Abstract 535: Proteomics Analysis Identifies Positive Transcription Elongation Factor b as a Novel GATA-4-binding Partner Involved in Hypertrophic Responses in Cardiomyocytes

Abstract

Introduction: A zinc finger protein GATA-4 is one of the factors involved in transcriptional regulation during myocardial cell hypertrophy. In response to hypertrophic stimuli, GATA-4 forms a large complex with ERK, NFATc and an intrinsic histone acetyltransferase, p300. Disruption of this complex results in the inhibition of hypertrophic responses in cardiomyocytes. Hypothesis: We assessed the hypothesis that proteomics analysis may identify novel GATA-4-binding partners required for myocardial cell hypertrophy. Methods: GATA-4 was stably expressed as FLAG-HA-epitope fusion in HeLa cells by retroviral transduction. GATA-4 complex was purified from nuclear extracts of these cells by sequential immunoprecipitation with an anti-FLAG antibody followed by an anti-HA antibody (Tandem Affinity Purification). Results: By mass spectrometric analyses, we identified 73 GATA-4 binding proteins, including histone modifying factors, methyltransferase, HDAC and unknown proteins. One was cyclin-dependent kinases-9 (cdk9), a component of Positive Transcription Elongation Factor b (P-TEFb). Immunoblotting analyses demonstrated that GATA-4 formed a complex with p300, cdk9 and cyclin T1, another component of P-TEFb. A dominant-negative form of p300 disrupted the interaction of GATA-4 with cdk9 or cyclin T1. Conversely, a dominant-negative form of cdk9 and a cdk9 kinase inhibitor, 5,6-dichloro-1-h-ribofuranosyl-benzimidazole (DRB) inhibited p300-induced activation of GATA-4-dependent transcription as well as the acetylation of GATA-4. Stimulation of cardiomyocytes with phenylephrine (PE) increased the binding of GATA-4/p300 with cdk9 or cyclin T1. DN-cdk9 and DRB not only disrupted the complex of GATA-4/p300 with cdk9 or cyclin T1, but also repressed PE-induced hypertrophic responses such as myofibrillar organization, increase in cell size and promoter activation of the endothelin-1 and atrial natriuretic factors in cardiomyocytes. Conclusion: These findings demonstrate that cdk9 and cyclin T1 form a functional protein complex with GATA-4/p300 and that this large complex is a prerequisite for hypertrophic responses in cardiac myocytes.