Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis

Although accumulating evidence suggests that the heart develops in a segmen tal fashion, the molecular mechanisms that control regional specification o f cardiomyocytes in the developing heart remain largely unknown. In this st udy, we have used the mouse cardiac-restricted ankyrin repeat protein (CARP ) gene as a model system to study these mechanisms. The CARP gene encodes a nuclear coregulator for cardiac gene expression, which lies downstream of the cardiac homeobox gene, Nkx 2.5, and is an early marker of the cardiac m uscle cell lineage, We have demonstrated that the expression of the gene is developmentally down regulated and dramatically induced as part of the emb ryonic gene program during cardiac hypertrophy, Using a lacZ/knock-in mouse and three lines of transgenic mouse harboring various CARP promoter/lacZ r eporters, we have identified distinct 5' cis regulatory elements of the gen e that can direct heart segment-specific transgene expression, such as atri al versus ventricular and left versus right. Most interestingly, a 213 base pair sequence element of the gene was found to confer conotruncal segment- specific transgene expression. Using the transgene as a conotruncal segment -specific marker, we were able to document the developmental fate of a subs et of cardiomyocytes in the conotruncus during cardiogenesis, In addition, we have identified an essential GATA-4 binding site in the proximal upstrea m regulatory region of the gene and cooperative transcriptional regulation mediated by Nkx2.5 and GATA-4. We have shown that this cooperative regulati on is dependent on binding of GATA-4 to its cognate DNA sequence in the pro moter, which suggests that Nkx2.5 controls CARP expression, at least in par t, through GATA-4.