Abstract P255: Protein Arginine Methyltransferase 5 and Methylosome Protein 50, Novel Components of P300/gata4 Complex, Suppress Hypertrophic Gene Transcriptions in Cardiomyocytes

Abstract

Introduction: One of intrinsic histone acetyltransferases (HAT), p300, serves as a coactivator of hypertrophy-responsive transcriptional factors such as a cardiac zinc finger protein GATA4 and is involved in its hypertrophic stimulus-induced acetylation and DNA binding. Disruption of this complex results in the inhibition of hypertrophic responses in cardiomyocytes. By tandem affinity purification and mass spectrometric analyses, we identified protein arginine methyltransferase 5 (PRMT5) and methylosome protein 50 (MEP50), an activator of PRMT5, as novel p300/GATA4-binding partners. However, the precise functional relationships among p300/GATA4, PRMT5 and MEP50 remain unknown. Methods and Results: To confirm the binding of GATA4 with PRMT5, expression plasmids encoding GATA4 and PRMT5 were co-transfected into HEK293 cells. Nuclear extracts from these cells were subjected to immunoprecipitation, followed by western blotting. We observed that GATA4 formed a complex with p300, PRMT5 and MEP50 in HEK293 cells. Next, to investigate whether PRMT5 is required for p300-mediated activation of the GATA4-dependent promoter activities, we performed reporter assays in HEK293 cells. While overexpression of either PRMT5 or MEP50 inhibited p300/GATA4-induced atrial natriuretic factors (ANF) and endotheline-1 (ET-1) promoter activities, knockdown of PRMT5 by RNAi enhanced these promoter activities in HEK293 cells. Immunoprecipitation followed by western blotting demonstrated that PRMT5 repressed p300-induced acetylation of GATA4 in HEK293T cells. Finally, PRMT5 or MEP50 repressed phenylephrine-induced promoter activation of the ANF and ET-1 in cardiomyocytes. Conclusion: These findings demonstrate that PRMT5 and MEP50 form a functional protein complex with GATA4/p300 and regulated hypertrophic responses in cardiomyocytes.