MYOCYTE-SPECIFIC ENHANCER FACTOR-2 AND THYROID-HORMONE RECEPTOR ASSOCIATE AND SYNERGISTICALLY ACTIVATE THE ALPHA-CARDIAC MYOSIN HEAVY-CHAINGENE

The muscle-specific regulatory region of the alpha-cardiac myosin heav y-chain (MHC) gene contains the thyroid hormone response element (TRE) and two A/T-rich DNA sequences, designated A/T-1 and A/T-2, the putat ive myocyte-specific enhancer factor 2 (MEF2) binding sites. We invest igated the roles of the TRE and MEF2 binding sites and the potential i nteraction between thyroid hormone receptor (TR) and MEF2 proteins reg ulating the alpha-MHC promoter. Deletion mutation analysis indicated t hat both the A/T-2 motif and TRE were required for muscle-specific exp ression of the alpha-MHC gene. The alpha-MHC enhancer containing both the A/T-2 motif and TRE was synergistically activated by coexpression of MEF2 and TR in nonmuscle cells, whereas neither factor by itself ac tivated the alpha-MHC reporters. The reporter construct containing the A/T-2 sequence and the TRE linked to a heterologous promoter also sho wed synergistic activation by coexpression of MEF2 and TR in nonmuscle cells. Moreover, protein binding assays demonstrated that MEF2 and TR specifically bound to one another in vitro and in vivo. The MADS doma in of MEF2 and the DNA-binding domain of TR were necessary and suffici ent to mediate their physical interaction. Our results suggest that th e members of the MADS family (MEF2) and steroid receptor superfamily ( TR) interact,vith one another to synergistically activate the alpha-ca rdiac MHC gene expression.