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Sequential association of myogenic regulatory factors and E proteins at muscle-specific genes

Sequential association of myogenic regulatory factors and E proteins at muscle-specific genes Background: Gene expression in skeletal muscle is controlled by a family of basic helix-loop-helix transcription factors known as the myogenic regulatory factors (MRFs). The MRFs work in conjunction with E proteins to regulate gene expression during myogenesis. However, the precise mechanism by which the MRFs activate gene expression is unclear. In this work, we sought to define the binding profiles of MRFs and E proteins on muscle- specific genes throughout a time course of differentiation. Results: We performed chromatin immunoprecipitation (ChIP) assays for myogenin, MyoD, Myf5 and E proteins over a time course of C C differentiation, resulting in several surprising findings. The pattern of recruitment is 2 12 specific to each promoter tested. The recruitment of E proteins often coincides with the arrival of the MRFs, but the binding profile does not entirely overlap with the MRF binding profiles. We found that E12/E47 is bound to certain promoters during proliferation, but every gene tested is preferentially bound by HEB during differentiation. We also show that MyoD, myogenin and Myf5 have transient roles on each of these promoters during muscle differentiation. We also found that RNA polymerase II occupancy correlates with the transcription profile of these promoters. ChIP sequencing assays confirmed that MyoD, myogenin and Myf5 co-occupy promoters. Conclusions: Our data reveal the sequential association of MyoD, myogenin, Myf5 and HEB on muscle-specific promoters. These data suggest that each of the MRFs, including Myf5, contribute to gene expression at each of the geness analyzed here.. The dynamic binding profiles observed suggest that MRFs and E proteins are recruited independently to promoters. Background birth. The myogenin-nullmicehavemyoblasts,but very The entire process of skeletal muscle differentiation is few muscle fibers. This suggests that myogenin is not controlled by four highly related basic helix-loop-helix required for the specification of skeletal muscle, but is (bHLH) proteins referred to as the myogenic regulatory required for the later stages of myofiber fusion. factors (MRFs). The MRFs have distinct but overlapping MyoD and myogenin have been shown to bind highly patterns of gene expression during muscle development overlapping gene sets, although certain genes appear to [1]. Gene knockouts of each factor in the mouse have be selective for either factor [6,7]. However, the high revealed that each MRF has a unique role in skeletal degree of overlap in the binding patterns suggests that muscle differentiation. Myf5, Myf6 (also known as the majority of genes utilize both factors to activate gene MRF4) and MyoD are not required for viability, although expression. Previous work has shown that certain genes require the sequential expression of both MyoD and each mutant has a distinct phenotype [2]. In the com- bined absence of Myf5, Myf6 and MyoD, myoblasts are myogenin to activate gene expression [7]. The present not specified and no skeletal muscle forms, resulting in a work suggests that the activation of specific targets lethal phenotype [3]. Myogenin is the only MRF singly requires MyoD and its associated chromatin-modifying required for viability [4,5]. Mice heterozygous for the null activities before myogenin can activate transcription. allele appear normal, while mice lacking myogenin die at Why MyoD cannot activate transcription without myogenin on these genes is still unknown. Recent work on Myf5 has revealed unexpected roles * Correspondence: jdavie@siumed.edu Department of Biochemistry and Molecular Biology, Southern Illinois for this factor in adult animals. As mentioned above, University School of Medicine, 1245 Lincoln Drive, Carbondale, IL 62901, USA © 2011 Londhe and Davie; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Londhe and Davie Skeletal Muscle 2011, 1:14 Page 2 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 Myf5 functions as a determination gene in early myo- widely used cell culture model for myoblast differentia- genesis. The role of Myf5 in later stages is unclear. In tion. These binding profiles were correlated with the the absence of MyoD, Myf6 or myogenin, Myf5 is levels of mRNA present in the cells, the levels of RNA unable to promote differentiation from myoblasts [8]. polymerase II (RNAP II) occupancy and histone H3 This finding suggests that Myf5 functions only in mus- acetylation present at the promoters. We show several cle progenitor cells (MPCs) and myoblasts. However, novel findings. Surprisingly, we have found that the pat- recent work has shown that Myf5-null mice exhibit tern of recruitment is unique to each gene, although impaired muscle regeneration with a significant increase some common features arise. As others have observed, in muscle fiber hypertrophy and a delay in differentia- we saw an early association of MyoD with most of these tion [9]. However, satellite cell numbers were not signif- genes. In a cooperative pattern, myogenin then binds icantly altered in the Myf5-null animals, although a many of these promoters following MyoD. Unexpect- modest impaired proliferation was observed under some edly, we found that Myf5 is also associated with genes conditions in vitro. This work highlights the questions expressed late in differentiation and often colocalizes still remaining about the roles of the MRFs at distinct with myogenin. We show that this colocalization also stages in myogenesis. occurs in vivo at a late embryonic time point. The bind- All bHLH transcription factors function as either ing of each of the MRFs is transient. We also show that homodimers or heterodimers. The bHLH transcription the occupancy of the E proteins is transient and that the factors are loosely grouped into several categories: the occupancy often peaks concurrently with the peak in widely expressed E proteins, including the E2A gene pro- gene transcription. While the E2A gene products could ducts E12 and E47, HEB, E2-2 and Daughterless, are in be detected on a few genes in our study in proliferating the class I category and the MRF family is included in the cells, HEB does appear to be the dominant E protein tissue-specific class II category. Class II bHLH proteins used during differentiation. At the genes occupied by form weak homodimers and preferentially heterodimer- E12/E47 in early myogenesis, we detected a switch in ize with E proteins [10]. Prior in vitro experiments have occupancy for HEB during differentiation. Taken demonstrated that the class II MRFs form avid heterodi- together, our data suggest new models for the recruit- mers with class I E proteins, but homodimerize poorly in ment of MRFs and E proteins and support a novel role the presence of DNA sites [11-14]. Thus, it is thought for Myf5 during differentiation. that the MRFs function as heterodimers with ubiquitous E proteins. The E proteins suggested to be involved in Results skeletal muscle differentiation are the E2A gene products Time course of MRF and E protein expression E12 and E47, as well as HEB. Recent work has suggested To initiate this work, we first characterized the available that HEB may be the primary E protein that regulates antibodies for these studies and confirmed MRF and E skeletal muscle differentiation [15]. protein expression patterns over a time course of differ- The MRFs all bind the canonical E-box consensus entiation. We tested well-characterized antibodies for sequence, CANNTG. Genome-wide binding studies have MyoD and myogenin [17,18]. We found that antibodies revealed that both MyoD and myogenin preferentially against MyoD and myogenin immunoprecipitated the bind E boxes with a consensus sequence of CASCTG target protein (data not shown) and did not recognize (International Union of Pure and Applied Chemistry any of the other MRFs (Additional file 1 Figure S1). For nomenclature http://www.iupac.org), where S represents Myf5, we tested commercially available antibodies for G or C [7,16]. The site recognized at the highest fre- their ability to recognize and immunoprecipitate Myf5 quency is CAGCTG. The sequences flanking the E box specifically. We identified one antibody that immunopre- also make important contributions to the binding affinity cipitated Myf5 exclusively (Additional file 1 Figure S2), and contribute to the overall consensus sequence and this antibody was used for all the studies presented. elements determined for MyoD and myogenin [11,16]. We did note that the antibody does recognize recombi- Given the high degree of overlap detected in the gen- nant MyoD by using Western blot analysis, but we could ome occupancy of MyoD and myogenin, we were inter- not immunoprecipitate this protein (Additional file 1 Fig- ested in understanding the binding profile of these ure S2). For the E proteins, we used antibodies raised factors over a time course of differentiation. We were against HEB and the E2A gene products E12 and E47. also interested in the binding profile of Myf5, as binding TheHEB antibodyrecognized E12/E47 on the basis of data for this factor during differentiation have not been Western blot analysis, but immunoprecipitated HEB spe- reported. We also sought to compare the DNA-bound cifically (Additional file 1 Figure S3). The antibody profiles of the MRFs with the E proteins. Thus, we against E12/E47 did not recognize or immunoprecipitate initiated a temporal analysis of the binding of MyoD, recombinant HEB (Additional file 1 Figure S4). Next, we Myf5, myogenin and the E proteins in C examined the expression profile of the MRFs and HEB C cells, a 2 12 Londhe and Davie Skeletal Muscle 2011, 1:14 Page 3 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 over an extended time course of C C differentiation [19-25]. We also chose the fast-twitch skeletal muscle 2 12 (Figure 1). As previously observed, MyoD and Myf5 were troponin I, type 2 (Tnni2)and leiomodin 2 (Lmod2) expressed in proliferating myoblasts. MyoD levels genes, as these have been characterized as myogenin- increased upon differentiation, but then rapidly decreased dependent targets in embryonic skeletal muscle during after two days of differentiation. Unexpectedly, Myf5 was embryogenesis, and the promoter proximal MRF bind- expressed throughout the entire time course. Myogenin ing sites are known [26]. We also chose titin cap was not detectable in proliferating cells, but was rapidly (Tcap), also known as telethonin.Wehaverecently upregulated upon differentiation. HEB was also expressed characterized the promoter proximal regulatory ele- in proliferating myoblasts, but was steadily downregu- ments of the gene encoding Tcap and identified a pro- lated after about four days of differentiation. The E12/ moter proximal fragment that recapitulates the E47 proteins were also expressed in proliferating myo- expression pattern of Tcap in reporter assays and is blasts, but the expression decreased after two days of bound by myogenin in vivo [27]. For each of these differentiation. genes, we profiled the change in RNA expression pro- files over a time course of differentiation. Expression of genes chosen for analysis For each gene, we saw that expression increased when Several genes were chosen for this analysis. We chose cells began to differentiate, as expected (Figure 2A). muscle creatine kinase (Ckm) and desmin (Des), as both Unexpectedly, we also observed that the expression con- are well-characterized genes whose expression increases tinued to increase over several days of differentiationand during differentiation. As the regulatory regions of these reached very high levels of expression after six days of genes have been studied extensively by others, promoter differentiation. We also observed that the expression proximal binding sites for the MRFs are well defined levels significantly decreased for each gene, with the UD D1 D2 D3 D4 D6 D8 D9 D10 α-Myf5 α-MyoD α-myogenin α-HEB α-E12/47 α-GAPDH Figure 1 Expression of myogenic regulatory factors (MRFs) and E proteins over a time course of differentiation in C C cells.C C 2 12 2 12 cells were differentiated for the indicated number of days and harvested for protein (UD, undifferentiated cells; D1-D10, cells differentiated for the indicated number of days). Protein concentration was determined for each extract and used to normalize the sample loading. Parallel blots were probed for each of the indicated antibodies as described in Methods. Londhe and Davie Skeletal Muscle 2011, 1:14 Page 4 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 A. Desmin Tnni2 5 200 D2 D3 D4 D5 D6 D10 UD D2 D3 D4 D5 D6 D10 UD Lmod2 Ckm 50 20 0 0 UD D2 D3 D4 D5 D6 D10 UD D2 D3 D4 D5 D6 D10 B. Tcap 0.8 0.6 0.4 100 0.2 0 0 UD D2 D3 D4 D5 D6 D10 MB MT MB MT MB MT MB MT MB MT Desmin Tnni2 Lmod2 Tcap CKM Figure 2 Expression profiles of genes upregulated during skeletal muscle differentiation in C C cells. (A) Expression profiles of 2 12 individual gene expression. Shown are graphs representing the change in expression of each gene examined in this study observed at each day of differentiation with respect to the expression level detected in proliferating C C myoblast cells. (B) The relative transcription levels of each of 2 12 the genes in this study under conditions of proliferation or differentiation are shown. Expression of each gene was determined by quantitative real-time polymerase chain reaction (qRT-PCR) assay and normalized to HPRT levels. Numbers indicate the fold change between undifferentiated samples (MB, myoblasts) and samples differentiated for six days (MT, myotubes). (A and B) Calculations of the relative fold changes in gene expression and mRNA expression are described in Methods. qRT-PCR assays were performed in triplicate on cDNA samples derived from independent RNA isolations. All data are normalized to the expression level of HPRT. Error bars represent standard deviations of the mean. exception of Lmod2, after ten days in differentiation (Figure 2B). This analysis revealed that Des was conditions. We also wanted to compare the relative expressed at a much higher level in proliferating myo- expression levels of the chosen gene set. To perform blasts than any of the other genes examined in this this analysis, we compared the expression of each gene study. We also observed that Tnni2 and Des were to a constitutive housekeeping gene, HPRT, in both expressed at approximately the same high level in differ- proliferating myoblasts and differentiated myotubes entiated cells. The expression levels of Lmod2, Tcap and Relative Expression Relative Expression Relative Expression Relative Expression Relative Expression mRNA Expression Londhe and Davie Skeletal Muscle 2011, 1:14 Page 5 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 Ckm increased significantly but did not reach the level ratio peaking at six days of differentiation. Myf5 and of activation of Tnni2 or Des in this time course. HEB were also present on the Des promoter in prolifer- ating cells. A peak of Myf5 binding was observed after Binding of MRFs and E proteins to muscle-specific genes three days of differentiation, when a peak of myogenin Next, we profiled the binding of MyoD, myogenin, Myf5 binding was also detected. By six days of differentiation, and E proteins over the time course of differentiation Myf5 binding was still detectable, but at greatly reduced levels. The levels continued to decrease after ten days of on each of these gene promoters. Proliferating cells and differentiation. The level of HEB remained fairly con- cells differentiated for one, two, three, six and ten days stant during the first two days of differentiation, but were used for the analysis. Surprisingly, we found that the pattern of recruitment was specific to each gene began to increase after three days and steadily increased tested. First, we examined the well-characterized Ckm until six days of differentiation. HEB remained bound to promoter [20-22]. For this analysis, we chose to examine the promoter after ten days of differentiation. Myogenin the enhancer 1 element located upstream of the first was not detected at the promoter in proliferating cells. noncoding exon of Ckm that contains one E box with Myogenin was detectable at the promoter after two days thesequenceCAGCTG, thepreferredbinding site for of differentiation, and its levels were greatly increased MyoD and myogenin. In accordance with recent chro- after three days of differentiation. The levels remained matin immunoprecipitation sequencing (ChIP-seq) stu- high at six days of differentiation. After ten days of dif- dies for MyoD [16], we did not detect MyoD at the ferentiation, HEB levels remained relatively unchanged, Ckm enhancer in proliferating myoblasts (Figure 3). We but the binding ratios of the MRFs were greatly also did not detect myogenin, Myf5 or HEB at this time decreased. MyoD was no longer detected at the promo- point. After three days of differentiation, MyoD, myo- ter, and only low levels of both myogenin and Myf5 genin and HEB were detectable at the Ckm enhancer. remained associated with the promoter. We observed However, six days after differentiation, we observed that RNAP II and AcH3 were associated with the pro- greatly enriched binding of HEB and myogenin com- moter after two days of differentiation, and, surprisingly, pared to the relatively unchanged binding of MyoD. We those levels moderately decreased after six days of dif- also detected Myf5 at this enhancer at this time point. ferentiation. We note that the relative fold enrichment By ten days of differentiation, the MRFs and HEB of RNAP II and AcH3 was very high at Des,the most appeared to be departing the promoter. While the bind- highly expressed gene in this study. This indicates that the number of Des promoters bound by RNAP II was ing was significantly enrichedoverbackgroundfor all much higher than what was observed at other factors except MyoD, the levels at ten days of differen- tiation were greatly reduced from those observed at six promoters. days of differentiation. The dynamic profiles of the We next examined the Tnni2 promoter, which, like MRFs and E proteins were surprising to us, so we also Des, is highly expressed in differentiating C C cells 2 12 compared the levels of RNAP II occupancy as a measure (Figure 5). Previous work has shown that the expression of transcriptional activity. As histone H3 acetylation is of Tnni2 is highly dependent on myogenin in vivo [26]. also a marker of active genes and increases in histone The regulatory elements of Tnni2 are uncharacterized, so H3 acetylation are correlated with MyoD binding, we we chosetoanalyzeahighlyconserved noncoding also examined the level of histone H3 acetylation at sequence immediately upstream of the transcriptional these promoters. For these assays, we compared the start site. The transcriptional start site of Tnni2 is pre- binding profile of RNAP II and acetylated histone H3 dicted to encode a short 5’ untranslated region (5’ UTR) (AcH3) at two days of differentiation and at six days of that initiates 23 bp prior to the translational start site. differentiation. We found that both RNAP II binding The conserved noncoding region is approximately 300 bp and histone H3 acetylation increased at six days of dif- upstream of the start of transcription and contains two E ferentiation, consistent with the transcriptional profiling boxes. The sequence of the promoter distal E box is and the increased occupancy of all the MRFs and HEB. CACCTG, while the sequence of the promoter proximal At Des, the most robustly expressed gene examined in E box is CAGCTG. The E boxes are separated by only this study, we examined the previously characterized 35 bp, so binding to either box could not be distin- promoter proximal enhancer element [24]. This region guished in our assay. As was true of Des, we observed an immediately upstream of the transcriptional start site association of MyoD with the Tnni2 promoter in prolifer- contains one E box with the sequence CAGCTG. We ating cells. The levels remained relatively unchanged dur- observed that MyoD bound to the Des promoter in ing the time course of differentiation, although small proliferating cells, again consistent with the recent variations were observed. Myf5 was recruited to the pro- ChIP-seq study (Figure 4). MyoD remained bound to moter upon the first day of differentiation, and the levels the promoter as the cells differentiated, with the binding continued to increase until after day six. By day ten of Londhe and Davie Skeletal Muscle 2011, 1:14 Page 6 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 MyoD ChIP at Ckm myogenin ChIP at Ckm UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Myf5 ChIP Myf5 ChIP at Ckm at Ckm HEB ChIP at Ckm 4 20 2 10 0 0 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Pol II ChIP at Ckm Ac H3 ChIP at Ckm D2 D6 D2 D6 Figure 3 Chromatin immunoprecipitation (ChIP) analysis of the muscle creatine kinase (Ckm)promoter. Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) and myofibers in differentiation media for the indicated number of days (D1-D10) were immunoprecipitated with antibodies against myogenin, MyoD, Myf5, HEB, RNA polymerase II (RNAP II), histone H3 acetylated at lysine 9 and/or 18 (H3 Ac9/18) or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Ckm. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. differentiation, Myf5 levels were greatly reduced. The levels continued to increase at six days of differentiation, recruitment of HEB was particularly surprising at this but binding rapidly declined after this point. At this pro- promoter. Here, we saw that HEB was not recruited to moter, the binding pattern of HEB completely overlapped the promoter before two days of differentiation. The with the binding pattern of myogenin. Myogenin could Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 7 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 MyoD ChIP at Desmin myogenin ChIP at Desmin 6 15 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 HEB ChIP at Desmin Myf5 ChIP at Desmin UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Pol II ChIP at Desmin Ac H3 ChIP at Desmin 0 0 D2 D6 D2 D6 Figure 4 Chromatin immunoprecipitation (ChIP) analysis of the desmin (Des) promoter. Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) and myofibers in differentiation media for the indicated number of days (D1-D10) were immunoprecipitated with antibodies against myogenin, MyoD, Myf5, HEB, RNA polymerase II (RNAP II), histone H3 acetylated at lysine 9 and/or 18 (H3 Ac9/18) or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Des. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. be detected at the promoter at two days of differentiation, significantly increased at six days of differentiation. After but binding was greatly enhanced after three days of dif- six days of differentiation, AcH3 levels reached the very ferentiation. The levels continued to increase at six days high levels observed at Des. of differentiation, but rapidly decreased after this point. The next promoter analyzed was the Lmod2 promoter At Tnni2,weobservedthatwhile RNAP II andAcH3 (Figure 6). The expression of Lmod2 is dependent on were present at two days of differentiation, the levels myogenin during embryogenesis, and a promoter Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 8 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 MyoD ChIP at Tnni2 myogenin ChIP at Tnni2 1 10 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Myf5 ChIP at Tnni2 HEB ChIP at Tnni2 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Pol II ChIP at Tnni2 Ac H3 ChIP at Tnni2 D2 D6 D2 D6 Figure 5 Chromatin immunoprecipitation (ChIP) analysis of fast-twitch skeletal muscle troponin I, type 2 (Tnni2). Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) and myofibers in differentiation media for the indicated number of days (D1-D10) were immunoprecipitated with antibodies against myogenin, MyoD, Myf5, HEB, RNA polymerase II (RNAP II), histone H3 acetylated at lysine 9 and/or 18 (H3Ac9/18) or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Tnni2. Relative enrichments at the IgH locus were used to normalize the data. The fold enrichment values were calculated as described in Methods. site of Lmod2 is immediately upstream of the transla- proximal binding site for myogenin has been defined [26]. A highly conserved noncoding region approxi- tional start site, predicting a short 5’ UTR of 112 bp. mately 100 bp upstream of the predicted transcriptional The sequence of the promoter proximal E box is start site contains two E boxes. The transcriptional start CAGCTG, while the sequence of the promoter distal Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 9 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 MyoD ChIP at Lmod2 myogenin ChIP at Lmod2 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Myf5 ChIP at Lmod2 HEB ChIP at Lmod2 0 0 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Pol II ChIP at Lmod2 Ac H3 ChIP at Lmod2 D2 D6 D2 D6 Figure 6 Chromatin immunoprecipitation (ChIP) analysis of the leiomodin 2 (Lmod2)promoter. Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) and myofibers in differentiation media for the indicated number of days (D1-D10) were immunoprecipitated with antibodies against myogenin, MyoD, Myf5, HEB, RNA polymerase II (RNAP II), histone H3 acetylated at lysine 9 and/or 18 (H3 Ac9/18) or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Lmod2. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. box E box is CAAATG. The E boxes are separated by significantly present. MyoD and Myf5 were recruited 117 bp. Deletion of the promoter proximal E box in a with ahighaffinitytothepromoter on thefirst dayof luciferase reporter assay largely abolished the transacti- differentiation. Myogenin could be detected at this time vation of the Lmod2 promoter by myogenin or MyoD point, but the binding ratio was relatively low. The asso- [26]. At the Lmod2 promoter, we saw that HEB was pre- ciation of myogenin increased greatly by two days of dif- sent in proliferating cells, but none of the MRFs were ferentiation. Myogenin remained bound at six days of Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 10 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 differentiation, but dissociated from the promoter by the was recruited, followed by HEB, which then dissociated tenth day of differentiation. MyoD and Myf5, recruited as Myf5 arrived. At this promoter, we again observed on the first day of differentiation, gradually dissociated that RNAP II occupancy and H3 acetylation greatly from the promoter after this time point. MyoD was increased at six days of differentiation as compared to undetectable at three days of differentiation, while Myf5 two days of differentiation. remained associated with the promoter until sometime after six days of differentiation. The occupancy of HEB HEB replaces E12/E47 at specific promoters during continued to increase during the initial stages of differ- differentiation entiation, peaking at two days of differentiation. HEB At each of these promoters, we found the distinct pat- appeared to begin to dissociate from the promoter after tern of HEB binding very surprising. We had anticipated two days of differentiation. HEB was only weakly seeing HEB bound to the promoter whenever an MRF detected at three days of differentiation and the levels was bound. While there are overlaps, the pattern of continued to decrease at six days of differentiation, HEB binding is temporally controlled and unique to becoming undetectable by ten days of differentiation. each gene. We wondered if perhaps these findings could The levels of RNAP II and AcH3 were significantly be the result of the binding of the E2A gene products higher at six days of differentiation than at two days of E12 or E47 in a compensatory fashion. We reasoned differentiation. that E proteins might be associated at all time points, The final promoter analyzed was the Tcap promoter butthe Eprotein couldbeeither E12/E47 or HEB. To (Figure 7). The Tcap locus contains a highly conserved address this hypothesis, we repeated the ChIP assays on promoter proximal noncoding sequence that contains proliferating cells, on cells differentiated for two days two E boxes. Interestingly, neither the promoter proxi- and on cells differentiated for three days with antibodies mal E box (CATCTG) nor the promoter distal E box against the E2A gene products. In proliferating cells, (CATGTG) is a favored binding site for MyoD or myo- E12 or E47 could be detected at the Des and Lmod2 genin. However, MyoD and myogenin can activate this promoter binding sites (Figure 8A). For Lmod2,the promoter, and myogenin binds to the promoter during binding of E12/E47 was detected with HEB in proliferat- embryogenesis [27]. In proliferating cells, none of the ing cells, but E12/E47 was lost as cells began to differ- MRFs or HEB is bound. At day one of differentiation, entiate while HEB levels increased (Figure 8B). At two MyoD was recruited to the Tcap promoter. MyoD days of differentiation, E12/E47 could still be detected at remained associated with the promoter until two days of the Des promoter, but that binding was lost at three differentiation, but dissociated from the promoter by days of differentiation (Figure 8C). At both of these pro- threedaysofdifferentiation.Thisresultisconsistent moter binding sites, we observed an exchange of E12/ with our promoter characterization of Tcap,aswe have E47 and HEB as cells began to differentiate. E12/E47 shown that the promoter proximal E box is required for was not detected on any of the other promoters assayed both the activity in C C cells and the activation by in this study. We have analyzed additional promoter 2 12 MyoD in NIH3T3 cells [27]. Myogenin was not proximal elements of differentiation-specific genes and recruited to the Tcap promoter at any time point tested, have found that E12/E47 was associated with the myo- which was surprising to us, as we have detected myo- genin(myog), troponin C, type 2(Tnnc2) and myosin genin binding to the Tcap promoter in skeletal muscle heavy chain 3 (Myh3) promoters in undifferentiated tissue during embryogenesis [27]. The profile of Myf5 cells as well (Additional file 1 Figure S5A). We did not was particularly surprising. Myf5 was not associated observe an association with the troponin T, type 2 with the promoter at early stages of differentiation, but (Tnnt2) promoter. In each case, binding was lost by two could be weakly detected at the promoter after six days days of differentiation (data not shown). HEB was of differentiation. This binding increased greatly by ten observed on the Tnnt2, Myh3 and Tnnc2 promoters days of differentiation. HEB could be detected on the following two days of differentiation (Additional file 1 Tcap promoter after two days of differentiation, when Figure S5B). For the majority of the promoters analyzed MyoD was still present. However, by day three, MyoD here, it appears that HEB is the predominant E protein had departed the promoter and the occupancy of HEB recruited during differentiation and that the transient increased greatly at this time. The peak of HEB binding association of HEB is not compensated by an overlap- appears to occur at three days of differentiation. By day ping pattern of E12/E47. These data are consistent with six, HEB was at the same low level observed at two days previously reported data that have established an impor- of differentiation, and by ten days of differentiation it tant role for HEBb in inducing differentiation [15]. was undetectable. We found the pattern of MRF and Next, we asked whether the presence of HEB is required to displace E12/E47 from promoters as cells begin to HEB association at this gene particularly interesting, as differentiate. HEB levels were depleted with small there is almost no overlap between these factors. MyoD Londhe and Davie Skeletal Muscle 2011, 1:14 Page 11 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 MyoD ChIP at Tcap myogenin ChIP at Tcap 2 2 1 1 0 0 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Myf5 ChIP at Tcap HEB ChIP at Tcap 25 20 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Pol II ChIP at Tcap Ac H3 ChIP at Tcap D2 D6 D2 D6 Figure 7 Chromatin immunoprecipitation (ChIP) analysis of the titin cap (Tcap)promoter. Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) and myofibers in differentiation media for the indicated number of days (D1-D10) were immunoprecipitated with antibodies myogenin, MyoD, Myf5, HEB, RNA polymerase II (RNAP II), histone H3 acetylated at lysine 9 and/or 18 (H3 Ac9/18) or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Tcap. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. hairpin (shRNA) constructs targeting HEB. Stable cell 8E). No change in E12/E47 expression was detected by lines expressing these constructs were screened for gene expression analysis (data not shown). ChIP assays expression of HEB and E12/E47. We proceeded with a with antibodies against E12/E47 were performed on construct that showed a 73% knockdown of HEB cells differentiated for two days. We saw no enhanced expression by RNA analysis (Figure 8D) and a reduction association of E12/E47 in cells reduced in expression of in protein expression by Western blot analysis (Figure HEB (Figure 8F), indicating that HEB is not required for Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 12 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 A. E12/47 ChIP in proliferating myoblasts Ckm Desmin Tnni2 Lmod2 Tcap B. C. E protein ChIP at Lmod2 E protein ChIP at Desmin 8 4 6 3 HEB E12/47 HEB E12/47 HEB E12/47 HEB E12/47 UD Day2 Day 2 Day3 D. E. F. E12/47 ChIP in myotubes (D2) HE B 1.5 1.5 0.5 0.5 probe: α HEB scr H E B scr K D scr K D scr K D scr K D scr K D sh RNA C mk D e smin Tn n i2 L mo d 2 Tca p Figure 8 E12/E47 and HEB exchange at the leiomodin 2 (Lmod2) and desmin (Des) promoters. (A) E12/E47 binds to the promoters of Des and Lmod2 in myoblasts. Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) were immunoprecipitated with antibodies against the E2A gene products. Immunoprecipitated DNA was purified and amplified with primers specific to the promoters of Ckm, Des, Tnni2, Lmod2 and Tcap. (B) E12/E47 and HEB exchange at the Lmod2 promoter. Cross-linked extracts from proliferating myoblasts and myofibers in differentiation media for two days were immunoprecipitated with antibodies against the E2A gene products, HEB or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Lmod2. (C) E12/E47 and HEB exchange at the Des promoter. Cross- linked extracts from myofibers in differentiation media for two or three days were immunoprecipitated with antibodies against the E2A gene products, HEB or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Des. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. (D) Gene expression analysis of HEB in cells expressing a small hairpin RNA (shRNA) construct targeting HEB or a scrambled control (scr). (E) Western blot analysis of the cells described in Figure 8D. The Western blot was probed with antibodies against HEB. (F) HEB is not required to displace E12/E47 at promoters. Results of chromatin immunoprecipitation assays performed after two days of differentiation on HEB-depleted cells and the scr control are shown. R el ati v e gen e ex pres s i on Fold Enrichment Fold Enrichment scr HEB shRNA Fo l d e n ri c h me n t Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 13 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 the displacement of E12/E47 from promoters during co-occupied the Des and Tnni2 promoters (Figure 9B). differentiation. These data confirm that MyoD, myogenin and Myf5 are bound to the same promoter elements. Myogenin, MyoD and Myf5 co-occupy promoters We were particularly interested in the binding profile of Discussion Myf5. For each gene tested, Myf5 had a unique binding We have found that each muscle gene assayed showed a pattern that was distinct from the binding patterns of unique temporal association of the MRFs and E pro- MyoD and myogenin. We were surprised to observe teins. We were surprised to observe the dynamic and that the profile of Myf5 overlapped with myogenin at transient roles of the MRFs on each of these promoters. several genes. To understand whether Myf5 and myo- MyoD has been proposed to be a “pioneer” transcription genin also colocalized during embryonic muscle devel- factor required to initiate the cascade of regulatory opment, we repeated the ChIP experiments in skeletal events required to initiate expression of muscle-specific muscle tissue derived from embryonic day 18.5 (E18.5) genes [29]. MyoD recruits chromatin-modifying activ- embryos. We chose E18.5 as it is late in embryogenesis ities that alter both the regional histone modifications and is a time point at which myogenin is assumed to be and the chromatin remodeling at promoter binding sites highly active. Myf5 is known to function only during the [7,30]. It is thought that these events then allow the early stages of myogenesis and thus would not be subsequent binding and transcriptional activity of myo- expected to contribute to gene expression at this stage. genin. Our data are consistent with this model, as we Moreover, Myf5 transcripts are not observed in embryos observed early associations of MyoD followed by the after E14 [28]. However, we detected Myf5 protein in association of myogenin. Our gene expression data also hindlimb samples at late embryonic stages (Additional show that for most genes examined in this study, the file 1 Figure S6). As expected, we detected myogenin at recruitment of myogenin is coincident with high levels several muscle-specific promoters, including Lmod2 and of transcription. Our data are consistent with those Des (Figure 9A). Consistent with our C C data, Myf5 reported in other studies that showed that at the genes 2 12 colocalized with myogenin at both of these promoters whose expression marks late myogenesis, Ckm and Des, in vivo (Figure 9A). After confirming that myogenin and MyoD is bound first, followed by the appearance of Myf5 appear to bind to the same sequences at a late myogenin, Mef2 (myocyte enhancer factor 2) and Brg1 time point in embryogenesis, we were very interested in (Brahma-related gene 1), the catalytic subunit of the understanding whether the overlapping pattern was Swi/Snf chromatin remodeling complex [25]. In this occurring in two cell populations or whether Myf5, prior study, it was also shown that the recruitment of myogenin was coincident with high levels of transcrip- myogenin and MyoD co-occupy promoters. To address this question, we performed sequential ChIP(ChIP-seq) tion of these genes in embryonic tissue. In our study, assays for myogenin, MyoD and Myf5 in C C cells. To the peak of transcription and myogenin binding corre- 2 12 address whether Myf5 and myogenin co-occupy promo- lated with high levels of RNAP II promoter occupancy ters, cells were differentiated for three days, immunopre- and histone H3 acetylation at the majority of genes cipitated with Myf5 antibodies and subsequently assayed. We note that the histone H3 acetylation levels immunoprecipitated with antibodies against myogenin. continued to rise following the departure of MyoD at We detected co-occupancy of Myf5 and myogenin at several genes. These data suggest that while MyoD may the Tnni2 and Des promoters (Figure 9B). We also per- be the initiating factor for chromatin modifications at formed the experiment with antibodies against myo- the promoter, the continued presence of MyoD is not genin first, followed by immunoprecipitation with Myf5 required for further increases in histone H3 acetylation. antibodies, and, again, co-occupancy of Myf5 and myo- The pattern of Myf5 binding was highly surprising. On geninonthe Tnni2 and Des promoters was confirmed certain genes, the Myf5 binding pattern overlapped with (data not shown). We next assayed for co-occupancy of MyoD, but at other genes, the binding pattern over- MyoD and myogenin. Cells were differentiated for three lapped with myogenin. In each case, the binding profile days, immunoprecipitated with antibodies against MyoD of Myf5 suggests that Myf5 has a previously uncharac- and subsequently immunoprecipitated with antibodies terized role in mediating gene expression in differentiat- against myogenin. Again, we observed co-occupancy of ing cells. While it is known that Myf5 cannot mediate MyoD andmyogeninonthe Des and Tnni2 promoters differentiation without myogenin or MyoD [8], our data (Figure 9B). Finally, we asked whether Myf5 and MyoD suggest that Myf5 does cooperate with both MyoD and co-occupy promoters. Differentiated cell extract was myogenin. Other groups have suggested that C C 2 12 immunoprecipitated with antibodies against Myf5 and cells, or the C cells used to derive them, have two subsequently immunoprecipitated with antibodies populations of myoblasts: a MyoD-expressing population against MyoD. We observed that Myf5 and MyoD thought to be the differentiating population and a Londhe and Davie Skeletal Muscle 2011, 1:14 Page 14 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 A. E18.5 ChIP at Lmod2 E18.5 ChIP at Desmin IgG myog Myf5 IgG myog Myf5 B. ChIP at Desmin ChIP at Desmin ChIP at Desmin 20 10 0 0 1st IP: IgG Myf5 1st IP: IgG 1st IP: IgG MyoD Myf5 2nd IP: MyoD MyoD 2nd IP: myog myog 2nd IP: myog myog ChIP at Tnni2 ChIP at Tnni2 ChIP at Tnni2 50 25 40 20 30 15 20 10 0 0 0 1st IP: IgG Myf5 1st IP: IgG MyoD 1st IP: IgG Myf5 2nd IP: myog myog 2nd IP: myog myog 2nd IP: MyoD MyoD Figure 9 Myogenin and Myf5 colocalize in vivo and co-occupy promoters with MyoD. (A) Myf5 and myogenin colocalize on promoters during embryonic development. Chromatin immunoprecipitation (ChIP) analysis was performed on skeletal muscle from E18.5 embryos. The immunoprecipitated DNA was purified and amplified with primers specific to the promoter of leiomodin 2 (Lmod2) and desmin (Des). The fold enrichment values were calculated as described in Methods. (B) Myf5, myogenin and MyoD co-occupy promoters. Cross-linked extracts from myofibers in differentiation media for two days were immunoprecipitated with antibodies against the indicated antibody (1st IP) or IgG. The immunoprecipitated complexes were released and immunoprecipitated again with antibodies against the indicated antibody (2nd IP). The immunoprecipitated DNA was purified and amplified with primers specific to the promoters of Tnni2 and Des. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 15 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 nondifferentiating or reserve population that expresses recruitment and release ofHEB.Thispattern wasnot Myf5 [31,32]. However, in our studies, we can conclude compensated by E12/E47, as we observed E12/E47 bind- that Myf5 is expressed in differentiating cells and that it ing to only two of the promoters in this study at early colocalizes with MyoD and myogenin on specific pro- time points. At three days of differentiation, E12/E47 moters in C C cells. Our data are highly suggestive was not detected at any of the promoters analyzed. At 2 12 that Myf5 plays a role in differentiation, but additional Des, the only gene highly expressed during proliferation examined in this study, we did observe an association experiments are required to confirm this hypothesis. with both E12/E47 and HEB in proliferating cells. At The binding pattern of myogenin was surprising as Lmod2, we also observed an early recruitment of E12/ well. The association of myogenin with muscle-specific genes as cells began to differentiate was expected, as E47 and HEB, whereas we observed only late recruit- myogenin is greatly upregulated at this time. However, ment of HEB at genes such as Tnni2.Wehypothesize the relatively brief association of myogenin with target that E12/E47 might be required at a subset of genes genes was unexpected. A transient role of myogenin on whose expression is immediately required as cells begin target genes has previously been suggested, as myogenin to differentiate. While Lmod2 is not significantly appears to have distinct target gene sets during embryo- expressed in proliferating cells, Lmod2 is upregulated genesis and in adult satellite cells and adult tissue very rapidly upon differentiation, and while the expres- [26,33]. Our data suggest that myogenin may mediate sion does continue to increase over an extended time changes at the promoter that maintain high levels of course, the expression increases only two fold. This is in expression without the continued presence of myogenin. contrast to genes such as Tnni2, where the expression Candidates for such a change include the switching of level increases ten fold over the extended time course. core promoter complexes, which has been observed in Lmod2 does not reach the high levels of transcription skeletal muscle differentiation. A TATA-binding protein seen at Tnni2 that coincide with the peak of HEB bind- (TBP)-related factor, TRF3, and an associated TBP-asso- ing. It is possible that the early recruitment of E12/E47 ciated factor, TAF3, have been shown to be targeted by and HEB at Lmod2 helps to support a relatively constant MyoD to the myogenin promoter following differentia- level of expression that initiates immediately upon dif- tion [31,34]. TBP is expressed in proliferating myoblasts, ferentiation.Itisstrikingthatatbothgenes wherewe but following differentiation, TBP is downregulated and observed the binding of E12/E47, we also observed that TRF3 and TAF3 are upregulated. It is also possible that HEB appeared to replace E12/E47 as cells began to dif- ferentiate. The binding pattern of HEB at Tcap is parti- myogenin may direct epigenetic changes that maintain cularly interesting. HEB binding peaks at a time point gene expression. The binding pattern of HEB was very surprising to us when no MRFs are detected. Reduced levels of binding as well. Detailed biophysical experiments have shown are detected at two additional time points when MyoD that MRF and E protein heterodimers are highly stable and Myf5 are bound on the individual days. Thus, while when bound to DNA. These studies have also indicated the HEB binding profile does overlap with MRF binding that heterodimers likely form on the DNA. MyoD and as predicted by the biophysical studies, the occupancy of E47 heterodimers are not detected in diluted conditions HEB does not always overlap with the occupancy of the without DNA. However, in the presence of DNA, het- MRFs. erodimeric complexes are formed almost exclusively While these data have revealed many novel findings [35]. Additional work has shown that the weak MyoD regarding the recruitment of the MRFs and E proteins, homodimers and heterodimers that can form in the many questions remain. The additional factors and absence of DNA are equally stable [36]. This suggests DNA elements that mediate the individual recruitment that the MRFs and E proteins are likely to be mono- and release of each of these factors remain to be charac- meric in the cell. In this work {Maleki, 2002 #376}, it terized. Many elegant studies of the role of chromatin was also shown that while MyoD or myogenin E protein modification in muscle differentiation have suggested heterodimers on DNA were the most energetically that epigenetic events are important mediators in the favorable, MyoD and myogenin homodimers can bind activation of muscle genes. The Swi/Snf chromatin E boxes with considerable positive cooperativity, while remodeling complex promotes muscle differentiation, E12 homodimers exhibited negative cooperativity. The and it is known that the Swi/Snf complex is recruited to negative cooperativity of E12 suggests that the heterodi- both the Des and Ckm promoters studied here [25,37]. Important questions for future studies include how chro- mer may form on DNA by binding of the E12 monomer matin remodelers and chromatin-modifying enzymes followed by binding of the MRF monomer. contribute to the recruitment and release of the myogenic Given these data, we anticipated detecting E proteins ontheDNAthroughout thetimecourseof differentia- regulatory factors and E proteins to regulate muscle gene tion. Instead, we found a highly dynamic pattern of expression. Londhe and Davie Skeletal Muscle 2011, 1:14 Page 16 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 anti-Myf5 (C-20; Santa Cruz Biotechnology), anti-MyoD Conclusions (5.8A; Santa Cruz Biotechnology), anti-GAPDH (anti-gly- Here we have shown that MyoD, myogenin and Myf5 ceraldehyde 3-phosphate dehydrogenase; Chemicon have sequential and transient roles on each of the pro- International, Billerica, MA USA) and anti-MyoG (F5D).. moters assayed. For almost every gene assayed, we The F5D antibody developed by W. E. Wright was found that the binding of myogenin and HEB correlated obtained from the Developmental Studies Hybridoma with high levels of RNAP II occupancy, histone H3 acet- Bank under the auspices of the NICHD and maintained ylation and the peak of transcription as assayed by by the University of Iowa, Department of Biology, Iowa mRNA levels. We found that the primary E protein City, IA USA. Normal rabbit immunoglobulin G (IgG) recruited to late differentiation genes is HEB. At the few (Santa Cruz Biotechnology, Santa Cruz, CA USA) was promoters where E12/E47 was detected at early stages, used as a nonspecific control. HEB replaced E12/E47 during differentiation. Finally, we have shown that MyoD, myogenin and Myf5 colocalize Quantitative reverse transcriptase-polymerase chain on promoters, suggesting that Myf5 contributes to the reaction assays gene expression of late differentiation genes. RNA was isolated from C C cells by TRIzol reagent 2 12 extraction (Invitrogen, Carlsbad, CA. Two micrograms Methods of total RNA were reverse-transcribed with Multi- Cell culture Scribe™ Reverse Transcriptase (Applied Biosystems, Cells were grown in a humidified chamber at 37°C with Carlsbad, CA USA. cDNA equivalent to 40 ng was used 5% CO . Proliferating C C myoblasts (American Type 2 2 12 for quantitative reverse transcriptase-polymerase chain Culture Collection, Manassas, VA, USA) were grown in reaction (qRT-PCR) amplification (Applied Biosystems, Dulbecco’s modified Eagle’s medium (DMEM) supple- Foster City,CAUSA)withSYBRGreenPCRMaster mented with 10% fetal bovine serum (Thermo Scientific Mix (Applied Biosystems, Foster City, CA USA). Sam- HyClone, Logan, UT USA. To induce differentiation ples in which no RT was added were included for each into myotubes, cells were grown to 70% confluence and RNA sample. For measurements of relative gene expres- media were switched to DMEM supplemented with 2% sion, a fold change was calculated for each sample pair horse serum (Thermo Scientific HyClone, Logan, UT and normalized to the fold change observed at HPRT. USA). C C cells were grown in differentiation medium 2 12 mRNA expression levels were quantitated using a cali- for the number of days indicated in each experiment. bration curve based on known dilutions of concentrated cDNA. Each mRNA value was normalized to that of Western blot analysis HPRT. Fold change was calculated by dividing the Cell extracts were made by lysing phosphate-buffered sal- mRNA expression values of each sample pair. qRT-PCR ine-washed cell pellets in radioimmunoprecipitation data were calculated using the comparative C method assay buffer supplemented with protease inhibitors (Applied Biosystems, Foster City, CA USA). Standard (Complete Protease Inhibitor Cocktail Tablets; Roche deviations from the mean of the ΔC values were calcu- Applied Science, Indianapolis, IN USA. Following incu- lated from three independent RNA samples and used to bation on ice, clear lysates were obtained by performing generate error bars. Intron-spanning primers to the cod- centrifugation. Protein concentrations were determined ing region of Lmod2, Des, Tnni2, Ckm and Tcap are by using the Bio-Rad Protein Assay (Bio-Rad, Hercules, described in Additional file 1 Table S1. All qPCR assays CA USA. For each sample, 30 μg of protein were loaded were performed in triplicate, and at least two indepen- onto each gel. Proteins were transferred onto a nitrocel- dent RNA samples were assayed for each time point. lulose membrane using a tank blotter (Bio-Rad, Hercules, CA USA), then blocked using 5% milk and 1× Tris-buf- ChIP assays fered saline plus Tween 20 (TBST) and incubated with Cell culture ChIP assays were performed and quantified primary antibody overnight at 4°C. Membranes were as described previously [38] with the following modifica- then washed with 1× TBST and incubated with the cor- tions: 1 × 10 cells were used for each immunoprecipita- responding secondary antibody. Membranes were again tion, and protein A agarose beads (Invitrogen) were washed with 1× TBST, incubated with chemilumines- used to immunoprecipitate the antibody-antigen com- cence substrate according to the manufacturer’s protocol plexes. ChIP assays of embryonic tissue were performed (SuperSignal West Pico Chemiluminescent Substrate; as previously described [26]. Limb tissue from wild-type Pierce Biotechnology, Rockford, IL USA and visualized C57BL/6 mice{Jackson Laboratory, Bar Harbor, ME by autoradiography. The antibodies used include anti- USA) was isolated, cross-linked and enriched for nuclei. HEB (A-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E12/E47 (Yae; Santa Cruz Biotechnology), Nuclear extracts of limb tissue were precleared using Londhe and Davie Skeletal Muscle 2011, 1:14 Page 17 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 Acknowledgements incubation with protein A agarose beads (Invitrogen, We thank Meiling Zhang for assistance in establishing stable cell lines Carlsbad, CA USA), which were also used to immuno- expressing shRNA constructs against HEB. This work was supported by precipitate the antibody complexes from tissue extracts grants from the Central Research Committee, Southern Illinois University School of Medicine, and by grant 159609 from the American Cancer Society, following antibody addition to the incubation mix. Anti- Illinois Division, awarded to JD. bodies against the following proteins were used: MyoD (5.8A; Santa Cruz Biotechnology, Santa Cruz, CA USA), Authors’ contributions PL and JD designed the experimental approach. PL performed the described HEB (A-20; Santa Cruz Biotechnology, Santa Cruz, CA experiments, and JD wrote the manuscript with assistance from PL. Both USA), Myf5 (C-20; Santa Cruz Biotechnology, Santa authors read and approved the final manuscript. Cruz, CA USA), E proteins (Yae; Santa Cruz Biotechnol- Competing interests ogy, Santa Cruz, CA), myogenin (F5D; Developmental The authors declare that they have no competing interests. Studies Hybridoma Bank), RNAP II (H-224; Santa Cruz Biotechnology, Santa Cruz, CA USA) and histone H3 Received: 8 October 2010 Accepted: 4 April 2011 Published: 4 April 2011 acetylated at lysine 9 and/or 18 (H3.Ac9/18; Millipore, References Billerica, MA USAUpstate Biotechnology. Primers span- 1. Kablar B, Rudnicki MA: Skeletal muscle development in the mouse ning the described promoter elements of Lmod2, Des, embryo. Histol Histopathol 2000, 15:649-656. Tnni2, Ckm, Tcap, Myog, Tnnt2, Myh3 and Tnnc2 are 2. Parker MH, Seale P, Rudnicki MA: Looking back to the embryo: defining -[Δ][Δ]Ct transcriptional networks in adult myogenesis. Nat Rev Genet 2003, described in Additional file 1 Table S1. 2 values 4:497-507. were calculated using the following formula based on 3. 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Tapscott SJ: The circuitry of a master switch: Myod and the regulation of skeletal muscle gene transcription. Development 2005, 132:2685-2695. 30. Gerber AN, Klesert TR, Bergstrom DA, Tapscott SJ: Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin: a mechanism for lineage determination in myogenesis. Genes Dev 1997, 11:436-450. 31. Deato MD, Tjian R: Switching of the core transcription machinery during myogenesis. Genes Dev 2007, 21:2137-2149. 32. Lindon C, Montarras D, Pinset C: Cell cycle-regulated expression of the muscle determination factor Myf5 in proliferating myoblasts. J Cell Biol 1998, 140:111-118. 33. Meadows E, Cho JH, Flynn JM, Klein WH: Myogenin regulates a distinct genetic program in adult muscle stem cells. Dev Biol 2008, 322:406-414. 34. Deato MD, Marr MT, Sottero T, Inouye C, Hu P, Tjian R: MyoD targets TAF3/ TRF3 to activate myogenin transcription. Mol Cell 2008, 32:96-105. 35. 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Cell 2003, • Convenient online submission 115:751-763. • Thorough peer review doi:10.1186/2044-5040-1-14 • No space constraints or color figure charges Cite this article as: Londhe and Davie: Sequential association of myogenic regulatory factors and E proteins at muscle-specific genes. • Immediate publication on acceptance Skeletal Muscle 2011 1:14. • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Sequential association of myogenic regulatory factors and E proteins at muscle-specific genes

Skeletal Muscle , Volume 1 (1) – Apr 4, 2011

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Copyright © 2011 by Londhe and Davie; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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10.1186/2044-5040-1-14
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Abstract

Background: Gene expression in skeletal muscle is controlled by a family of basic helix-loop-helix transcription factors known as the myogenic regulatory factors (MRFs). The MRFs work in conjunction with E proteins to regulate gene expression during myogenesis. However, the precise mechanism by which the MRFs activate gene expression is unclear. In this work, we sought to define the binding profiles of MRFs and E proteins on muscle- specific genes throughout a time course of differentiation. Results: We performed chromatin immunoprecipitation (ChIP) assays for myogenin, MyoD, Myf5 and E proteins over a time course of C C differentiation, resulting in several surprising findings. The pattern of recruitment is 2 12 specific to each promoter tested. The recruitment of E proteins often coincides with the arrival of the MRFs, but the binding profile does not entirely overlap with the MRF binding profiles. We found that E12/E47 is bound to certain promoters during proliferation, but every gene tested is preferentially bound by HEB during differentiation. We also show that MyoD, myogenin and Myf5 have transient roles on each of these promoters during muscle differentiation. We also found that RNA polymerase II occupancy correlates with the transcription profile of these promoters. ChIP sequencing assays confirmed that MyoD, myogenin and Myf5 co-occupy promoters. Conclusions: Our data reveal the sequential association of MyoD, myogenin, Myf5 and HEB on muscle-specific promoters. These data suggest that each of the MRFs, including Myf5, contribute to gene expression at each of the geness analyzed here.. The dynamic binding profiles observed suggest that MRFs and E proteins are recruited independently to promoters. Background birth. The myogenin-nullmicehavemyoblasts,but very The entire process of skeletal muscle differentiation is few muscle fibers. This suggests that myogenin is not controlled by four highly related basic helix-loop-helix required for the specification of skeletal muscle, but is (bHLH) proteins referred to as the myogenic regulatory required for the later stages of myofiber fusion. factors (MRFs). The MRFs have distinct but overlapping MyoD and myogenin have been shown to bind highly patterns of gene expression during muscle development overlapping gene sets, although certain genes appear to [1]. Gene knockouts of each factor in the mouse have be selective for either factor [6,7]. However, the high revealed that each MRF has a unique role in skeletal degree of overlap in the binding patterns suggests that muscle differentiation. Myf5, Myf6 (also known as the majority of genes utilize both factors to activate gene MRF4) and MyoD are not required for viability, although expression. Previous work has shown that certain genes require the sequential expression of both MyoD and each mutant has a distinct phenotype [2]. In the com- bined absence of Myf5, Myf6 and MyoD, myoblasts are myogenin to activate gene expression [7]. The present not specified and no skeletal muscle forms, resulting in a work suggests that the activation of specific targets lethal phenotype [3]. Myogenin is the only MRF singly requires MyoD and its associated chromatin-modifying required for viability [4,5]. Mice heterozygous for the null activities before myogenin can activate transcription. allele appear normal, while mice lacking myogenin die at Why MyoD cannot activate transcription without myogenin on these genes is still unknown. Recent work on Myf5 has revealed unexpected roles * Correspondence: jdavie@siumed.edu Department of Biochemistry and Molecular Biology, Southern Illinois for this factor in adult animals. As mentioned above, University School of Medicine, 1245 Lincoln Drive, Carbondale, IL 62901, USA © 2011 Londhe and Davie; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Londhe and Davie Skeletal Muscle 2011, 1:14 Page 2 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 Myf5 functions as a determination gene in early myo- widely used cell culture model for myoblast differentia- genesis. The role of Myf5 in later stages is unclear. In tion. These binding profiles were correlated with the the absence of MyoD, Myf6 or myogenin, Myf5 is levels of mRNA present in the cells, the levels of RNA unable to promote differentiation from myoblasts [8]. polymerase II (RNAP II) occupancy and histone H3 This finding suggests that Myf5 functions only in mus- acetylation present at the promoters. We show several cle progenitor cells (MPCs) and myoblasts. However, novel findings. Surprisingly, we have found that the pat- recent work has shown that Myf5-null mice exhibit tern of recruitment is unique to each gene, although impaired muscle regeneration with a significant increase some common features arise. As others have observed, in muscle fiber hypertrophy and a delay in differentia- we saw an early association of MyoD with most of these tion [9]. However, satellite cell numbers were not signif- genes. In a cooperative pattern, myogenin then binds icantly altered in the Myf5-null animals, although a many of these promoters following MyoD. Unexpect- modest impaired proliferation was observed under some edly, we found that Myf5 is also associated with genes conditions in vitro. This work highlights the questions expressed late in differentiation and often colocalizes still remaining about the roles of the MRFs at distinct with myogenin. We show that this colocalization also stages in myogenesis. occurs in vivo at a late embryonic time point. The bind- All bHLH transcription factors function as either ing of each of the MRFs is transient. We also show that homodimers or heterodimers. The bHLH transcription the occupancy of the E proteins is transient and that the factors are loosely grouped into several categories: the occupancy often peaks concurrently with the peak in widely expressed E proteins, including the E2A gene pro- gene transcription. While the E2A gene products could ducts E12 and E47, HEB, E2-2 and Daughterless, are in be detected on a few genes in our study in proliferating the class I category and the MRF family is included in the cells, HEB does appear to be the dominant E protein tissue-specific class II category. Class II bHLH proteins used during differentiation. At the genes occupied by form weak homodimers and preferentially heterodimer- E12/E47 in early myogenesis, we detected a switch in ize with E proteins [10]. Prior in vitro experiments have occupancy for HEB during differentiation. Taken demonstrated that the class II MRFs form avid heterodi- together, our data suggest new models for the recruit- mers with class I E proteins, but homodimerize poorly in ment of MRFs and E proteins and support a novel role the presence of DNA sites [11-14]. Thus, it is thought for Myf5 during differentiation. that the MRFs function as heterodimers with ubiquitous E proteins. The E proteins suggested to be involved in Results skeletal muscle differentiation are the E2A gene products Time course of MRF and E protein expression E12 and E47, as well as HEB. Recent work has suggested To initiate this work, we first characterized the available that HEB may be the primary E protein that regulates antibodies for these studies and confirmed MRF and E skeletal muscle differentiation [15]. protein expression patterns over a time course of differ- The MRFs all bind the canonical E-box consensus entiation. We tested well-characterized antibodies for sequence, CANNTG. Genome-wide binding studies have MyoD and myogenin [17,18]. We found that antibodies revealed that both MyoD and myogenin preferentially against MyoD and myogenin immunoprecipitated the bind E boxes with a consensus sequence of CASCTG target protein (data not shown) and did not recognize (International Union of Pure and Applied Chemistry any of the other MRFs (Additional file 1 Figure S1). For nomenclature http://www.iupac.org), where S represents Myf5, we tested commercially available antibodies for G or C [7,16]. The site recognized at the highest fre- their ability to recognize and immunoprecipitate Myf5 quency is CAGCTG. The sequences flanking the E box specifically. We identified one antibody that immunopre- also make important contributions to the binding affinity cipitated Myf5 exclusively (Additional file 1 Figure S2), and contribute to the overall consensus sequence and this antibody was used for all the studies presented. elements determined for MyoD and myogenin [11,16]. We did note that the antibody does recognize recombi- Given the high degree of overlap detected in the gen- nant MyoD by using Western blot analysis, but we could ome occupancy of MyoD and myogenin, we were inter- not immunoprecipitate this protein (Additional file 1 Fig- ested in understanding the binding profile of these ure S2). For the E proteins, we used antibodies raised factors over a time course of differentiation. We were against HEB and the E2A gene products E12 and E47. also interested in the binding profile of Myf5, as binding TheHEB antibodyrecognized E12/E47 on the basis of data for this factor during differentiation have not been Western blot analysis, but immunoprecipitated HEB spe- reported. We also sought to compare the DNA-bound cifically (Additional file 1 Figure S3). The antibody profiles of the MRFs with the E proteins. Thus, we against E12/E47 did not recognize or immunoprecipitate initiated a temporal analysis of the binding of MyoD, recombinant HEB (Additional file 1 Figure S4). Next, we Myf5, myogenin and the E proteins in C examined the expression profile of the MRFs and HEB C cells, a 2 12 Londhe and Davie Skeletal Muscle 2011, 1:14 Page 3 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 over an extended time course of C C differentiation [19-25]. We also chose the fast-twitch skeletal muscle 2 12 (Figure 1). As previously observed, MyoD and Myf5 were troponin I, type 2 (Tnni2)and leiomodin 2 (Lmod2) expressed in proliferating myoblasts. MyoD levels genes, as these have been characterized as myogenin- increased upon differentiation, but then rapidly decreased dependent targets in embryonic skeletal muscle during after two days of differentiation. Unexpectedly, Myf5 was embryogenesis, and the promoter proximal MRF bind- expressed throughout the entire time course. Myogenin ing sites are known [26]. We also chose titin cap was not detectable in proliferating cells, but was rapidly (Tcap), also known as telethonin.Wehaverecently upregulated upon differentiation. HEB was also expressed characterized the promoter proximal regulatory ele- in proliferating myoblasts, but was steadily downregu- ments of the gene encoding Tcap and identified a pro- lated after about four days of differentiation. The E12/ moter proximal fragment that recapitulates the E47 proteins were also expressed in proliferating myo- expression pattern of Tcap in reporter assays and is blasts, but the expression decreased after two days of bound by myogenin in vivo [27]. For each of these differentiation. genes, we profiled the change in RNA expression pro- files over a time course of differentiation. Expression of genes chosen for analysis For each gene, we saw that expression increased when Several genes were chosen for this analysis. We chose cells began to differentiate, as expected (Figure 2A). muscle creatine kinase (Ckm) and desmin (Des), as both Unexpectedly, we also observed that the expression con- are well-characterized genes whose expression increases tinued to increase over several days of differentiationand during differentiation. As the regulatory regions of these reached very high levels of expression after six days of genes have been studied extensively by others, promoter differentiation. We also observed that the expression proximal binding sites for the MRFs are well defined levels significantly decreased for each gene, with the UD D1 D2 D3 D4 D6 D8 D9 D10 α-Myf5 α-MyoD α-myogenin α-HEB α-E12/47 α-GAPDH Figure 1 Expression of myogenic regulatory factors (MRFs) and E proteins over a time course of differentiation in C C cells.C C 2 12 2 12 cells were differentiated for the indicated number of days and harvested for protein (UD, undifferentiated cells; D1-D10, cells differentiated for the indicated number of days). Protein concentration was determined for each extract and used to normalize the sample loading. Parallel blots were probed for each of the indicated antibodies as described in Methods. Londhe and Davie Skeletal Muscle 2011, 1:14 Page 4 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 A. Desmin Tnni2 5 200 D2 D3 D4 D5 D6 D10 UD D2 D3 D4 D5 D6 D10 UD Lmod2 Ckm 50 20 0 0 UD D2 D3 D4 D5 D6 D10 UD D2 D3 D4 D5 D6 D10 B. Tcap 0.8 0.6 0.4 100 0.2 0 0 UD D2 D3 D4 D5 D6 D10 MB MT MB MT MB MT MB MT MB MT Desmin Tnni2 Lmod2 Tcap CKM Figure 2 Expression profiles of genes upregulated during skeletal muscle differentiation in C C cells. (A) Expression profiles of 2 12 individual gene expression. Shown are graphs representing the change in expression of each gene examined in this study observed at each day of differentiation with respect to the expression level detected in proliferating C C myoblast cells. (B) The relative transcription levels of each of 2 12 the genes in this study under conditions of proliferation or differentiation are shown. Expression of each gene was determined by quantitative real-time polymerase chain reaction (qRT-PCR) assay and normalized to HPRT levels. Numbers indicate the fold change between undifferentiated samples (MB, myoblasts) and samples differentiated for six days (MT, myotubes). (A and B) Calculations of the relative fold changes in gene expression and mRNA expression are described in Methods. qRT-PCR assays were performed in triplicate on cDNA samples derived from independent RNA isolations. All data are normalized to the expression level of HPRT. Error bars represent standard deviations of the mean. exception of Lmod2, after ten days in differentiation (Figure 2B). This analysis revealed that Des was conditions. We also wanted to compare the relative expressed at a much higher level in proliferating myo- expression levels of the chosen gene set. To perform blasts than any of the other genes examined in this this analysis, we compared the expression of each gene study. We also observed that Tnni2 and Des were to a constitutive housekeeping gene, HPRT, in both expressed at approximately the same high level in differ- proliferating myoblasts and differentiated myotubes entiated cells. The expression levels of Lmod2, Tcap and Relative Expression Relative Expression Relative Expression Relative Expression Relative Expression mRNA Expression Londhe and Davie Skeletal Muscle 2011, 1:14 Page 5 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 Ckm increased significantly but did not reach the level ratio peaking at six days of differentiation. Myf5 and of activation of Tnni2 or Des in this time course. HEB were also present on the Des promoter in prolifer- ating cells. A peak of Myf5 binding was observed after Binding of MRFs and E proteins to muscle-specific genes three days of differentiation, when a peak of myogenin Next, we profiled the binding of MyoD, myogenin, Myf5 binding was also detected. By six days of differentiation, and E proteins over the time course of differentiation Myf5 binding was still detectable, but at greatly reduced levels. The levels continued to decrease after ten days of on each of these gene promoters. Proliferating cells and differentiation. The level of HEB remained fairly con- cells differentiated for one, two, three, six and ten days stant during the first two days of differentiation, but were used for the analysis. Surprisingly, we found that the pattern of recruitment was specific to each gene began to increase after three days and steadily increased tested. First, we examined the well-characterized Ckm until six days of differentiation. HEB remained bound to promoter [20-22]. For this analysis, we chose to examine the promoter after ten days of differentiation. Myogenin the enhancer 1 element located upstream of the first was not detected at the promoter in proliferating cells. noncoding exon of Ckm that contains one E box with Myogenin was detectable at the promoter after two days thesequenceCAGCTG, thepreferredbinding site for of differentiation, and its levels were greatly increased MyoD and myogenin. In accordance with recent chro- after three days of differentiation. The levels remained matin immunoprecipitation sequencing (ChIP-seq) stu- high at six days of differentiation. After ten days of dif- dies for MyoD [16], we did not detect MyoD at the ferentiation, HEB levels remained relatively unchanged, Ckm enhancer in proliferating myoblasts (Figure 3). We but the binding ratios of the MRFs were greatly also did not detect myogenin, Myf5 or HEB at this time decreased. MyoD was no longer detected at the promo- point. After three days of differentiation, MyoD, myo- ter, and only low levels of both myogenin and Myf5 genin and HEB were detectable at the Ckm enhancer. remained associated with the promoter. We observed However, six days after differentiation, we observed that RNAP II and AcH3 were associated with the pro- greatly enriched binding of HEB and myogenin com- moter after two days of differentiation, and, surprisingly, pared to the relatively unchanged binding of MyoD. We those levels moderately decreased after six days of dif- also detected Myf5 at this enhancer at this time point. ferentiation. We note that the relative fold enrichment By ten days of differentiation, the MRFs and HEB of RNAP II and AcH3 was very high at Des,the most appeared to be departing the promoter. While the bind- highly expressed gene in this study. This indicates that the number of Des promoters bound by RNAP II was ing was significantly enrichedoverbackgroundfor all much higher than what was observed at other factors except MyoD, the levels at ten days of differen- tiation were greatly reduced from those observed at six promoters. days of differentiation. The dynamic profiles of the We next examined the Tnni2 promoter, which, like MRFs and E proteins were surprising to us, so we also Des, is highly expressed in differentiating C C cells 2 12 compared the levels of RNAP II occupancy as a measure (Figure 5). Previous work has shown that the expression of transcriptional activity. As histone H3 acetylation is of Tnni2 is highly dependent on myogenin in vivo [26]. also a marker of active genes and increases in histone The regulatory elements of Tnni2 are uncharacterized, so H3 acetylation are correlated with MyoD binding, we we chosetoanalyzeahighlyconserved noncoding also examined the level of histone H3 acetylation at sequence immediately upstream of the transcriptional these promoters. For these assays, we compared the start site. The transcriptional start site of Tnni2 is pre- binding profile of RNAP II and acetylated histone H3 dicted to encode a short 5’ untranslated region (5’ UTR) (AcH3) at two days of differentiation and at six days of that initiates 23 bp prior to the translational start site. differentiation. We found that both RNAP II binding The conserved noncoding region is approximately 300 bp and histone H3 acetylation increased at six days of dif- upstream of the start of transcription and contains two E ferentiation, consistent with the transcriptional profiling boxes. The sequence of the promoter distal E box is and the increased occupancy of all the MRFs and HEB. CACCTG, while the sequence of the promoter proximal At Des, the most robustly expressed gene examined in E box is CAGCTG. The E boxes are separated by only this study, we examined the previously characterized 35 bp, so binding to either box could not be distin- promoter proximal enhancer element [24]. This region guished in our assay. As was true of Des, we observed an immediately upstream of the transcriptional start site association of MyoD with the Tnni2 promoter in prolifer- contains one E box with the sequence CAGCTG. We ating cells. The levels remained relatively unchanged dur- observed that MyoD bound to the Des promoter in ing the time course of differentiation, although small proliferating cells, again consistent with the recent variations were observed. Myf5 was recruited to the pro- ChIP-seq study (Figure 4). MyoD remained bound to moter upon the first day of differentiation, and the levels the promoter as the cells differentiated, with the binding continued to increase until after day six. By day ten of Londhe and Davie Skeletal Muscle 2011, 1:14 Page 6 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 MyoD ChIP at Ckm myogenin ChIP at Ckm UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Myf5 ChIP Myf5 ChIP at Ckm at Ckm HEB ChIP at Ckm 4 20 2 10 0 0 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Pol II ChIP at Ckm Ac H3 ChIP at Ckm D2 D6 D2 D6 Figure 3 Chromatin immunoprecipitation (ChIP) analysis of the muscle creatine kinase (Ckm)promoter. Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) and myofibers in differentiation media for the indicated number of days (D1-D10) were immunoprecipitated with antibodies against myogenin, MyoD, Myf5, HEB, RNA polymerase II (RNAP II), histone H3 acetylated at lysine 9 and/or 18 (H3 Ac9/18) or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Ckm. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. differentiation, Myf5 levels were greatly reduced. The levels continued to increase at six days of differentiation, recruitment of HEB was particularly surprising at this but binding rapidly declined after this point. At this pro- promoter. Here, we saw that HEB was not recruited to moter, the binding pattern of HEB completely overlapped the promoter before two days of differentiation. The with the binding pattern of myogenin. Myogenin could Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 7 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 MyoD ChIP at Desmin myogenin ChIP at Desmin 6 15 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 HEB ChIP at Desmin Myf5 ChIP at Desmin UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Pol II ChIP at Desmin Ac H3 ChIP at Desmin 0 0 D2 D6 D2 D6 Figure 4 Chromatin immunoprecipitation (ChIP) analysis of the desmin (Des) promoter. Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) and myofibers in differentiation media for the indicated number of days (D1-D10) were immunoprecipitated with antibodies against myogenin, MyoD, Myf5, HEB, RNA polymerase II (RNAP II), histone H3 acetylated at lysine 9 and/or 18 (H3 Ac9/18) or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Des. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. be detected at the promoter at two days of differentiation, significantly increased at six days of differentiation. After but binding was greatly enhanced after three days of dif- six days of differentiation, AcH3 levels reached the very ferentiation. The levels continued to increase at six days high levels observed at Des. of differentiation, but rapidly decreased after this point. The next promoter analyzed was the Lmod2 promoter At Tnni2,weobservedthatwhile RNAP II andAcH3 (Figure 6). The expression of Lmod2 is dependent on were present at two days of differentiation, the levels myogenin during embryogenesis, and a promoter Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 8 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 MyoD ChIP at Tnni2 myogenin ChIP at Tnni2 1 10 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Myf5 ChIP at Tnni2 HEB ChIP at Tnni2 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Pol II ChIP at Tnni2 Ac H3 ChIP at Tnni2 D2 D6 D2 D6 Figure 5 Chromatin immunoprecipitation (ChIP) analysis of fast-twitch skeletal muscle troponin I, type 2 (Tnni2). Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) and myofibers in differentiation media for the indicated number of days (D1-D10) were immunoprecipitated with antibodies against myogenin, MyoD, Myf5, HEB, RNA polymerase II (RNAP II), histone H3 acetylated at lysine 9 and/or 18 (H3Ac9/18) or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Tnni2. Relative enrichments at the IgH locus were used to normalize the data. The fold enrichment values were calculated as described in Methods. site of Lmod2 is immediately upstream of the transla- proximal binding site for myogenin has been defined [26]. A highly conserved noncoding region approxi- tional start site, predicting a short 5’ UTR of 112 bp. mately 100 bp upstream of the predicted transcriptional The sequence of the promoter proximal E box is start site contains two E boxes. The transcriptional start CAGCTG, while the sequence of the promoter distal Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 9 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 MyoD ChIP at Lmod2 myogenin ChIP at Lmod2 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Myf5 ChIP at Lmod2 HEB ChIP at Lmod2 0 0 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Pol II ChIP at Lmod2 Ac H3 ChIP at Lmod2 D2 D6 D2 D6 Figure 6 Chromatin immunoprecipitation (ChIP) analysis of the leiomodin 2 (Lmod2)promoter. Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) and myofibers in differentiation media for the indicated number of days (D1-D10) were immunoprecipitated with antibodies against myogenin, MyoD, Myf5, HEB, RNA polymerase II (RNAP II), histone H3 acetylated at lysine 9 and/or 18 (H3 Ac9/18) or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Lmod2. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. box E box is CAAATG. The E boxes are separated by significantly present. MyoD and Myf5 were recruited 117 bp. Deletion of the promoter proximal E box in a with ahighaffinitytothepromoter on thefirst dayof luciferase reporter assay largely abolished the transacti- differentiation. Myogenin could be detected at this time vation of the Lmod2 promoter by myogenin or MyoD point, but the binding ratio was relatively low. The asso- [26]. At the Lmod2 promoter, we saw that HEB was pre- ciation of myogenin increased greatly by two days of dif- sent in proliferating cells, but none of the MRFs were ferentiation. Myogenin remained bound at six days of Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 10 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 differentiation, but dissociated from the promoter by the was recruited, followed by HEB, which then dissociated tenth day of differentiation. MyoD and Myf5, recruited as Myf5 arrived. At this promoter, we again observed on the first day of differentiation, gradually dissociated that RNAP II occupancy and H3 acetylation greatly from the promoter after this time point. MyoD was increased at six days of differentiation as compared to undetectable at three days of differentiation, while Myf5 two days of differentiation. remained associated with the promoter until sometime after six days of differentiation. The occupancy of HEB HEB replaces E12/E47 at specific promoters during continued to increase during the initial stages of differ- differentiation entiation, peaking at two days of differentiation. HEB At each of these promoters, we found the distinct pat- appeared to begin to dissociate from the promoter after tern of HEB binding very surprising. We had anticipated two days of differentiation. HEB was only weakly seeing HEB bound to the promoter whenever an MRF detected at three days of differentiation and the levels was bound. While there are overlaps, the pattern of continued to decrease at six days of differentiation, HEB binding is temporally controlled and unique to becoming undetectable by ten days of differentiation. each gene. We wondered if perhaps these findings could The levels of RNAP II and AcH3 were significantly be the result of the binding of the E2A gene products higher at six days of differentiation than at two days of E12 or E47 in a compensatory fashion. We reasoned differentiation. that E proteins might be associated at all time points, The final promoter analyzed was the Tcap promoter butthe Eprotein couldbeeither E12/E47 or HEB. To (Figure 7). The Tcap locus contains a highly conserved address this hypothesis, we repeated the ChIP assays on promoter proximal noncoding sequence that contains proliferating cells, on cells differentiated for two days two E boxes. Interestingly, neither the promoter proxi- and on cells differentiated for three days with antibodies mal E box (CATCTG) nor the promoter distal E box against the E2A gene products. In proliferating cells, (CATGTG) is a favored binding site for MyoD or myo- E12 or E47 could be detected at the Des and Lmod2 genin. However, MyoD and myogenin can activate this promoter binding sites (Figure 8A). For Lmod2,the promoter, and myogenin binds to the promoter during binding of E12/E47 was detected with HEB in proliferat- embryogenesis [27]. In proliferating cells, none of the ing cells, but E12/E47 was lost as cells began to differ- MRFs or HEB is bound. At day one of differentiation, entiate while HEB levels increased (Figure 8B). At two MyoD was recruited to the Tcap promoter. MyoD days of differentiation, E12/E47 could still be detected at remained associated with the promoter until two days of the Des promoter, but that binding was lost at three differentiation, but dissociated from the promoter by days of differentiation (Figure 8C). At both of these pro- threedaysofdifferentiation.Thisresultisconsistent moter binding sites, we observed an exchange of E12/ with our promoter characterization of Tcap,aswe have E47 and HEB as cells began to differentiate. E12/E47 shown that the promoter proximal E box is required for was not detected on any of the other promoters assayed both the activity in C C cells and the activation by in this study. We have analyzed additional promoter 2 12 MyoD in NIH3T3 cells [27]. Myogenin was not proximal elements of differentiation-specific genes and recruited to the Tcap promoter at any time point tested, have found that E12/E47 was associated with the myo- which was surprising to us, as we have detected myo- genin(myog), troponin C, type 2(Tnnc2) and myosin genin binding to the Tcap promoter in skeletal muscle heavy chain 3 (Myh3) promoters in undifferentiated tissue during embryogenesis [27]. The profile of Myf5 cells as well (Additional file 1 Figure S5A). We did not was particularly surprising. Myf5 was not associated observe an association with the troponin T, type 2 with the promoter at early stages of differentiation, but (Tnnt2) promoter. In each case, binding was lost by two could be weakly detected at the promoter after six days days of differentiation (data not shown). HEB was of differentiation. This binding increased greatly by ten observed on the Tnnt2, Myh3 and Tnnc2 promoters days of differentiation. HEB could be detected on the following two days of differentiation (Additional file 1 Tcap promoter after two days of differentiation, when Figure S5B). For the majority of the promoters analyzed MyoD was still present. However, by day three, MyoD here, it appears that HEB is the predominant E protein had departed the promoter and the occupancy of HEB recruited during differentiation and that the transient increased greatly at this time. The peak of HEB binding association of HEB is not compensated by an overlap- appears to occur at three days of differentiation. By day ping pattern of E12/E47. These data are consistent with six, HEB was at the same low level observed at two days previously reported data that have established an impor- of differentiation, and by ten days of differentiation it tant role for HEBb in inducing differentiation [15]. was undetectable. We found the pattern of MRF and Next, we asked whether the presence of HEB is required to displace E12/E47 from promoters as cells begin to HEB association at this gene particularly interesting, as differentiate. HEB levels were depleted with small there is almost no overlap between these factors. MyoD Londhe and Davie Skeletal Muscle 2011, 1:14 Page 11 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 MyoD ChIP at Tcap myogenin ChIP at Tcap 2 2 1 1 0 0 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Myf5 ChIP at Tcap HEB ChIP at Tcap 25 20 UD D1 D2 D3 D6 D10 UD D1 D2 D3 D6 D10 Pol II ChIP at Tcap Ac H3 ChIP at Tcap D2 D6 D2 D6 Figure 7 Chromatin immunoprecipitation (ChIP) analysis of the titin cap (Tcap)promoter. Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) and myofibers in differentiation media for the indicated number of days (D1-D10) were immunoprecipitated with antibodies myogenin, MyoD, Myf5, HEB, RNA polymerase II (RNAP II), histone H3 acetylated at lysine 9 and/or 18 (H3 Ac9/18) or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Tcap. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. hairpin (shRNA) constructs targeting HEB. Stable cell 8E). No change in E12/E47 expression was detected by lines expressing these constructs were screened for gene expression analysis (data not shown). ChIP assays expression of HEB and E12/E47. We proceeded with a with antibodies against E12/E47 were performed on construct that showed a 73% knockdown of HEB cells differentiated for two days. We saw no enhanced expression by RNA analysis (Figure 8D) and a reduction association of E12/E47 in cells reduced in expression of in protein expression by Western blot analysis (Figure HEB (Figure 8F), indicating that HEB is not required for Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 12 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 A. E12/47 ChIP in proliferating myoblasts Ckm Desmin Tnni2 Lmod2 Tcap B. C. E protein ChIP at Lmod2 E protein ChIP at Desmin 8 4 6 3 HEB E12/47 HEB E12/47 HEB E12/47 HEB E12/47 UD Day2 Day 2 Day3 D. E. F. E12/47 ChIP in myotubes (D2) HE B 1.5 1.5 0.5 0.5 probe: α HEB scr H E B scr K D scr K D scr K D scr K D scr K D sh RNA C mk D e smin Tn n i2 L mo d 2 Tca p Figure 8 E12/E47 and HEB exchange at the leiomodin 2 (Lmod2) and desmin (Des) promoters. (A) E12/E47 binds to the promoters of Des and Lmod2 in myoblasts. Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) were immunoprecipitated with antibodies against the E2A gene products. Immunoprecipitated DNA was purified and amplified with primers specific to the promoters of Ckm, Des, Tnni2, Lmod2 and Tcap. (B) E12/E47 and HEB exchange at the Lmod2 promoter. Cross-linked extracts from proliferating myoblasts and myofibers in differentiation media for two days were immunoprecipitated with antibodies against the E2A gene products, HEB or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Lmod2. (C) E12/E47 and HEB exchange at the Des promoter. Cross- linked extracts from myofibers in differentiation media for two or three days were immunoprecipitated with antibodies against the E2A gene products, HEB or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Des. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. (D) Gene expression analysis of HEB in cells expressing a small hairpin RNA (shRNA) construct targeting HEB or a scrambled control (scr). (E) Western blot analysis of the cells described in Figure 8D. The Western blot was probed with antibodies against HEB. (F) HEB is not required to displace E12/E47 at promoters. Results of chromatin immunoprecipitation assays performed after two days of differentiation on HEB-depleted cells and the scr control are shown. R el ati v e gen e ex pres s i on Fold Enrichment Fold Enrichment scr HEB shRNA Fo l d e n ri c h me n t Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 13 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 the displacement of E12/E47 from promoters during co-occupied the Des and Tnni2 promoters (Figure 9B). differentiation. These data confirm that MyoD, myogenin and Myf5 are bound to the same promoter elements. Myogenin, MyoD and Myf5 co-occupy promoters We were particularly interested in the binding profile of Discussion Myf5. For each gene tested, Myf5 had a unique binding We have found that each muscle gene assayed showed a pattern that was distinct from the binding patterns of unique temporal association of the MRFs and E pro- MyoD and myogenin. We were surprised to observe teins. We were surprised to observe the dynamic and that the profile of Myf5 overlapped with myogenin at transient roles of the MRFs on each of these promoters. several genes. To understand whether Myf5 and myo- MyoD has been proposed to be a “pioneer” transcription genin also colocalized during embryonic muscle devel- factor required to initiate the cascade of regulatory opment, we repeated the ChIP experiments in skeletal events required to initiate expression of muscle-specific muscle tissue derived from embryonic day 18.5 (E18.5) genes [29]. MyoD recruits chromatin-modifying activ- embryos. We chose E18.5 as it is late in embryogenesis ities that alter both the regional histone modifications and is a time point at which myogenin is assumed to be and the chromatin remodeling at promoter binding sites highly active. Myf5 is known to function only during the [7,30]. It is thought that these events then allow the early stages of myogenesis and thus would not be subsequent binding and transcriptional activity of myo- expected to contribute to gene expression at this stage. genin. Our data are consistent with this model, as we Moreover, Myf5 transcripts are not observed in embryos observed early associations of MyoD followed by the after E14 [28]. However, we detected Myf5 protein in association of myogenin. Our gene expression data also hindlimb samples at late embryonic stages (Additional show that for most genes examined in this study, the file 1 Figure S6). As expected, we detected myogenin at recruitment of myogenin is coincident with high levels several muscle-specific promoters, including Lmod2 and of transcription. Our data are consistent with those Des (Figure 9A). Consistent with our C C data, Myf5 reported in other studies that showed that at the genes 2 12 colocalized with myogenin at both of these promoters whose expression marks late myogenesis, Ckm and Des, in vivo (Figure 9A). After confirming that myogenin and MyoD is bound first, followed by the appearance of Myf5 appear to bind to the same sequences at a late myogenin, Mef2 (myocyte enhancer factor 2) and Brg1 time point in embryogenesis, we were very interested in (Brahma-related gene 1), the catalytic subunit of the understanding whether the overlapping pattern was Swi/Snf chromatin remodeling complex [25]. In this occurring in two cell populations or whether Myf5, prior study, it was also shown that the recruitment of myogenin was coincident with high levels of transcrip- myogenin and MyoD co-occupy promoters. To address this question, we performed sequential ChIP(ChIP-seq) tion of these genes in embryonic tissue. In our study, assays for myogenin, MyoD and Myf5 in C C cells. To the peak of transcription and myogenin binding corre- 2 12 address whether Myf5 and myogenin co-occupy promo- lated with high levels of RNAP II promoter occupancy ters, cells were differentiated for three days, immunopre- and histone H3 acetylation at the majority of genes cipitated with Myf5 antibodies and subsequently assayed. We note that the histone H3 acetylation levels immunoprecipitated with antibodies against myogenin. continued to rise following the departure of MyoD at We detected co-occupancy of Myf5 and myogenin at several genes. These data suggest that while MyoD may the Tnni2 and Des promoters (Figure 9B). We also per- be the initiating factor for chromatin modifications at formed the experiment with antibodies against myo- the promoter, the continued presence of MyoD is not genin first, followed by immunoprecipitation with Myf5 required for further increases in histone H3 acetylation. antibodies, and, again, co-occupancy of Myf5 and myo- The pattern of Myf5 binding was highly surprising. On geninonthe Tnni2 and Des promoters was confirmed certain genes, the Myf5 binding pattern overlapped with (data not shown). We next assayed for co-occupancy of MyoD, but at other genes, the binding pattern over- MyoD and myogenin. Cells were differentiated for three lapped with myogenin. In each case, the binding profile days, immunoprecipitated with antibodies against MyoD of Myf5 suggests that Myf5 has a previously uncharac- and subsequently immunoprecipitated with antibodies terized role in mediating gene expression in differentiat- against myogenin. Again, we observed co-occupancy of ing cells. While it is known that Myf5 cannot mediate MyoD andmyogeninonthe Des and Tnni2 promoters differentiation without myogenin or MyoD [8], our data (Figure 9B). Finally, we asked whether Myf5 and MyoD suggest that Myf5 does cooperate with both MyoD and co-occupy promoters. Differentiated cell extract was myogenin. Other groups have suggested that C C 2 12 immunoprecipitated with antibodies against Myf5 and cells, or the C cells used to derive them, have two subsequently immunoprecipitated with antibodies populations of myoblasts: a MyoD-expressing population against MyoD. We observed that Myf5 and MyoD thought to be the differentiating population and a Londhe and Davie Skeletal Muscle 2011, 1:14 Page 14 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 A. E18.5 ChIP at Lmod2 E18.5 ChIP at Desmin IgG myog Myf5 IgG myog Myf5 B. ChIP at Desmin ChIP at Desmin ChIP at Desmin 20 10 0 0 1st IP: IgG Myf5 1st IP: IgG 1st IP: IgG MyoD Myf5 2nd IP: MyoD MyoD 2nd IP: myog myog 2nd IP: myog myog ChIP at Tnni2 ChIP at Tnni2 ChIP at Tnni2 50 25 40 20 30 15 20 10 0 0 0 1st IP: IgG Myf5 1st IP: IgG MyoD 1st IP: IgG Myf5 2nd IP: myog myog 2nd IP: myog myog 2nd IP: MyoD MyoD Figure 9 Myogenin and Myf5 colocalize in vivo and co-occupy promoters with MyoD. (A) Myf5 and myogenin colocalize on promoters during embryonic development. Chromatin immunoprecipitation (ChIP) analysis was performed on skeletal muscle from E18.5 embryos. The immunoprecipitated DNA was purified and amplified with primers specific to the promoter of leiomodin 2 (Lmod2) and desmin (Des). The fold enrichment values were calculated as described in Methods. (B) Myf5, myogenin and MyoD co-occupy promoters. Cross-linked extracts from myofibers in differentiation media for two days were immunoprecipitated with antibodies against the indicated antibody (1st IP) or IgG. The immunoprecipitated complexes were released and immunoprecipitated again with antibodies against the indicated antibody (2nd IP). The immunoprecipitated DNA was purified and amplified with primers specific to the promoters of Tnni2 and Des. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Fold Enrichment Londhe and Davie Skeletal Muscle 2011, 1:14 Page 15 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 nondifferentiating or reserve population that expresses recruitment and release ofHEB.Thispattern wasnot Myf5 [31,32]. However, in our studies, we can conclude compensated by E12/E47, as we observed E12/E47 bind- that Myf5 is expressed in differentiating cells and that it ing to only two of the promoters in this study at early colocalizes with MyoD and myogenin on specific pro- time points. At three days of differentiation, E12/E47 moters in C C cells. Our data are highly suggestive was not detected at any of the promoters analyzed. At 2 12 that Myf5 plays a role in differentiation, but additional Des, the only gene highly expressed during proliferation examined in this study, we did observe an association experiments are required to confirm this hypothesis. with both E12/E47 and HEB in proliferating cells. At The binding pattern of myogenin was surprising as Lmod2, we also observed an early recruitment of E12/ well. The association of myogenin with muscle-specific genes as cells began to differentiate was expected, as E47 and HEB, whereas we observed only late recruit- myogenin is greatly upregulated at this time. However, ment of HEB at genes such as Tnni2.Wehypothesize the relatively brief association of myogenin with target that E12/E47 might be required at a subset of genes genes was unexpected. A transient role of myogenin on whose expression is immediately required as cells begin target genes has previously been suggested, as myogenin to differentiate. While Lmod2 is not significantly appears to have distinct target gene sets during embryo- expressed in proliferating cells, Lmod2 is upregulated genesis and in adult satellite cells and adult tissue very rapidly upon differentiation, and while the expres- [26,33]. Our data suggest that myogenin may mediate sion does continue to increase over an extended time changes at the promoter that maintain high levels of course, the expression increases only two fold. This is in expression without the continued presence of myogenin. contrast to genes such as Tnni2, where the expression Candidates for such a change include the switching of level increases ten fold over the extended time course. core promoter complexes, which has been observed in Lmod2 does not reach the high levels of transcription skeletal muscle differentiation. A TATA-binding protein seen at Tnni2 that coincide with the peak of HEB bind- (TBP)-related factor, TRF3, and an associated TBP-asso- ing. It is possible that the early recruitment of E12/E47 ciated factor, TAF3, have been shown to be targeted by and HEB at Lmod2 helps to support a relatively constant MyoD to the myogenin promoter following differentia- level of expression that initiates immediately upon dif- tion [31,34]. TBP is expressed in proliferating myoblasts, ferentiation.Itisstrikingthatatbothgenes wherewe but following differentiation, TBP is downregulated and observed the binding of E12/E47, we also observed that TRF3 and TAF3 are upregulated. It is also possible that HEB appeared to replace E12/E47 as cells began to dif- ferentiate. The binding pattern of HEB at Tcap is parti- myogenin may direct epigenetic changes that maintain cularly interesting. HEB binding peaks at a time point gene expression. The binding pattern of HEB was very surprising to us when no MRFs are detected. Reduced levels of binding as well. Detailed biophysical experiments have shown are detected at two additional time points when MyoD that MRF and E protein heterodimers are highly stable and Myf5 are bound on the individual days. Thus, while when bound to DNA. These studies have also indicated the HEB binding profile does overlap with MRF binding that heterodimers likely form on the DNA. MyoD and as predicted by the biophysical studies, the occupancy of E47 heterodimers are not detected in diluted conditions HEB does not always overlap with the occupancy of the without DNA. However, in the presence of DNA, het- MRFs. erodimeric complexes are formed almost exclusively While these data have revealed many novel findings [35]. Additional work has shown that the weak MyoD regarding the recruitment of the MRFs and E proteins, homodimers and heterodimers that can form in the many questions remain. The additional factors and absence of DNA are equally stable [36]. This suggests DNA elements that mediate the individual recruitment that the MRFs and E proteins are likely to be mono- and release of each of these factors remain to be charac- meric in the cell. In this work {Maleki, 2002 #376}, it terized. Many elegant studies of the role of chromatin was also shown that while MyoD or myogenin E protein modification in muscle differentiation have suggested heterodimers on DNA were the most energetically that epigenetic events are important mediators in the favorable, MyoD and myogenin homodimers can bind activation of muscle genes. The Swi/Snf chromatin E boxes with considerable positive cooperativity, while remodeling complex promotes muscle differentiation, E12 homodimers exhibited negative cooperativity. The and it is known that the Swi/Snf complex is recruited to negative cooperativity of E12 suggests that the heterodi- both the Des and Ckm promoters studied here [25,37]. Important questions for future studies include how chro- mer may form on DNA by binding of the E12 monomer matin remodelers and chromatin-modifying enzymes followed by binding of the MRF monomer. contribute to the recruitment and release of the myogenic Given these data, we anticipated detecting E proteins ontheDNAthroughout thetimecourseof differentia- regulatory factors and E proteins to regulate muscle gene tion. Instead, we found a highly dynamic pattern of expression. Londhe and Davie Skeletal Muscle 2011, 1:14 Page 16 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 anti-Myf5 (C-20; Santa Cruz Biotechnology), anti-MyoD Conclusions (5.8A; Santa Cruz Biotechnology), anti-GAPDH (anti-gly- Here we have shown that MyoD, myogenin and Myf5 ceraldehyde 3-phosphate dehydrogenase; Chemicon have sequential and transient roles on each of the pro- International, Billerica, MA USA) and anti-MyoG (F5D).. moters assayed. For almost every gene assayed, we The F5D antibody developed by W. E. Wright was found that the binding of myogenin and HEB correlated obtained from the Developmental Studies Hybridoma with high levels of RNAP II occupancy, histone H3 acet- Bank under the auspices of the NICHD and maintained ylation and the peak of transcription as assayed by by the University of Iowa, Department of Biology, Iowa mRNA levels. We found that the primary E protein City, IA USA. Normal rabbit immunoglobulin G (IgG) recruited to late differentiation genes is HEB. At the few (Santa Cruz Biotechnology, Santa Cruz, CA USA) was promoters where E12/E47 was detected at early stages, used as a nonspecific control. HEB replaced E12/E47 during differentiation. Finally, we have shown that MyoD, myogenin and Myf5 colocalize Quantitative reverse transcriptase-polymerase chain on promoters, suggesting that Myf5 contributes to the reaction assays gene expression of late differentiation genes. RNA was isolated from C C cells by TRIzol reagent 2 12 extraction (Invitrogen, Carlsbad, CA. Two micrograms Methods of total RNA were reverse-transcribed with Multi- Cell culture Scribe™ Reverse Transcriptase (Applied Biosystems, Cells were grown in a humidified chamber at 37°C with Carlsbad, CA USA. cDNA equivalent to 40 ng was used 5% CO . Proliferating C C myoblasts (American Type 2 2 12 for quantitative reverse transcriptase-polymerase chain Culture Collection, Manassas, VA, USA) were grown in reaction (qRT-PCR) amplification (Applied Biosystems, Dulbecco’s modified Eagle’s medium (DMEM) supple- Foster City,CAUSA)withSYBRGreenPCRMaster mented with 10% fetal bovine serum (Thermo Scientific Mix (Applied Biosystems, Foster City, CA USA). Sam- HyClone, Logan, UT USA. To induce differentiation ples in which no RT was added were included for each into myotubes, cells were grown to 70% confluence and RNA sample. For measurements of relative gene expres- media were switched to DMEM supplemented with 2% sion, a fold change was calculated for each sample pair horse serum (Thermo Scientific HyClone, Logan, UT and normalized to the fold change observed at HPRT. USA). C C cells were grown in differentiation medium 2 12 mRNA expression levels were quantitated using a cali- for the number of days indicated in each experiment. bration curve based on known dilutions of concentrated cDNA. Each mRNA value was normalized to that of Western blot analysis HPRT. Fold change was calculated by dividing the Cell extracts were made by lysing phosphate-buffered sal- mRNA expression values of each sample pair. qRT-PCR ine-washed cell pellets in radioimmunoprecipitation data were calculated using the comparative C method assay buffer supplemented with protease inhibitors (Applied Biosystems, Foster City, CA USA). Standard (Complete Protease Inhibitor Cocktail Tablets; Roche deviations from the mean of the ΔC values were calcu- Applied Science, Indianapolis, IN USA. Following incu- lated from three independent RNA samples and used to bation on ice, clear lysates were obtained by performing generate error bars. Intron-spanning primers to the cod- centrifugation. Protein concentrations were determined ing region of Lmod2, Des, Tnni2, Ckm and Tcap are by using the Bio-Rad Protein Assay (Bio-Rad, Hercules, described in Additional file 1 Table S1. All qPCR assays CA USA. For each sample, 30 μg of protein were loaded were performed in triplicate, and at least two indepen- onto each gel. Proteins were transferred onto a nitrocel- dent RNA samples were assayed for each time point. lulose membrane using a tank blotter (Bio-Rad, Hercules, CA USA), then blocked using 5% milk and 1× Tris-buf- ChIP assays fered saline plus Tween 20 (TBST) and incubated with Cell culture ChIP assays were performed and quantified primary antibody overnight at 4°C. Membranes were as described previously [38] with the following modifica- then washed with 1× TBST and incubated with the cor- tions: 1 × 10 cells were used for each immunoprecipita- responding secondary antibody. Membranes were again tion, and protein A agarose beads (Invitrogen) were washed with 1× TBST, incubated with chemilumines- used to immunoprecipitate the antibody-antigen com- cence substrate according to the manufacturer’s protocol plexes. ChIP assays of embryonic tissue were performed (SuperSignal West Pico Chemiluminescent Substrate; as previously described [26]. Limb tissue from wild-type Pierce Biotechnology, Rockford, IL USA and visualized C57BL/6 mice{Jackson Laboratory, Bar Harbor, ME by autoradiography. The antibodies used include anti- USA) was isolated, cross-linked and enriched for nuclei. HEB (A-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E12/E47 (Yae; Santa Cruz Biotechnology), Nuclear extracts of limb tissue were precleared using Londhe and Davie Skeletal Muscle 2011, 1:14 Page 17 of 18 http://www.skeletalmusclejournal.com/content/1/1/14 Acknowledgements incubation with protein A agarose beads (Invitrogen, We thank Meiling Zhang for assistance in establishing stable cell lines Carlsbad, CA USA), which were also used to immuno- expressing shRNA constructs against HEB. This work was supported by precipitate the antibody complexes from tissue extracts grants from the Central Research Committee, Southern Illinois University School of Medicine, and by grant 159609 from the American Cancer Society, following antibody addition to the incubation mix. Anti- Illinois Division, awarded to JD. bodies against the following proteins were used: MyoD (5.8A; Santa Cruz Biotechnology, Santa Cruz, CA USA), Authors’ contributions PL and JD designed the experimental approach. PL performed the described HEB (A-20; Santa Cruz Biotechnology, Santa Cruz, CA experiments, and JD wrote the manuscript with assistance from PL. Both USA), Myf5 (C-20; Santa Cruz Biotechnology, Santa authors read and approved the final manuscript. Cruz, CA USA), E proteins (Yae; Santa Cruz Biotechnol- Competing interests ogy, Santa Cruz, CA), myogenin (F5D; Developmental The authors declare that they have no competing interests. Studies Hybridoma Bank), RNAP II (H-224; Santa Cruz Biotechnology, Santa Cruz, CA USA) and histone H3 Received: 8 October 2010 Accepted: 4 April 2011 Published: 4 April 2011 acetylated at lysine 9 and/or 18 (H3.Ac9/18; Millipore, References Billerica, MA USAUpstate Biotechnology. Primers span- 1. Kablar B, Rudnicki MA: Skeletal muscle development in the mouse ning the described promoter elements of Lmod2, Des, embryo. Histol Histopathol 2000, 15:649-656. Tnni2, Ckm, Tcap, Myog, Tnnt2, Myh3 and Tnnc2 are 2. Parker MH, Seale P, Rudnicki MA: Looking back to the embryo: defining -[Δ][Δ]Ct transcriptional networks in adult myogenesis. Nat Rev Genet 2003, described in Additional file 1 Table S1. 2 values 4:497-507. were calculated using the following formula based on 3. 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Blais A, Tsikitis M, Acosta-Alvear D, Sharan R, Kluger Y, Dynlacht BD: An initial blueprint for myogenic differentiation. Genes Dev 2005, 19:553-569. All ChIP assays shown in the figures are representative 7. Cao Y, Kumar RM, Penn BH, Berkes CA, Kooperberg C, Boyer LA, Young RA, of at least three individual experiments. Tapscott SJ: Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters. EMBO J 2006, 25:502-511. ChIP-seq assay 8. Valdez MR, Richardson JA, Klein WH, Olson EN: Failure of Myf5 to support ChIP-seq analysis was performed as previously described myogenic differentiation without myogenin, MyoD, and MRF4. Dev Biol [39] with antibodies against myogenin (F5D; Develop- 2000, 219:287-298. 9. 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Cell 2003, • Convenient online submission 115:751-763. • Thorough peer review doi:10.1186/2044-5040-1-14 • No space constraints or color figure charges Cite this article as: Londhe and Davie: Sequential association of myogenic regulatory factors and E proteins at muscle-specific genes. • Immediate publication on acceptance Skeletal Muscle 2011 1:14. • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Apr 4, 2011

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