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. 2010 Apr 20;18(4):662-74.
doi: 10.1016/j.devcel.2010.02.014.

Genome-wide MyoD binding in skeletal muscle cells: a potential for broad cellular reprogramming

Yi Cao  1 , Zizhen Yao, Deepayan Sarkar, Michael Lawrence, Gilson J Sanchez, Maura H Parker, Kyle L MacQuarrie, Jerry Davison, Martin T Morgan, Walter L Ruzzo, Robert C Gentleman, Stephen J Tapscott
Affiliations

Genome-wide MyoD binding in skeletal muscle cells: a potential for broad cellular reprogramming

Yi Cao et al. Dev Cell. .

Abstract

Recent studies have demonstrated that MyoD initiates a feed-forward regulation of skeletal muscle gene expression, predicting that MyoD binds directly to many genes expressed during differentiation. We have used chromatin immunoprecipitation and high-throughput sequencing to identify genome-wide binding of MyoD in several skeletal muscle cell types. As anticipated, MyoD preferentially binds to a VCASCTG sequence that resembles the in vitro-selected site for a MyoD:E-protein heterodimer, and MyoD binding increases during differentiation at many of the regulatory regions of genes expressed in skeletal muscle. Unanticipated findings were that MyoD was constitutively bound to thousands of additional sites in both myoblasts and myotubes, and that the genome-wide binding of MyoD was associated with regional histone acetylation. Therefore, in addition to regulating muscle gene expression, MyoD binds genome wide and has the ability to broadly alter the epigenome in myoblasts and myotubes.

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Figures

Figure 1
Figure 1
MyoD binding in C2C12 myotubes. A) Average number of each E-box sequence under myotube peaks and control regions, where 1.0 indicates that each peak has on average one E-box of that sequence. Reverse complement sequences were combined. Compared to the controls, myotubes are enriched for CAGCTG and CAGGTG/CACCTG. B) Myotube peaks are enriched for 5’ A/G and depleted for 3’A at the positions immediately flanking the E-box (CAG[C/G]TG). The size of each nucleotide at a given position is proportional to the frequency of the nucleotide at that position. The darkness of the line connecting the two adjacent nucleotides is determined by the corresponding dinucleotide frequency. The counts of patterns on both strands are indicated above the graphs. C) Motifs enriched in E-box containing Myod binding regions in C2C12 myotube peaks. D) Motifs enriched in non-E-box containing MyoD binding regions in C2C12 myotube peaks.
Figure 2
Figure 2
MyoD binding in C2C12 myoblasts compared to C2C12 myotubes. A) 50% and 95% confluent C2C12 myoblasts share similar binding profiles. The X- and Y-axes of the scatter plot correspond to the Variance Stabilization and Normalization (vsn) transformed number of reads in 50% and 95% confluent C2C12 myoblasts in the combined peak regions. The middle diagonal line is the linear fit. The two flanking lines define the 95% confidence interval for differential peaks. Grey scale values represent a smoothed density estimate for the bulk of the data. Individual points shown at the fringes represent the outliers in low density regions. Peaks within +/−2 kb of the TSS of selected muscle genes (Myh3, Acta, Myog, Cdh14, Mef2a, Des, and Ckm) are annotated by gene names. Note that some genes have multiple peaks within the promoter regions. B) 95% C2C12 myoblasts and C2C12 myotubes have overlapping but different binding profiles. Scatter plot is similar to Figure 2A. C)) MyoD binding profile at the Desmin locus. The Y-axis scale was adjusted by the total number of reads in each cell type, so that non-differentially bound peaks appear roughly the same heights in all three tracks. The two peaks match the two previously characterized MyoD binding sites (Tam et al., 2006). D) MyoD binding profile at the MyoD locus identifies three previously characterized MyoD binding sites (stars) (Asakura et al., 1995; Goldhamer et al., 1992; Tapscott et al., 1992) and several new MyoD binding sites. E) MyoD binding profile at the Myog locus identifies the known MyoD binding site in the promoter region, as well as a new binding site at the 3’ end of the gene. F) MyoD binding profile at the Ckm Locus identifies all known MyoD binding sites.
Figure 3
Figure 3
Characteristics of differentially bound regions between C2C12 myoblasts and myotubes. A) Motifs enriched (↑) and depleted (↓) in peaks that increase in C2C12 myotubes comparing to myoblasts. B) Motifs enriched in peaks that decrease in C2C12 myotubes comparing to myoblasts. C) and D) MyoD binding profiles at the Id2 and Id3 locus showed decreased binding of MyoD after differentiation. The characterized RP58 binding sites (Yokoyama et al, 2009) are indicated by vertical bars. E) Differentially bound peaks positively correlate with differentially expressed genes in 95% C2C12 myoblasts vs myotubes. The X and Y-axes correspond to the vsn-transformed number of reads in 95% confluent C2C12 myoblasts and myotubes in the combined peaks. Downregulated and upregulated genes are plotted in the left and right panels respectively. Note that in the left panel, data points are concentrated below the diagonal line, indicating that genes downregulated in myotubes have lower binding affinity in myotubes. In contrast, data points are concentrated above the diagonal line in the right panel, indicating that genes up-regulated in myotubes have stronger binding in myotubes. F) Differentially bound peaks do not correlate with differentially expressed genes in 50% vs 95% C2C12 myoblasts. Only the peaks associated with differentially expressed genes are plotted. The X and Y-axes correspond to the vsn-transformed number of reads in 50% and 95% myoblasts in the combined peaks. Downregulated and upregulated genes are plotted in the left and right panels respectively. Note that the points are relatively symmetrically distributed around the diagonal line, indicating that there is no correlation between the binding affinity and the mRNA expression levels.
Figure 4
Figure 4
MyoD binding in primary myotubes and fibroblasts. A) The MyoD binding regions are largely shared between C2C12 Myotubes (open circle) and primary myotubes (open triangle), or mouse embryonic fibroblasts expressing MyoD (cross). To assess the concordance between C2C12 myotubes and the other two types of myotubes, MyoD bound regions in C2C12 myotubes with at least 12 coverage were treated as reference, and the proportion of peaks that were also present in primary myotubes or fibroblasts expressing MyoD at each given cutoff was plotted. B) Plot is similar to A), except that the analysis was restricted to the peaks present in C2C12 myotubes that increased in coverage during differentiation, indicating a high similarity in this set of peaks that are associated with differentially expressed genes (see Fig. 4A). C), D), E) and F) show the MyoD binding profiles at the Acta1, Tnnc2, Actc1 and Mef2a loci.
Figure 5
Figure 5
MyoD induces histone H4 acetylation at binding sites genome-wide. A) Expression of MyoD does not induce changes in H3K4me3 at the majority of binding sites genome-wide. The difference of total H3K4me3 reads between MyoD-MEFs and MEFs in a 500 nt sliding window (Y-axis) was plotted against the square root of maximal MyoD peak height (X-axis) within the same sliding window across the genome. See Methods section for details. B) Expression of MyoD induces H4Ac at binding sites genome-wide. The plot is similar to A), except that the Y-axis is the H4Ac differences. C) Expression of MyoD increases H4Ac in MyoD bound regions. For each MyoD bound peak, the difference of total H4Ac reads between MyoD-MEFs and MEFs in a 500nt window centered at the peak summit (Y-axis) was plotted against the square root of maximal MyoD peak height (X-axis) D) Similar to C except that only the intergenic MyoD binding peaks are considered. E) C2 myoblasts and myotubes have increased H4Ac at MyoD binding sites. Quantitative real-time PCRs were performed at 20 loci on H4Ac ChIP samples from C2C12 myoblasts and myotubes. Hbb promoter region was used as a baseline control. The fold enrichments were calculated relative to the amount of input chromatin. The error bars indicate the difference between the duplicate measurements for the particular experiments. Hbb, Hemoglobin-beta; AMY2, Amylase 2; A1-A12, intergenic regions; B1-B4, promoter regions; C1-C4, intron regions. See Suppl. Table S1 for ChIP-seq and qPCR measurement of MyoD binding at these regions.
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