Abstract 12648: Retinoblastoma Protein (Rb)-Associated Protein 48 and 46 Form a Functional Complex With p300/GATA4 and Suppress Hypertrophic Responses in Cardiomyocytes
Introduction: The zinc finger protein GATA4 is one of the hypertrophy-responsive transcription factors, and it increases its DNA-binding and transcriptional activities in response to hypertrophic stimuli in cardiomyocytes. The activation of GATA4 during this process is mediated, in part, through acetylation by intrinsic histone acetyltransferases such as the transcriptional coactivator p300. Retinoblastoma protein (Rb)-associated protein 48 and 46 (RbAp48 and RbAp46), components of the NuRD (nucleosome remodeling and deacetylase) complex have been implicated in chromatin remodeling and transcriptional repression associated with histone deacetylation. Through tandem affinity purification and mass spectrometric analyses, we showed that RbAp48 and RbAp46 are novel components of the GATA4 complex. However, the precise functional relationships among p300, GATA4 and RbAp48/46 remain unknown.
Methods and Results: By employing glutathione S-transferase pull-down assays, we showed that RbAp48/46 physically bound not only to GATA4 but also to p300. Immunoprecipitation followed by Western-blot analysis in HEK293T cells demonstrated that RbAp48/46 associated with the p300/GATA4 complex. The DNA pull down assay showed that RbAp48/46 were recruited onto the GATA element of the endothelin-1 (ET-1) promoter through GATA4. The overexpression of RbAp48/46 inhibited p300-induced acetylation of GATA4 as well as histones H3 and H4, and inhibited p300/GATA4-induced activities of the atrial natriuretic factor (ANF) and ET-1 promoters. The stimulation of cardiomyocytes with phenylephrine (PE) decreased the association between GATA4 and RbAp48/46. The overexpression of RbAp48/46 in cardiomyocytes significantly inhibited PE-induced hypertrophic responses such as myofibrillar organization, an increase in the cell size and the activation of ANF and ET-1 promoters. Conversely, knockdown of both RbAp48 and RbAp46 by shRNA significantly induced myocardial cell hypertrophy and increased ANF promoter activity.
Conclusion: RbAp48 and RbAp46 form a functional complex with p300/GATA4 and suppress hypertrophic responses in cardiomyocytes.
- © 2011 by American Heart Association, Inc.