Developmental regulation of MyoD expression: Mapping the cis regulatory domains of the human MyoD core enhancer

Abstract

The basic helix-loop-helix transcription factor, MyoD, plays a critical role in the determination of skeletal muscle. While the function of this factor is beginning to be understood, little is known about the regulatory mechanisms that drive expression of MyoD in only those embryonic cells destined to become skeletal muscle. Recently, a highly conserved 258 bp human core enhancer element was identified that is capable of recapitulating MyoD expression in embryonic skeletal muscle. This enhancer is a candidate target for the embryonic signaling events that initiate MyoD activation in the embryo, providing a unique tool for dissecting the inductive events leading to skeletal myogenesis. In this dissertation, I present a detailed functional characterization of sequences present within the MyoD core enhancer, using linker-scanner mutagenesis, together with transfection and transgenic assays, to define the cis motifs required for enhancer activity. Comparison of cell culture and in vivo expression data indicates that the enhancer is regulated by distinct signaling or transcriptional systems in embryos and in culture. Given the complexity of the signaling pathways that ultimately lead to expression of MyoD and the other myogenic genes in vivo, it is not surprising that cell culture is not an accurate model for embryonic myogenesis. Transciptional regulation in vivo appears to involve activation through multiple cis regulatory elements, the majority of which are required for expression in all skeletal muscle domains. Analysis of one of these cis elements suggests that this element may mediate chromatin accessibility of core enhancer-driven transgenes. A second class of cis element was defined by two adjacent linker mutations. This 30 bp element is specifically required for expression in developing epaxial muscle, indicating that activation of MyoD expression in the different skeletal muscle lineages involves distinct transcriptional regulatory mechanisms. Ongoing experiments described herein will address the hypothesis that epaxial expression of the core enhancer is dependent on Myf5, and will determine whether this regulation occurs directly through E-box sequences present in this 30 bp region