Identification of GATA4 Regulatory Mechanisms of Heart Development and Disease

Abstract

The development and function of the heart is governed by a conserved set of transcription factors (TFs) that regulate gene expression in a cell-type, time point and stimulus driven manner. Of these core cardiac TFs, the most ubiquitously expressed is the zinc finger protein GATA4. In cardiomyocytes, GATA4 is central to proliferation, differentiation, hypertrophy and induction of pro-survival pathways. In cardiac endothelial cells, it is required for valve and septal development, although the exact mechanisms remain unclear. To regulate such a wide array of functions in a spatially and temporally controlled manner, GATA4 interacts with specific protein partners, the majority of whom have been identified in cardiomyocytes. However, a complete understanding of the protein interactome of GATA4, particularly in cardiac endothelial cells, has not yet been achieved. Using a mass spectrometry-based approach, we have identified a series of novel GATA4 interacting partners in cardiac endothelial cells. 3xFlag GATA4 was stably overexpressed via retroviral transduction in the TC13 cardiac endothelial precursor cell line, immunoprecipitated from nuclear protein extracts and sent for HPLC-ESI-MS/MS. Several novel GATA4 interacting partners were identified including the chaperone protein Heat Shock Protein 70 (HSP70), the inducible orphan nuclear receptor Nerve Growth Factor 1β (NGFIβ, NUR77) and the Drosophila-Binding/Human Splicing protein family members Non-POU Domain Containing Octamer Binding Protein (NONO) and Paraspeckle 1 (PSPC1). Chapter 1 discusses the interaction between GATA4 and HSP70 and its role in cardiomyocyte survival upon exposure to chemotherapeutic agent Doxorubicin (DOX). HSP70 binds directly to GATA4, preventing DOX-mediated cleavage and degradation by Caspase-1, cardiomyocyte cell death and heart failure. Chapter 2 focuses on the cooperative interaction between GATA4 and NUR77 in cardiac microvascular endothelial cells and its central role in myocardial angiogenesis in response to pressure overload. The GATA4-NUR77 complex transactivates the promoter of Angiopoietin-Like 7 (ANGPTL7), a secreted pro-angiogenic chemotactic factor, triggering endothelial cell proliferation and tube formation in cultured cardiac endothelial cells and increasing myocardial capillary density in vivo. Chapter 3 discusses the interaction between GATA4 and the DBHS proteins NONO and PSPC1 in the regulation of cardiac development. These proteins play opposing roles when bound to GATA4 as PSPC1 enhances GATA4 activation of critical cardiac promoter targets and NONO acts as a rheostat to repress GATA4 activity. In vivo, loss of NONO results in left ventricular non-compaction consistent with humans with loss-of-function mutations. However, simultaneous Gata4 haploinsufficiency partially rescues this phenotype. Together, this data identifies multiple novel cell type and time point specific GATA4 protein partners and sheds light on GATA4 regulatory mechanisms in cardiac development and disease.