P4998Novel GATA4 binding proteins, RbAp48/46, regulate cardiomyocyte hypertrophy with depending on the phosphorylate State of GATA4
Authors: Sunagawa Y , Funamoto M , Shimizu K , Shimizu S , Katanasaka Y , Miyazaki Y , Wada H , Hasegawa K , Morimoto T 
Published: October 2019

Abstract

Introduction
Cardiac hypertrophy is being recognized as a critical event during the development of heart failure. A zinc finger protein GATA4 associates with an intrinsic histone acetyltransferase p300 and regulates myocardial transcriptional activities in response to hypertrophic stimuli. Here, we show that Retinoblastoma protein (Rb)-associated protein 48 and 46 (RbAp48, RbAp46) are novel components of p300/GATA4 complex. Both RbAp48 and 46 form a repressor complex with HDACs and has been implicated in chromatin remodeling and transcriptional repression. However, the precise functional relationships among p300, GATA4, RbAp48, and RbAp46 remain unknown.

Hypothesis
We assessed the hypothesis that RbAp48/46 form a functional complex with p300/GATA4 and regulated hypertrophic responses in cardiomyocytes.

Methods and results
IP-WB using nuclear extract from rat heart demonstrated that GATA4 formed a complex with RbAp48, RbAp46, HDAC1, and HDAC2. GST pull down assay using recombinant proteins showed that GATA4 physically interacted with both RbAp48 and RbAp46 but not HDAC1 and HDAC2. Deletion mutant assay revealed that N-terminal domain of GATA4 interacted with RbAp48/46. In HEK293T cell, overexpression of RbAp48/46 recruited HDAC1/2 to GATA4, inhibited p300-induced GATA4 acetylation and suppressed p300/GATA4-dependent ANF and ET-1 promoter activations. Conversely, the knockdown of RbAp48/46 reversed these changes. Although overexpression of HDAC1/2 did not change p300/GATA4-induced these promoter activities, co-expression of HDAC1 or HDAC2 with RbAp48/46 enhanced RbAp48/46-mediated inhibitory actions. In cardiomyocytes, overexpression of RbAp48/46 significantly inhibited phenylephrine (PE)-induced GATA4 acetylation, activation of ANF and ET-1 promoters, and cardiomyocyte hypertrophy. The knockdown of RbAp48/46 reversed these changes. Moreover, the knockdown of HDAC1/2 deteriorated PE-induced hypertrophy-responsive events and did not exhibit RbAp48/46-induced inhibitory actions. Finally, MEK1/ERK-mediated S105 phosphorylation of GATA4 by PE stimulus induced the dissociation of RbAp48/46 with GATA4, the increase of p300-induced GATA4-acetylation, the synergistic activation of ANF and ET-1 promoters with p300/GATA4, and the decrease of RbAp48/46 recruitments onto the GATA element of the ANF promoter. Conversely, PD98059, a MEK1 inhibitor, treatment inhibited GATA4-phosphorylation and these changes.

Conclusion
In this study, we demonstrate that RbAp48/46 mediate the binding between GATA4 and HDAC1/2 and regulate p300/GATA4 axis. The phosphorylation of S105 GATA4 has a critical role on the dissociation of GATA4/RbAp48/46/HDAC repressor complex, the formation of 300/GATA4 activator complex, and the increase of GATA4 acetylation and hypertrophic responses. These findings suggest that RbAp48/46 may regulate hypertrophic responses involved in modulating the posttranslational modification crosstalk of GATA4.

Acknowledgement/Funding
This work was supported by JSPS KAKENHI Grant.