CARP, A CARDIAC ANKYRIN REPEAT PROTEIN, IS DOWNSTREAM IN THE NKX2-5 HOMEOBOX GENE PATHWAY

To identify the molecular pathways that guide cardiac ventricular cham ber specification, maturation and morphogenesis, we have sought to cha racterize factors that regulate the expression of the ventricular myos in light chain-2 gene, one of the earliest markers of ventricular regi onalization during mammalian cardiogenesis. Previously, our laboratory identified a 28 bp HF-1a/MEF-2 element in the MLC-2v promoter region, which confers cardiac ventricular chamber-specific gene expression du ring murine cardiogenesis, and showed that the ubiquitous transcriptio n factor YB-1 binds to the HF-la site in conjunction with a co-factor. In a search for interacting co-factors, a nuclear ankyrin-like repeat protein CARP (cardiac ankyrin repeat protein) was isolated from a rat neonatal heart cDNA library by yeast two-hybrid screening, using YB-1 as the bait. Co-immunoprecipitation and GST-CARP pulldown studies rev eal that CARP forms a physical complex with YB-1 in cardiac myocytes a nd immunostaining shows that endogenous CARP is localized in the cardi ac myocyte nucleus. Go-transfection assays indicate that CARP can nega tively regulate an HF-1-TK minimal promoter in an HF-1 sequence-depend ent manner in cardiac myocytes, and CARP displays a transcriptional in hibitory activity when fused to a GAL4 DNA-binding domain in both card iac and noncardiac cell context. Northern analysis revealed that carp mRNA is highly enriched in the adult heart, with only trace levels in skeletal muscle. During murine embryogenesis, endogenous carp expressi on was first clearly detected as early as E8.5 specifically in heart a nd is regulated temporally and spatially in the myocardium. Nkx2-5, th e murine homologue of Drosophila gene tinman was previously shown to b e required for heart tube looping morphogenesis and ventricular chambe r-specific myosin light chain-2 expression during mammalian heart deve lopment. In Nkx2-5(-/-) embryos, carp expression was found to be signi ficantly and selectively reduced as assessed by both whole-mount in si tu hybridizations and RNase protection assays, suggesting that carp is downstream of the homeobox gene Nkx2-5 in the cardiac regulatory netw ork. Co-transfection assays using a dominant negative mutant Nkx2-5 co nstruct with CARP promoter-luciferase reporter constructs in cardiac m yocytes confirms that Nkx2-5 either directly or indirectly regulates c arp at the transcriptional level. Finally, a carp promoter-lacZ transg ene, which displays cardiac-specific expression in wild-type and Nkx2- 5(+/-) background, was also significantly reduced in Nkx2-5(-/-) embry os, indicating that Nkx2-5 either directly or indirectly regulates car p promoter activity during in vivo cardiogenesis as well as in culture d cardiac myocytes. Thus, CARP is a YB-1 associated factor and represe nts the first identified cardiac-restricted downstream regulatory gene in the homeobox gene Nkx2-5 pathway and may serve as a negative regul ator of HF-1-dependent pathways for ventricular muscle gene expression .