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 expres sion to the atrial chambers of the heart. Understanding its regulation prov ides a basis for determination of the mechanisms controlling chamber-specif ic 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 v itamin D receptor (VDR) and retinoic X receptor alpha (RXR alpha) within th e slow MyHC3 promoter mediates chamber-specific expression of the gene. Irx 4, an Iroquois family homeobox gene whose expression is restricted to the v entricular 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 re quires the VDRE. Unlike VDR and RXR alpha, Irx4 does not bind directly to t he VDRE. Instead two-hybrid and co-immunoprecipitation assays show that Irx 4 interacts with the RXR alpha component of the VDR/RXR alpha heterodimer a nd that the amino terminus of the Irx4 protein is required for its inhibito ry action. These observations indicate that the mechanism of atrial chamber -specific expression requires the formation of an inhibitory protein comple x composed of VDR, RXR alpha, and Irx4 that binds at the VDRE inhibiting sl ow MyHC3 expression in the ventricles.