Developmental expression of the skeletal muscle determination gene, MyoD, is regulated by novel enhancer elements that interact with the core enhancer and distal regulatory region

<jats:p>MyoD plays a central role in determining the skeletal muscle lineage in vertebrate embryos. The core enhancer (CE) and distal regulatory region (DRR) are the only known MyoD enhancers, and together, they recapitulate all major aspects of MyoD expression in the embryo. However, knocking out each enhancer individually has only modest effects on MyoD expression. Here, we show that embryos lacking both enhancers maintain muscle-specific MyoD expression, indicating the existence of unknown MyoD regulatory elements. Precision run-on sequencing together with available ChIP-seq and DNase I hypersensitivity datasets identified three new candidate enhancer regions within 96 kb of MyoD 5' flanking sequences. Transgenic analysis revealed that DNA elements at -36 and -60 kb are active in all muscle-forming regions, each recapitulating aspects of endogenous MyoD expression. Enhancer activities in muscle regulatory factor-deficient mice suggest that they are components of the auto- and cross-regulatory circuitry that maintains MyoD expression. Analysis of Hi-C data showed that the entire -96 kb region constitutes a loop domain, within which multiple interactions between elements and with the MyoD gene were detected. A larger loop domain delimited by CTCF sites was also identified from -96 kb to +220 kb relative to the MyoD transcriptional start site. These data indicate that the newly identified enhancers are key components of a cis regulatory network that controls the activation and maintenance of MyoD expression in the embryo.</jats:p>