Identification of GATA-4 acetylation targets by p300 during myocardial cell hypertrophy

Identification of GATA-4 acetylation targets by p300 during myocardial cell hypertrophy

Tomohide Takaya1, Teruhisa Kawamura1, Tatsuya Morimoto1, Koh Ono2, Toru Kita2, Akira Shimatsu1, Koji Hasegawa1.

  1. Kyoto Medical Center, National Hospital Organization, Kyoto, Japan.
  2. Graduate School of Medicine, Kyoto University, Kyoto, Japan.

American Heart Association Scientific Sessions 2006 (Chicago, USA), 2006/11/14 (Talk).

Abstract

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.