Irx4 forms an inhibitory complex with the vitamin D and retinoic X receptors to regulate cardiac chamber-specific slow MyHC3 expression

The slow myosin heavy chain 3 gene (slow MyHC3) is restricted in its expression to the atrial chambers of the heart. Understanding its regulation provides a basis for determination of the mechanisms controlling chamber-specific gene expression in heart development. The observed chamber distribution results from repression of slow MyHC3 gene expression in the ventricles. A binding site, the vitamin D response element (VDRE), for a heterodimer of vitamin D receptor (VDR) and retinoic X receptor alpha (RXR alpha) within the slow MyHC3 promoter mediates chamber-specific expression of the gene. Irx4, an Iroquois family homeobox gene whose expression is restricted to the ventricular chambers at all stages of development, inhibits AMHC1, the chick homolog of quail slow MyHC3, gene expression within developing ventricles. Repression of the slow MyHC3 gene in ventricular cardiomyocytes by Irx4 requires the VDRE. Unlike VDR and RXR alpha, Irx4 does not bind directly to the VDRE. Instead two-hybrid and co-immunoprecipitation assays show that Irx4 interacts with the RXR alpha component of the VDR/RXR alpha heterodimer and that the amino terminus of the Irx4 protein is required for its inhibitory action. These observations indicate that the mechanism of atrial chamber-specific expression requires the formation of an inhibitory protein complex composed of VDR, RXR alpha, and Irx4 that binds at the VDRE inhibiting slow MyHC3 expression in the ventricles.