Abstract P362: Gata4 Dimerization May Be A Target For Heart Failure Therapy

Abstract

Introduction: Cardiac hypertrophy is regulated by activation of GATA4. Although GATA4 post-translational modification such as acetylation by p300 is well examined, the details of the activation mechanism of GATA4 are still unclear. The purpose of this study is to investigate whether GATA4 dimerization involved in transcriptional activation and cardiomyocyte hypertrophic responses. Methods and Results: A GST pull-down assay using GST fusion GATA4 full-length and deletion mutants demonstrated that GATA4 308-326, including the acetylation site, was required for the dimerization of GATA4. A DNA pull down assay showed that the C-zinc finger motif (256-295) and the acetylation site were required for the DNA binding capacity of GATA4. IP-WB using nuclear extract from HEK293T cells expressing FLAG- or HA-tagged GATA4 showed that co-expression of p300 increased the formation of the homo-dimer as well as acetylation of GATA4. The GATA4 homo-dimer was disrupted by both acetyl-deficient GATA4 and HAT-deficient p300. This result indicates that acetylation of GATA4 is important for dimerization of GATA4. Overexpression of the deletion mutant containing a GATA4 308-326 (G4D) prevented p300-induced GATA4 dimerization but not the p300 binding nor acetylation of GATA4. ChIP assay and DNA pulldown assay showed that G4D did not inhibit the p300-induced DNA binding of GATA4. In cardiomyocytes, the G4D inhibited phenylephrine-induced ANF and ET-1 promoter activities and cardiomyocyte hypertrophy. To perform the X-ray crystal structure analysis, recombinant GATA4 fragment including GATA4 308-326 was highly purified. The X-ray diffraction data of obtained crystals was collected. Resolution of the crystal was 3.1Å, which was insufficient for phase determine. To obtain a high-quality crystal, GATA4 fragment was crystallized in international space station, in collaboration with JAXA. Resolution of the crystal was 3.16Å, which was similar to the best data before obtained. Conclusions: These results suggest that GATA4 dimerization may play an important role in hypertrophy-response gene transcription. It is expected to elucidate the GATA4 dimerization mechanism and targeted this dimerization will lead to the development of a noble heart failure therapy.