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

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.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.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,