MYOGENIN AND MEF2 FUNCTION SYNERGISTICALLY TO ACTIVATE THE MRF4 PROMOTER DURING MYOGENESIS

The basic helix-loop-helix muscle regulatory factor (MRF) gene family encodes four distinct muscle-specific transcription factors known as M yoD, myogenin, Myf-5, and MRF4. These proteins represent key regulator y factors that control many aspects of skeletal myogenesis. Although t he MRFs often exhibit overlapping functional activities, their distinc t expression patterns during embryogenesis suggest that each protein p lays a unique role in controlling aspects of muscle development. As a first step in determining how MRF4 gene expression is developmentally regulated, we examined the ability of the MRF4 gene to be expressed in a muscle-specific fashion in vitro. Our studies show that the proxima l MRF4 promoter contains sufficient information to direct muscle-speci fic expression. Located within the proximal promoter are a single MEF2 site and E box that are required for maximum MRF4 expression. Mutatio n of the MEF2 site or E box severely impairs the ability of this promo ter to produce a muscle-specific response. In addition, the MEF2 site and E box function in concert to synergistically activate the MRF4 gen e in nonmuscle cells coexpressing MEF2 and myogenin proteins. Thus, th e MRF4 promoter is regulated by the MEF2 and basic helix-loop-helix MR F protein family through a cross-regulatory circuitry. Surprisingly, t he MRF4 promoter itself is not transactivated by MRF4, suggesting that this MRF gene is not subject to an autoregulatory pathway as previous ly implied by other studies. Understanding the molecular mechanisms re gulating expression of each MRF gene is central to fully understanding how these factors control developmental events.