In skeletal myogenesis, the transcription factor MyoD activates distinct transcriptional programs in progenitors compared to terminally differentiated cells. Using ChIP-Seq and gene expression analyses, we show that in primary myoblasts, Snail-HDAC1/2 repressive complex binds and excludes MyoD from its targets. Notably, Snail binds E box motifs that are G/C rich in their central dinucleotides, and such sites are almost exclusively associated with genes expressed during differentiation. By contrast, Snail does not bind the A/T-rich E boxes associated with MyoD targets in myoblasts. Thus, Snai1-HDAC1/2 prevent MyoD occupancy on differentiation-specific regulatory elements, and the change from Snail to MyoD binding often results in enhancer switching during differentiation. Furthermore, we show that a regulatory network involving myogenic regulatory factors (MRFs), Snai1/2, miR-30a, and miR-206 acts as a molecular switch that controls entry into myogenic differentiation. Together, these results reveal a regulatory paradigm that directs distinct gene expression programs in progenitors versus terminally differentiated cells.

Muscle-specific transcription factor MyoD orchestrates the myogenic gene expression program by binding to short DNA motifs called E-boxes within myogenic cis-regulatory elements (CREs). Genome-wide analyses of MyoD cistrome by chromatin immnunoprecipitation sequencing shows that MyoD-bound CREs contain multiple E-boxes of various sequences. However, how E-box numbers, sequences and their spatial arrangement within CREs collectively regulate the binding affinity and transcriptional activity of MyoD remain largely unknown. Here, by an integrative analysis of MyoD cistrome combined with genome-wide analysis of key regulatory histones and gene expression data we show that the affinity landscape of MyoD is driven by multiple E-boxes, and that the overall binding affinity-and associated nucleosome positioning and epigenetic features of the CREs-crucially depend on the variant sequences and positioning of the E-boxes within the CREs. By comparative genomic analysis of single nucleotide polymorphism (SNPs) across publicly available data from 17 strains of laboratory mice, we show that variant sequences within the MyoD-bound motifs, but not their genome-wide counterparts, are under selection. At last, we show that the quantitative regulatory effect of MyoD binding on the nearby genes can, in part, be predicted by the motif composition of the CREs to which it binds. Taken together, our data suggest that motif numbers, sequences and their spatial arrangement within the myogenic CREs are important determinants of the cis-regulatory code of myogenic CREs.