Abstract 901: Identification of GATA-4 Acetylation Targets by p300 During Myocardial Cell Hypertrophy
A zinc finger protein GATA-4 is one of hypertrophy-responsive transcription factors, and increases its DNA-binding and transcriptional activities in response to hypertrophic stimuli in cardiac myocytes. Activation of GATA-4 during this process is mediated, in part, through acetylation by intrinsic histone acetyltransferases such as a transcriptional coactivator p300. However, precise roles of GATA-4 acetylation in myocardial cell hypertrophy have not been determined. Within GATA-4, nine lysine residues, possible acetylation sites, exist near the two zinc fingers that serve as cofactor- and DNA-binding domains. We prepared seven mutants, in each of which one or two acetylation-target-lysines are substituted with alanines, and examined their effects on GATA-4-dependent atrial natriuretic factor and endothelin-1-promoters. We have found that four lysine residues located at amino acid sequences between 311–322 are required for synergistic activation by GATA-4 and p300 as well as GATA-4-dependent transcription. Then, we have generated a tetra-mutant GATA-4 in which these four lysine residues are simultaneously substituted with alanines. This tetra-mutant not only showed lack of p300-induced acetylation, DNA-binding and transcriptional activities, but also inhibited transcriptional activation by wild-type GATA-4. To determine the role of GATA-4 acetylation in myocardial cell hypertrophy, we expressed this tetra-mutant GATA-4 in primary cardiac myocytes from neonatal rats in culture. Transient transfection assays demonstrate that this tetra-mutant repressed phenylephrine-induced activation of the atrial natriuretic factor promoters. In addition, expression of this mutant using a lenti virus vector supressed phenylephrine-induced increase in cell size and myofibrillar organization. However, the expression did not affect cardiac myocytes at a basal state. Thus, we have identified the most critical lysine residues of p300-mediated acetylation target in GATA-4 and demonstrate that GATA-4 with simultaneous mutation of these sites supresses hypertrophic responses as a dominant-negative form in cardiac myocytes. These findings provide further evidence for the role of GATA-4 acetylation in myocardial cell hypertrophy.