Myogenic differentiation proceeds through a highly coordinated cascade of gene 
activation that necessitates epigenomic changes in chromatin structure. Using a 
screen of small molecule epigenetic probes we identified three compounds which 
inhibited myogenic differentiation in C2C12 myoblasts; (+)-JQ1, PFI-1, and 
Bromosporine. These molecules target Bromodomain and Extra Terminal domain (BET) 
proteins, which are epigenetic readers of acetylated histone lysine tail 
residues. BETi-mediated anti-myogenic effects were also observed in a model of 
MYOD1-mediated myogenic conversion of human fibroblasts, and in primary mouse 
and human myoblasts. All three BET proteins BRD2, BRD3 and BRD4 exhibited 
distinct and dynamic patterns of protein expression over the course of 
differentiation without concomitant changes in mRNA levels, suggesting that BET 
proteins are regulated at the post-transcriptional level. Specific BET protein 
knockdown by RNA interference revealed that BRD4 was required for myogenic 
differentiation, whereas BRD3 down-regulation resulted in enhanced myogenic 
differentiation. ChIP experiments revealed a preferential binding of BRD4 to the 
Myog promoter during C2C12 myoblast differentiation, co-incident with increased 
levels of H3K27 acetylation. These results have identified an essential role for 
BET proteins in the regulation of skeletal myogenesis, and assign distinct 
functions to BRD3 and BRD4.