EMBRYONIC ACTIVATION OF THE MYOD GENE IS REGULATED BY A HIGHLY CONSERVED DISTAL CONTROL ELEMENT

MyoD belongs to a small family of basic helix-loop-helix transcription factors implicated in skeletal muscle lineage determination and diffe rentiation. Previously, we identified a transcriptional enhancer that regulates the embryonic expression of the human myoD gene. This enhanc er had been localized to a 4 kb fragment located 18 to 22 kb upstream of the myoD transcriptional start site. We now present a molecular cha racterization of this enhancer. Transgenic and transfection analyses l ocalize the myoD enhancer to a core sequence of 258 bp. In transgenic mice, this enhancer directs expression of a lacZ reporter gene to skel etal muscle compartments in a spatiotemporal pattern indistinguishable from the normal myoD expression domain, and distinct from expression patterns reported for the other myogenic factors. In contrast to the m yoD promoter, the myoD enhancer shows striking conservation between hu mans and mice both in its sequence and its distal position. Furthermor e, a myoD enhancer/heterologous promoter construct exhibits muscle-spe cific expression in transgenic mice, demonstrating that the myoD promo ter is dispensable for myoD activation. With the exception of E-boxes, the myoD enhancer has no apparent sequence similarity with regulatory regions of other characterized muscle-specific structural or regulato ry genes. Mutation of these E-boxes, however, does not affect the patt ern of lacZ transgene expression, suggesting that myoD activation in t he embryo is E-box-independent, DNase I protection assays reveal multi ple nuclear protein binding sites in the core enhancer, although none are strictly muscle-specific. Interestingly, extracts from myoblasts a nd 10T1/2 fibroblasts yield identical protection profiles, indicating a similar complement of enhancer-binding factors in muscle and this no n-muscle cell type. However, a clear difference exists between myoblas ts and 10T1/2 cells (and other non-muscle cell types) in the chromatin structure of the chromosomal myoD core enhancer, suggesting that the myoD enhancer is repressed by epigenetic mechanisms in 10T1/2 cells. T hese data indicate that myoD activation is regulated at multiple level s by mechanisms that are distinct from those controlling other charact erized muscle-specific genes.