MYOD represses gene expression from non-E-box motifs

We report here on the identification of a previously unrecognized property of MYOD as a repressor of gene expression via E-box-independent chromatin binding during the process of somatic cell trans-differentiation into skeletal muscle. When ectopically expressed in proliferating human fibroblasts or endogenously induced in activated muscle stem cells (MuSCs), MYOD was detected at accessible regulatory elements of expressed genes, invariably leading to reduced chromatin accessibility and gene repression. At variance with conventional E-box-driven increased chromatin accessibility and H3K27 acetylation at previously silent loci of MYOD-activated genes, MYOD-mediated chromatin compaction and repression of transcription was associated with high occurrence of non-E-box motifs and did not lead to reduced levels of H3K27ac but coincided with reduced levels of H4 acetyl-methyl lysine modification (Kacme). Using MYOD mutants, we dissected the molecular mechanism of MYOD-mediated repression, whereby repression of mitogen-responsive and growth factor-responsive genes occurred via promoter binding, which requires a conserved domain within the first helix; conversely, repression of cell of origin/alternative lineage genes occurred via binding and decommissioning of distal regulatory elements such as superenhancers (SEs), required either the N-terminal activation domain or the two chromatin remodeling domains, and coincided with reduced strength of CTCF-mediated chromatin interactions. These data extend MYOD biological properties beyond the current dogma that restricts MYOD function to a monotone transcriptional activator. They also reveal an unprecedented functional versatility arising from alternative chromatin recruitment through E-box or non-E-box sequences, whereby genetic determinants dictate differential usage of MYOD functional domains.