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.

Highlights

  • Cell hypertrophy is characterized by an increase in cell size, accumulation of contractile proteins, and activation of hypertrophy-responsive transcription factors [1]

  • 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

  • The results demonstrate that GATA4 with simultaneous mutation of these sites 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

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Summary

EXPERIMENTAL PROCEDURES

Plasmid Constructs—pcDNAG4 [22, 23], pCMVp300wt (a gift from Dr Richard Eckner, University of Medicine and Dentistry New Jersey, Newark, NJ) [24], pwtFOG-2 PLentiGFP-G4 and pLentiGFP-G4M456, which express wild-type and M456GATA4 fused with enhanced GFP, were constructed by inserting GATA4 cDNA from pcDNAG4 and pcDNAG4M456 into pLentiGFP, respectively. COS7 cells were resuspended in the medium containing 200 ␮Ci/ml [1-14C]acetic acid sodium salt (GE Healthcare) and incubated for 12 h Nuclear extracts from these cells were precipitated with anti-GATA4 antibody as described above, resolved by SDS-PAGE, fixed, and autoradiographed using a bioimaging analyzer, BAS 3000 (FUJIX). D, lysates from COS7 cells used for luciferase assays were subjected to Western blotting with anti-p300, anti-GATA4, and anti-␤-actin antibodies. These data demonstrate that the lysine residues within the C-motif of GATA4 are required for p300-induced transcriptional activation. The M4, M5, and M6 where appropriate, with a probability value Ͻ0.05 taken to indi- mutations markedly reduced the transcriptional activity of cate significance

RESULTS
HAT activity and is able to acetylate
Findings
DISCUSSION
Full Text
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