Identification of p300-targeted Acetylated Residues in GATA4 during Hypertrophic Responses in Cardiac Myocytes

Abstract

A zinc finger protein, GATA4, is one of the hypertrophy-responsive transcription factors and increases its DNA binding and transcriptional activities in response to hypertrophic stimuli in cardiac myocytes. Activation of GATA4 during this process is mediated, in part, through acetylation by intrinsic histone acetyltransferases such as a transcriptional coactivator p300. However, p300-targeted acetylated sites of GATA4 during myocardial cell hypertrophy have not been identified. By mutational analysis, we showed that 4 lysine residues located between amino acids 311 and 322 are required for synergistic activation of atrial natriuretic factor and endothelin-1 promoters by GATA4 and p300. A tetra-mutant GATA4, in which these 4 lysine residues were simultaneously mutated, retained the ability to localize in nuclei and to interact with cofactors including FOG-2, GATA6, and p300 but lacked p300-induced acetylation, DNA binding, and transcriptional activities. Furthermore, coexpression of the tetra-mutant GATA4 with wild-type GATA4 impaired the p300-induced acetylation, DNA binding, and transcriptional activities of the wild type. When we expressed the tetra-mutant GATA4 in neonatal rat cardiac myocytes using a lentivirus vector, this mutant suppressed phenylephrine-induced increases in cell size, protein synthesis, and expression of hypertrophy-responsive genes. However, its expression did not affect the basal state. Thus, we have identified the most critical lysine residues acting as p300-mediated acetylation targets in GATA4 during hypertrophic responses in cardiac myocytes. The results also demonstrate that GATA4 with simultaneous mutation of these sites specifically suppresses hypertrophic responses as a dominant-negative form, providing further evidence for the acetylation of GATA4 as one of critical nuclear events in myocardial cell hypertrophy.

Kruppel-like Factor 15 Is a Critical Regulator of Cardiac Lipid Metabolism

“…Previous studies have implicated p300 acetyltransferase as a positive regulator of pathologic cardiomyocyte hypertrophy, possibly via its ability to acetylate prohypertrophic transcription factors, such as GATA4, MEF2, and p53 (21,(47)(48)(49)(50). Prior work from our group has shown that Klf15 expression is dramatically reduced during pathologic cardiac hypertrophy, an event that releases repression of GATA4, MEF2, and the p300-p53 transcriptional module (20,21).…”

Inhibition of Cardiomyocyte Hypertrophy by Protein Arginine Methyltransferase 5

“…However, mutation of Arg-317 completely abolished the inhibition of GATA4 activity by PRMT5, thus implicating a critical role of PRMT5-mediated GATA4 methylation at Arg-317 in regulating GATA4 transcriptional activity. Indeed, p300 has been to shown to induce lysine acetylation of GATA4 at positions of 311, 318, 320, and 322 (39). Considering the close proximity of Arg-317 to these acetylated residues, it is reasonable to speculate that methylation of Arg-317 in GATA4 might induce a conformational change and create a steric hindrance that blocks the lysine acetylation of GATA4 by p300.…”

Regulation of Cardiac Transcription Factor GATA4 by Post-Translational Modification in Cardiomyocyte Hypertrophy and Heart Failure

“…We analyzed the sites of p300-induced GATA4 acetylation, and found that 4 lysine resides (K311, K318, K320, and K322) were acetylated by p300; additionally, point mutations of these residues to alanine inhibited cardiomyocyte hypertrophy as a dominant-negative mutant. 26) We have identified cyclin-dependent kinase 9 (Cdk9), which is a component of positive transcription elongation factor b, as a novel GATA4 binding partner. We reported that Cdk9 promotes cardiomyocyte hypertrophy and p300/GATA4 pathway activation by phosphorylation of p300 and promotion of the HAT activity.…”