- Research
- Open Access
Sequential association of myogenic regulatory factors and E proteins at muscle-specific genes
- Priya Londhe1 and
- Judith K Davie1Email author
https://doi.org/10.1186/2044-5040-1-14
© Londhe and Davie; licensee BioMed Central Ltd. 2011
- Received: 8 October 2010
- Accepted: 4 April 2011
- Published: 4 April 2011
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 C2C12 differentiation, resulting in several surprising findings. The pattern of recruitment is 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.
Keywords
- C2C12 Cell
- Transcriptional Start Site
- Binding Pattern
- Binding Profile
- Proliferate Myoblast
Background
The entire process of skeletal muscle differentiation is controlled by four highly related basic helix-loop-helix (bHLH) proteins referred to as the myogenic regulatory factors (MRFs). The MRFs have distinct but overlapping patterns of gene expression during muscle development [1]. Gene knockouts of each factor in the mouse have revealed that each MRF has a unique role in skeletal muscle differentiation. Myf5, Myf6 (also known as MRF4) and MyoD are not required for viability, although each mutant has a distinct phenotype [2]. In the combined absence of Myf5, Myf6 and MyoD, myoblasts are not specified and no skeletal muscle forms, resulting in a lethal phenotype [3]. Myogenin is the only MRF singly required for viability [4, 5]. Mice heterozygous for the null allele appear normal, while mice lacking myogenin die at birth. The myogenin-null mice have myoblasts, but very few muscle fibers. This suggests that myogenin is not required for the specification of skeletal muscle, but is required for the later stages of myofiber fusion.
MyoD and myogenin have been shown to bind highly overlapping gene sets, although certain genes appear to be selective for either factor [6, 7]. However, the high degree of overlap in the binding patterns suggests that the majority of genes utilize both factors to activate gene expression. Previous work has shown that certain genes require the sequential expression of both MyoD and myogenin to activate gene expression [7]. The present work suggests that the activation of specific targets requires MyoD and its associated chromatin-modifying activities before myogenin can activate transcription. Why MyoD cannot activate transcription without myogenin on these genes is still unknown.
Recent work on Myf5 has revealed unexpected roles for this factor in adult animals. As mentioned above, Myf5 functions as a determination gene in early myogenesis. The role of Myf5 in later stages is unclear. In the absence of MyoD, Myf6 or myogenin, Myf5 is unable to promote differentiation from myoblasts [8]. This finding suggests that Myf5 functions only in muscle progenitor cells (MPCs) and myoblasts. However, recent work has shown that Myf5-null mice exhibit impaired muscle regeneration with a significant increase in muscle fiber hypertrophy and a delay in differentiation [9]. However, satellite cell numbers were not significantly altered in the Myf5-null animals, although a modest impaired proliferation was observed under some conditions in vitro. This work highlights the questions still remaining about the roles of the MRFs at distinct stages in myogenesis.
All bHLH transcription factors function as either homodimers or heterodimers. The bHLH transcription factors are loosely grouped into several categories: the widely expressed E proteins, including the E2A gene products E12 and E47, HEB, E2-2 and Daughterless, are in the class I category and the MRF family is included in the tissue-specific class II category. Class II bHLH proteins form weak homodimers and preferentially heterodimerize with E proteins [10]. Prior in vitro experiments have demonstrated that the class II MRFs form avid heterodimers with class I E proteins, but homodimerize poorly in the presence of DNA sites [11–14]. Thus, it is thought that the MRFs function as heterodimers with ubiquitous E proteins. The E proteins suggested to be involved in skeletal muscle differentiation are the E2A gene products E12 and E47, as well as HEB. Recent work has suggested that HEB may be the primary E protein that regulates skeletal muscle differentiation [15].
The MRFs all bind the canonical E-box consensus sequence, CANNTG. Genome-wide binding studies have revealed that both MyoD and myogenin preferentially bind E boxes with a consensus sequence of CASCTG (International Union of Pure and Applied Chemistry nomenclature http://www.iupac.org), where S represents G or C [7, 16]. The site recognized at the highest frequency is CAGCTG. The sequences flanking the E box also make important contributions to the binding affinity and contribute to the overall consensus sequence elements determined for MyoD and myogenin [11, 16].
Given the high degree of overlap detected in the genome occupancy of MyoD and myogenin, we were interested in understanding the binding profile of these factors over a time course of differentiation. We were also interested in the binding profile of Myf5, as binding data for this factor during differentiation have not been reported. We also sought to compare the DNA-bound profiles of the MRFs with the E proteins. Thus, we initiated a temporal analysis of the binding of MyoD, Myf5, myogenin and the E proteins in C2C12 cells, a widely used cell culture model for myoblast differentiation. These binding profiles were correlated with the levels of mRNA present in the cells, the levels of RNA polymerase II (RNAP II) occupancy and histone H3 acetylation present at the promoters. We show several novel findings. Surprisingly, we have found that the pattern of recruitment is unique to each gene, although some common features arise. As others have observed, we saw an early association of MyoD with most of these genes. In a cooperative pattern, myogenin then binds many of these promoters following MyoD. Unexpectedly, we found that Myf5 is also associated with genes expressed late in differentiation and often colocalizes with myogenin. We show that this colocalization also occurs in vivo at a late embryonic time point. The binding of each of the MRFs is transient. We also show that the occupancy of the E proteins is transient and that the occupancy often peaks concurrently with the peak in gene transcription. While the E2A gene products could be detected on a few genes in our study in proliferating cells, HEB does appear to be the dominant E protein used during differentiation. At the genes occupied by E12/E47 in early myogenesis, we detected a switch in occupancy for HEB during differentiation. Taken together, our data suggest new models for the recruitment of MRFs and E proteins and support a novel role for Myf5 during differentiation.
Results
Time course of MRF and E protein expression
Expression of myogenic regulatory factors (MRFs) and E proteins over a time course of differentiation in C 2 C 12 cells. C2C12 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.
Expression of genes chosen for analysis
Several genes were chosen for this analysis. We chose muscle creatine kinase (Ckm) and desmin (Des), as both are well-characterized genes whose expression increases during differentiation. As the regulatory regions of these genes have been studied extensively by others, promoter proximal binding sites for the MRFs are well defined [19–25]. We also chose the fast-twitch skeletal muscle troponin I, type 2 (Tnni2) and leiomodin 2 (Lmod2) genes, as these have been characterized as myogenin-dependent targets in embryonic skeletal muscle during embryogenesis, and the promoter proximal MRF binding sites are known [26]. We also chose titin cap (Tcap), also known as telethonin. We have recently characterized the promoter proximal regulatory elements of the gene encoding Tcap and identified a promoter proximal fragment that recapitulates the expression pattern of Tcap in reporter assays and is bound by myogenin in vivo[27]. For each of these genes, we profiled the change in RNA expression profiles over a time course of differentiation.
Expression profiles of genes upregulated during skeletal muscle differentiation in C 2 C 12 cells. (A) Expression profiles of 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 C2C12 myoblast cells. (B) The relative transcription levels of each of 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.
Binding of MRFs and E proteins to muscle-specific genes
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.
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.
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.
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.
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.
HEB replaces E12/E47 at specific promoters during differentiation
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.
Myogenin, MyoD and Myf5 co-occupy promoters
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.
Discussion
We have found that each muscle gene assayed showed a unique temporal association of the MRFs and E proteins. We were surprised to observe the dynamic and transient roles of the MRFs on each of these promoters. MyoD has been proposed to be a "pioneer" transcription factor required to initiate the cascade of regulatory events required to initiate expression of muscle-specific genes [29]. MyoD recruits chromatin-modifying activities that alter both the regional histone modifications and the chromatin remodeling at promoter binding sites [7, 30]. It is thought that these events then allow the subsequent binding and transcriptional activity of myogenin. Our data are consistent with this model, as we observed early associations of MyoD followed by the association of myogenin. Our gene expression data also show that for most genes examined in this study, the recruitment of myogenin is coincident with high levels of transcription. Our data are consistent with those reported in other studies that showed that at the genes whose expression marks late myogenesis, Ckm and Des, MyoD is bound first, followed by the appearance of myogenin, Mef2 (myocyte enhancer factor 2) and Brg1 (Brahma-related gene 1), the catalytic subunit of the Swi/Snf chromatin remodeling complex [25]. In this prior study, it was also shown that the recruitment of myogenin was coincident with high levels of transcription of these genes in embryonic tissue. In our study, the peak of transcription and myogenin binding correlated with high levels of RNAP II promoter occupancy and histone H3 acetylation at the majority of genes assayed. We note that the histone H3 acetylation levels continued to rise following the departure of MyoD at several genes. These data suggest that while MyoD may be the initiating factor for chromatin modifications at the promoter, the continued presence of MyoD is not required for further increases in histone H3 acetylation.
The pattern of Myf5 binding was highly surprising. On certain genes, the Myf5 binding pattern overlapped with MyoD, but at other genes, the binding pattern overlapped with myogenin. In each case, the binding profile of Myf5 suggests that Myf5 has a previously uncharacterized role in mediating gene expression in differentiating cells. While it is known that Myf5 cannot mediate differentiation without myogenin or MyoD [8], our data suggest that Myf5 does cooperate with both MyoD and myogenin. Other groups have suggested that C2C12 cells, or the C2 cells used to derive them, have two populations of myoblasts: a MyoD-expressing population thought to be the differentiating population and a nondifferentiating or reserve population that expresses Myf5 [31, 32]. However, in our studies, we can conclude that Myf5 is expressed in differentiating cells and that it colocalizes with MyoD and myogenin on specific promoters in C2C12 cells. Our data are highly suggestive that Myf5 plays a role in differentiation, but additional experiments are required to confirm this hypothesis.
The binding pattern of myogenin was surprising as well. The association of myogenin with muscle-specific genes as cells began to differentiate was expected, as myogenin is greatly upregulated at this time. However, the relatively brief association of myogenin with target genes was unexpected. A transient role of myogenin on target genes has previously been suggested, as myogenin appears to have distinct target gene sets during embryogenesis and in adult satellite cells and adult tissue [26, 33]. Our data suggest that myogenin may mediate changes at the promoter that maintain high levels of expression without the continued presence of myogenin. Candidates for such a change include the switching of core promoter complexes, which has been observed in skeletal muscle differentiation. A TATA-binding protein (TBP)-related factor, TRF3, and an associated TBP-associated factor, TAF3, have been shown to be targeted by MyoD to the myogenin promoter following differentiation [31, 34]. TBP is expressed in proliferating myoblasts, but following differentiation, TBP is downregulated and TRF3 and TAF3 are upregulated. It is also possible that myogenin may direct epigenetic changes that maintain gene expression.
The binding pattern of HEB was very surprising to us as well. Detailed biophysical experiments have shown that MRF and E protein heterodimers are highly stable when bound to DNA. These studies have also indicated that heterodimers likely form on the DNA. MyoD and E47 heterodimers are not detected in diluted conditions without DNA. However, in the presence of DNA, heterodimeric complexes are formed almost exclusively [35]. Additional work has shown that the weak MyoD homodimers and heterodimers that can form in the absence of DNA are equally stable [36]. This suggests that the MRFs and E proteins are likely to be monomeric in the cell. In this work {Maleki, 2002 #376}, it was also shown that while MyoD or myogenin E protein heterodimers on DNA were the most energetically favorable, MyoD and myogenin homodimers can bind E boxes with considerable positive cooperativity, while E12 homodimers exhibited negative cooperativity. The negative cooperativity of E12 suggests that the heterodimer may form on DNA by binding of the E12 monomer followed by binding of the MRF monomer.
Given these data, we anticipated detecting E proteins on the DNA throughout the time course of differentiation. Instead, we found a highly dynamic pattern of recruitment and release of HEB. This pattern was not compensated by E12/E47, as we observed E12/E47 binding to only two of the promoters in this study at early time points. At three days of differentiation, E12/E47 was not detected at any of the promoters analyzed. At Des, the only gene highly expressed during proliferation examined in this study, we did observe an association with both E12/E47 and HEB in proliferating cells. At Lmod2, we also observed an early recruitment of E12/E47 and HEB, whereas we observed only late recruitment of HEB at genes such as Tnni2. We hypothesize that E12/E47 might be required at a subset of genes whose expression is immediately required as cells begin to differentiate. While Lmod2 is not significantly expressed in proliferating cells, Lmod2 is upregulated very rapidly upon differentiation, and while the expression does continue to increase over an extended time course, the expression increases only two fold. This is in contrast to genes such as Tnni2, where the expression level increases ten fold over the extended time course. Lmod2 does not reach the high levels of transcription seen at Tnni2 that coincide with the peak of HEB binding. It is possible that the early recruitment of E12/E47 and HEB at Lmod2 helps to support a relatively constant level of expression that initiates immediately upon differentiation. It is striking that at both genes where we observed the binding of E12/E47, we also observed that HEB appeared to replace E12/E47 as cells began to differentiate. The binding pattern of HEB at Tcap is particularly interesting. HEB binding peaks at a time point when no MRFs are detected. Reduced levels of binding are detected at two additional time points when MyoD and Myf5 are bound on the individual days. Thus, while the HEB binding profile does overlap with MRF binding as predicted by the biophysical studies, the occupancy of HEB does not always overlap with the occupancy of the MRFs.
While these data have revealed many novel findings regarding the recruitment of the MRFs and E proteins, many questions remain. The additional factors and DNA elements that mediate the individual recruitment and release of each of these factors remain to be characterized. Many elegant studies of the role of chromatin modification in muscle differentiation have suggested that epigenetic events are important mediators in the activation of muscle genes. The Swi/Snf chromatin remodeling complex promotes muscle differentiation, and it is known that the Swi/Snf complex is recruited to both the Des and Ckm promoters studied here [25, 37]. Important questions for future studies include how chromatin remodelers and chromatin-modifying enzymes contribute to the recruitment and release of the myogenic regulatory factors and E proteins to regulate muscle gene expression.
Conclusions
Here we have shown that MyoD, myogenin and Myf5 have sequential and transient roles on each of the promoters assayed. For almost every gene assayed, we found that the binding of myogenin and HEB correlated with high levels of RNAP II occupancy, histone H3 acetylation and the peak of transcription as assayed by mRNA levels. We found that the primary E protein recruited to late differentiation genes is HEB. At the few promoters where E12/E47 was detected at early stages, HEB replaced E12/E47 during differentiation. Finally, we have shown that MyoD, myogenin and Myf5 colocalize on promoters, suggesting that Myf5 contributes to the gene expression of late differentiation genes.
Methods
Cell culture
Cells were grown in a humidified chamber at 37°C with 5% CO2. Proliferating C2C12 myoblasts (American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Thermo Scientific HyClone, Logan, UT USA. To induce differentiation into myotubes, cells were grown to 70% confluence and media were switched to DMEM supplemented with 2% horse serum (Thermo Scientific HyClone, Logan, UT USA). C2C12 cells were grown in differentiation medium for the number of days indicated in each experiment.
Western blot analysis
Cell extracts were made by lysing phosphate-buffered saline-washed cell pellets in radioimmunoprecipitation assay buffer supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail Tablets; Roche Applied Science, Indianapolis, IN USA. Following incubation on ice, clear lysates were obtained by performing centrifugation. Protein concentrations were determined by using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA USA. For each sample, 30 μg of protein were loaded onto each gel. Proteins were transferred onto a nitrocellulose membrane using a tank blotter (Bio-Rad, Hercules, CA USA), then blocked using 5% milk and 1× Tris-buffered saline plus Tween 20 (TBST) and incubated with primary antibody overnight at 4°C. Membranes were then washed with 1× TBST and incubated with the corresponding secondary antibody. Membranes were again washed with 1× TBST, incubated with chemiluminescence substrate according to the manufacturer's protocol (SuperSignal West Pico Chemiluminescent Substrate; Pierce Biotechnology, Rockford, IL USA and visualized by autoradiography. The antibodies used include anti-HEB (A-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E12/E47 (Yae; Santa Cruz Biotechnology), anti-Myf5 (C-20; Santa Cruz Biotechnology), anti-MyoD (5.8A; Santa Cruz Biotechnology), anti-GAPDH (anti-glyceraldehyde 3-phosphate dehydrogenase; Chemicon International, Billerica, MA USA) and anti-MyoG (F5D).. The F5D antibody developed by W. E. Wright was obtained from the Developmental Studies Hybridoma Bank under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA USA. Normal rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology, Santa Cruz, CA USA) was used as a nonspecific control.
Quantitative reverse transcriptase-polymerase chain reaction assays
RNA was isolated from C2C12 cells by TRIzol reagent extraction (Invitrogen, Carlsbad, CA. Two micrograms of total RNA were reverse-transcribed with MultiScribe™ Reverse Transcriptase (Applied Biosystems, Carlsbad, CA USA. cDNA equivalent to 40 ng was used for quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) amplification (Applied Biosystems, Foster City, CA USA) with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA USA). Samples in which no RT was added were included for each RNA sample. For measurements of relative gene expression, a fold change was calculated for each sample pair and normalized to the fold change observed at HPRT. mRNA expression levels were quantitated using a calibration curve based on known dilutions of concentrated cDNA. Each mRNA value was normalized to that of HPRT. Fold change was calculated by dividing the mRNA expression values of each sample pair. qRT-PCR data were calculated using the comparative Ct method (Applied Biosystems, Foster City, CA USA). Standard deviations from the mean of the ΔCt values were calculated from three independent RNA samples and used to generate error bars. Intron-spanning primers to the coding region of Lmod2, Des, Tnni2, Ckm and Tcap are described in Additional file 1 Table S1. All qPCR assays were performed in triplicate, and at least two independent RNA samples were assayed for each time point.
ChIP assays
Cell culture ChIP assays were performed and quantified as described previously [38] with the following modifications: 1 × 107 cells were used for each immunoprecipitation, and protein A agarose beads (Invitrogen) were used to immunoprecipitate the antibody-antigen complexes. ChIP assays of embryonic tissue were performed as previously described [26]. Limb tissue from wild-type C57BL/6 mice{Jackson Laboratory, Bar Harbor, ME USA) was isolated, cross-linked and enriched for nuclei. Nuclear extracts of limb tissue were precleared using incubation with protein A agarose beads (Invitrogen, Carlsbad, CA USA), which were also used to immunoprecipitate the antibody complexes from tissue extracts following antibody addition to the incubation mix. Antibodies against the following proteins were used: MyoD (5.8A; Santa Cruz Biotechnology, Santa Cruz, CA USA), HEB (A-20; Santa Cruz Biotechnology, Santa Cruz, CA USA), Myf5 (C-20; Santa Cruz Biotechnology, Santa Cruz, CA USA), E proteins (Yae; Santa Cruz Biotechnology, Santa Cruz, CA), myogenin (F5D; Developmental Studies Hybridoma Bank), RNAP II (H-224; Santa Cruz Biotechnology, Santa Cruz, CA USA) and histone H3 acetylated at lysine 9 and/or 18 (H3.Ac9/ 18; Millipore, Billerica, MA USAUpstate Biotechnology. Primers spanning the described promoter elements of Lmod2, Des, Tnni2, Ckm, Tcap, Myog, Tnnt2, Myh3 and Tnnc2 are described in Additional file 1 Table S1. 2-[Δ][Δ]C tvalues were calculated using the following formula based on the comparative Ct method: ΔCt, template (antibody) - ΔCt, template (IgG) = 2-[Δ][Δ]C t. Fold enrichments were determined using the formula: 2-[Δ][Δ]Ct (experimental)/2-[Δ][Δ]C t(reference, IgH). The standard error of the mean was calculated on the basis of replicate ΔCt values. The immunoglobulin H (IgH) locus was used to normalize the fold enrichments for the individual promoters. All ChIP assays shown in the figures are representative of at least three individual experiments.
ChIP-seq assay
ChIP-seq analysis was performed as previously described [39] with antibodies against myogenin (F5D; Developmental Studies Hybridoma Bank, Iowa City, IA USA) and Myf5 (C-20; Santa Cruz Biotechnology, Santa Cruz, CA USA).
shRNA knockdown
Cell lines depleted for HEB were constructed with shRNA constructs designed by the RNAi Consortium in the pLOK.1 plasmid (Open Biosystems, Huntsville, AL USA). Five constructs targeting murine HEB and one scrambled control were linearized, transfected into C2C12 cells and selected with 2 μg/ml puromyosin. Pooled clones were selected and propagated. Depletion was confirmed at the RNA and protein levels. For the HEB depletions, the expression of E12/E47 was also confirmed at the RNA and protein levels.
Declarations
Acknowledgements
We thank Meiling Zhang for assistance in establishing stable cell lines expressing shRNA constructs against HEB. This work was supported by grants from the Central Research Committee, Southern Illinois University School of Medicine, and by grant 159609 from the American Cancer Society, Illinois Division, awarded to JD.
Authors’ Affiliations
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