,22,24,25]. Implicit in this observation is that other instance, Msx1 binds to two sites on Six1 (Six1–3 and Six1–6), one homeoproteins may also recruit methyltransferase enzymes to of the sites (Six1–6) is enriched for H3K9me2 while the other site target genes as a means of regulating their expression. (Six1–3) displays an Msx1-dependent enrichment for H3K27me3 However, G9a is not recruited to all Msx1 target genes. [23,29,30]. In the third case, Msx1 only recruits the H3K27me3 Moreover, Msx1 also interacts via the homeodomain with another mark, as in the case for Snai2, Met, and Id3 [32,36,37]. The final

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Figure 5. Association of Msx1 with G9a is required for transcriptional repression. (A) ChIP-qPCR analyses showing relative Msx1 binding in C2C12 cells expressing or lacking Msx1 as well as a control or G9a siRNA. ChIP data are expressed as fold enrichment of Msx1 binding in C2C12 cells expressing Msx1 versus control cells lacking Msx1. (B) Gel shift analyses were done using nuclear extracts from C2C12 cells expressing Flag-Msx1 as well as the control or G9a siRNA with DNA sequences corresponding to the MyoD CER (MyoD-4) and Myf5-1. The * indicate the protein-DNA binding bands, the arrowhead indicates the free probes. (C) Expression levels of Msx1 target genes in Msx1-expressing or lacking Msx1 C2C12 cells also expressing the control or G9a siRNA. Data are expressed as the fold change of mRNA relative to that of the control siRNA cells. The * indicate the following: ***P,0.0001, **P,0.001, *P,0.01. (D) Western blots analyses showing levels of MyoD and Myf5 protein following by G9a knock-down in Msx1-expressing or lacking Msx1 C2C12 cells. doi:10.1371/journal.pone.0037647.g005

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Figure 6. Binding analyses of H3K9 methyl marks in G9a knock-down cells. ChIP-qPCR assays were done using C2C12 cells expressing Flag- Msx1 as well as the control or G9a siRNA using anti-H3K9me2 or anti-H3K9me3 or control IgG, as indicated. ChIP data are expressed as relative enrichment normalized to input in control siRNA cells or G9a knock-down cells. doi:10.1371/journal.pone.0037647.g006 case is target genes that are bound by Msx1, but are not enriched GFP plasmids for retroviral gene transfer. Flag-tagged G9a was for either H3K9me2 or H3K27me3, such as Clcn3 and Fgf7 generated using PCR amplification and cloned into BamH I – [38,39]. Xho I sites of pcDNA3. The complete sequences of all PCR- Implicit in this description is the important but yet unanswered amplified constructs were confirmed. question regarding how Msx1 recruits multiple histone methyl- transferases to distinct target genes in specific spatial contexts. Cell Culture Analyses Therefore, Msx1 may interact with multiple histone methyltrans- Cell culture studies were done using human 293T cells ferases to influence the expression of target gene in dynamic spatial (ATCC) or mouse C2C12 myoblast cells (ATCC). Cells were contexts. Furthermore, since the region of methyltransferases maintained in DMEM supplemented with 10% fetal bovine recruitment is the homeodomain, a conserved motif, our findings serum in humidified atmosphere with 5% CO2 at 37uC. For raise the possibility that other homeoproteins may also function by myoblast differentiation assays, undifferentiated C2C12 cells promoting the recruitment of histone modifying enzymes to target were grown in media containing 10% fetal bovine serum, and genes. Thus, our findings demonstrate a novel means by which differentiation was induced by shifting cells to media containing homeoproteins can regulate gene expression during development 2% horse serum [21,24,25]. Transient transfection was per- by interacting with histone modifying enzymes to directly formed using Lipofectamine 2000 reagent (Invitrogen). For influence the chromatin status of target genes. retroviral gene transfer, replication-defective retroviruses were made in ecotropic Phoenix retroviral packaging cells (ATCC) by Materials and Methods transfection of the relevant pLZRS-IRES-GFP plasmid deriva- tives using Lipofectamine 2000 reagent (Invitrogen). C2C12 Plasmids myoblast cells were seeded 1 day before infection and infected Most of the expression plasmids used in this study has been with viral supernatants for two consecutive days. For siRNA, described previously [24,25]. As indicated, pcDNA3 expression C2C12 cells were first infected with viruses expressing Msx1 or plasmids were used for transient transfection and pLZRS-IRES- the empty vector and then transfected with siRNA against G9a

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Figure 7. G9a is required for Msx1-induced redistribution of H3K27me3 by Msx1 and localization of Msx1 at the nuclear periphery in C2C12 myoblasts. (A) ChIP-qPCR analyses of H3K27me3 mark on Msx1 target genes in C2C12 cells lacking Msx1 or expressing exogenous Msx1 also expressing the control or G9a siRNA. ChIP data are expressed as relative enrichment of the H3K27me3 mark normalized to input. (B) ChIP-qPCR analyses of H3K27me3 mark on genes not bound by Msx1 in C2C12 cells lacking Msx1 or expressing exogenous Msx1 also expressing the control or G9a siRNA. ChIP data are expressed as relative enrichment of the H3K27me3 mark normalized to input. (C) Immunofluorescence assays were done on C2C12 cells expressing exogenous Msx1 together with the G9a siRNA or a control siRNA and detected using antibodies for Msx1 or by detection of the nuclear marker TOPRO3. Quantitative analyses of nuclear localization for Msx1 using ImageJ show representative data from 3 independent assays, each counting a minimum of 20 cells per variable. The scale bars represent 5 mm. doi:10.1371/journal.pone.0037647.g007

(Ambion) using the Lipofectamine RNAiMAX reagent (Invitro- Analyses of Msx1 Mutant Embryos gen) according to the manufacturer’s recommendations. For the All experiments using animals were performed according to control siRNA using SilencerH Select negative control #1 siRNA protocols approved by the Institutional Animal Care and Use (Ambion). The sequences of G9a siRNA (Ambion) used in these Committee at Columbia University Medical Center. The Msx1 studies are provided in Table S2. Quantitative analyses of mutant were performed using a compound Msx1; Msx2 differentiation of the G9a knock-down C2C12 cells were done conditional allele [40] crossed to a ubiquitously-expressed by immunofluorescence staining with MHC antibody (see the tamoxifen-inducible Rosa26CreERT2 allele [41] to generate mice below). Quantitative analyses of myogenic index were preformed of the genotype Rosa26CreERT2/+; Msx1lox/lox; Msx2lox/lox. Targeted on Day 2 after differentiation. Myogenic index calculation was deletion was induced by delivery of tamoxifen in corn oil done as described in [35]. At least 3 independent assays were (2 mg/40 grams; Sigma-Aldrich) by oral gavage at embryonic done per variable, counting a minimum of 500 cells per day 9.5 (9.5 days post-coitum, dpc); targeted deletion in the tissue variable for each independent assay. The percentage of MHC+ of interest (i.e., the limb) was confirmed by PCR analyses. The cells in vector cells with control siRNA was given a value of analyses described herein focused on mid-gestation embryos, 100%. from days 10.5 to 13.5 dpc, at which developmental stages the limb buds are maturing and Msx1 expression is robust in the limb mesenchyme [16,42]. Embryos were collected from timed mating with noon on the day of the plug considered to be

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gene. The average values are given as the mean 6 SD. The primer sequences for real-time PCR using in this study were provided in Table S2.

Chromatin Immunoprecipitation (ChIP) ChIP analysis was performed as described previously [24,25]. Briefly, C2C12 cells expressing or lacking Msx1 and/or siRNA for G9a were cross-linked using 1% formaldehyde and genomic DNA was sonicated and then the relevant protein-DNA complexes were isolated by immunoprecipitation. ChIP analysis from limb, the cross-linked limb tissue was sonicated and then the relevant protein-DNA complexes were isolated by immunoprecipitation. The antibodies were used for ChIP assays were provided in Table S3. Quantitative real-time PCR was performed in triplicate using SYBR green reagent (Qiagen) using a Realplex2 machine (Eppendorf). At least three independent experiments were done for each assay. Primer sequences for ChIP-qPCR analyses were provide in Table S2.

Immunoprecipitation and Western Blotting Analyses For Western blotting, C2C12 cells were lysed in RIPA buffer and proteins were analyzed using ECL plus Western Blotting Detection (GE Healthcare) by indicated antibodies (Table S3). For immunoprecipitation assays, C2C12 cells were lysed in RIPA buffer and proteins were immunoprecipitated by addition of antibodies [24,25]. Where indicated, nuclear extracts from embryonic limbs were obtained by homogenization in hypotonic buffer (20 mM HEPES [pH 7.4], 5 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 1 mM DTT, and protease inhibitor cocktail) to isolate nuclei followed by extraction of nuclear proteins in buffer BC1000 (25 mM HEPES [pH 7.9], 10% glycerol, 0.2 mM EDTA, 1000 mM KCl, protease inhibitor cocktail, and PMSF) containing 0.1% NP40. The samples were then sonicated, centrifuged at 16,000 g, and then dialyzed against BC200 Figure 8. Working model. As described in the text, we have proposed that binding of the Msx1 homeoprotein to specific target (25 mM HEPES [pH 7.9], 10% glycerol, 0.2 mM EDTA, genes brings G9a and/or Ezh2 to the regulatory regions of these genes 200 mM KCl, protease inhibitor cocktail, and PMSF) containing to influence histone modifications. According G9a and Ezh2 bound 0.1% NP40. Immunoprecipitations were done in BC200 contain- status, the Msx1 bound and down-regulated target genes were ing 0.1% NP40 and immunoprecipitated proteins were analyzed categorized in 4 categories. (A) Category I, Msx1 brings G9a and Ezh2 using ECL plus Western Blotting Detection (GE Healthcare). The to the same site on target genes. (B) Category II, Msx1 brings G9a and antibodies were used for immunoprecipitation and immunopre- Ezh2 to the same target genes but at different sites. (C) Category III, Msx1 only brings Ezh2 to the Msx1 bound site on target genes. (D) cipitation Western blotting were provided in Table S3. Category IV, Msx1 do not brings ether G9a or Ezh2 to the target genes, but Msx1 may brings other factors to Msx1 bound site to repress target Gel Shift Assay genes expression. The probe of MyoD CER (MyoD-4) and Myf5-1 were doi:10.1371/journal.pone.0037647.g008 generated fragment from ChIP DNA by using PCR method with ChIP-qPCR primer sets (Table S2). For nuclear extracts, the embryonic day 0.5; embryos were genotyped from yolk sac C2C12 cells were homogenized in hypotonic buffer. The nuclei DNA. For ChIP assays, freshly dissected limbs were collected in were incubated for 10 min on ice and then pelleted by PBS with protease inhibitor cocktail and disrupted using a centrifugation at 8,000 g, and resuspended in BC1000 buffer Polytron homogenizer. Tissue extracts were cross-linked with containing 0.1% NP40 to extract nuclear proteins. The samples 1% formaldehyde for 30 minutes at room temperature, cross- were then sonicated, centrifuged at 16,000 g, and dialyzed against linking was stopped with 0.1 M Glycine, and cross-linked limb BC200 containing 0.1% NP40. The binding reaction was perform was collected by centrifugation and processed for ChIP-qPCR in 16 binding buffer (10 mM Tris-HCl pH 7.5, 5 mM NaCl, analyses as below. 0.7 mM MgCl2, 0.1 mM EDTA, 5% Glycerol, 0.05% NP-40, and 50 mg/ml BSA, 2.5 mM DTT) with 2 mg poly d(I-C), 50 ng probe, Real-time PCR for Gene Expression and 5 mg of nuclear protein from C2C12 cells expressing Flag- Real-time PCR was done using RNA isolated from C2C12 cells, Msx1 as well as the control or G9a siRNA in total volume of 20 ml. using Trizol reagent (Invitrogen), and purified using an RNeasy kit Binding was done at room temperate for 20 min. Protein-DNA (Qiagen). First strand cDNA was synthesized using SuperScript III binding complexes were resolved by electrophoresis. After finished kit (Invitrogen) and quantitative real-time PCR was performed running the gel, the gel was stained by SYBR Safe DNA gel stain 2 using SYBR green reagent (Qiagen) in the Realplex machine (Invitrogen) and the digital imagines were captured by CCD (Eppendorf). Expression values were normalized to GAPDH.At camera. least three independent experiments were performed for each

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Immunofluorescence Analyses given as the mean 6 SD. Comparison of differences among the Immunofluorescence analyses were done as described previous- groups was carried out by Student’s t-test. Significance was defined ly [23]. Briefly C2C12 cells seeded on 1-well BD FalconTM as p,0.01. (***p,0.0001, **p,0.001, *p,0.01). CultureSlide and transfected with the indicated Msx1 plasmids and, where indicated, also with the siRNA for G9a. Cells were Supporting Information fixed in 4% PFA in PBS with 1% sucrose and permeabilized by incubation in an isotonic solution, 0.5% Triton X-100 (10% Table S1 Summary of ChIP data for Msx1 bound and down-regulated target genes. sucrose, 50 mM NaCl, 6 mM MgCl2, 20 mM HEPES [pH 7.2], 0.5% Triton X-100). After blocking with 1% BSA (bovine serum (DOCX) albumin) in PBS, cells were incubated for 1.5 hours at room Table S2 List of primers used for ChIP-qPCR, RT-PCR temperature with primary antibodies. Following incubation with and RNA interference. primary antibodies, samples were washed in PBS containing 0.1% (DOCX) Tween 20 followed by incubation for 1 hour with TOPRO 3 and AlexaFluor 488 and/or AlexaFluor 555 secondary antibodies Table S3 List of antibodies used in this study. (Molecular Probes). Immunofluorescence staining was visualized (DOCX) using a Leica TCS SP5 inverted confocal microscope equipped with argon/krypton and helium/neon lasers capable of excitation Acknowledgments wavelengths 488, 555, and 642 nm. Details of primary antibodies We thank Drs. Danny Reinberg and Raphael Margueron for providing are provided in Table S3. The Msx1 sub-nuclear localization was reagents and many helpful discussions. We are grateful to Dr. Robert quantified using ImageJ (http://rsb.info.nih.gov/ij/) [43]. A line Maxson for providing the Msx1 and Msx2 conditional mice, and Dr. Yoichi was drawn from the nuclear periphery to the nuclear center, and Shinkai for providing G9a antibodies. We thank members of the Abate- along this line, the fluorescence intensity was recorded; the pixel Shen laboratories and Dr. Michael Shen for many discussions and helpful values versus radial position were used to generate the quantitative comments on the manuscript. plot. Author Contributions Statistical Analysis Conceived and designed the experiments: JW CAS. Performed the At least three independent experiments were performed for experiments: JW. Analyzed the data: JW CAS. Wrote the paper: JW CAS. each assay. The average values of the parallel experiments are

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