GATA4 Directly Regulates <i>Runx2</i> Expression and Osteoblast Differentiation

Abstract

GATA4 is a zinc-finger transcription factor that is a pioneer factor in various tissues and regulates tissue-specific gene regulation. In vivo deletion of <i>Gata4</i> using Cre-recombinase under the control of the <i>Col1a1</i> 2.3 kb promoter showed significantly reduced values for trabecular bone properties by microCT analysis of femur and tibia of 14-week-old male and female mice, suggesting GATA4 is necessary for maintaining normal adult bone phenotype. Quantitative PCR analysis revealed higher expression of <i>Gata4</i> in trabecular bone compared with cortical bone, suggesting a role for GATA4 in maintaining normal trabecular bone mass. In vivo and in vitro, reduction of <i>Gata4</i> correlates with reduced <i>Runx2</i> gene expression, along with reduced osteoblast mineralization. To determine if <i>Runx2</i> is a direct target of GATA4, chromatin immunoprecipitation (ChIP) was performed, and it demonstrated that GATA4 is recruited to the two <i>Runx2</i> promoters and an enhancer region. Furthermore, when <i>Gata4</i> is knocked down, the chromatin at the <i>Runx2</i> region is not open, as detected by DNase assays and ChIP with antibodies to the open chromatin marks H3K4me2 (histone 3 lysine 4 dimethylation) and H3K27ac (histone 3 lysine 27 acetylation) and the closed chromatin mark H3K27me2 (histone 3 lysine 27 trimethylation). Together, the data suggest that GATA4 binds near the <i>Runx2</i> promoter and enhancer and helps maintain open chromatin to regulate <i>Runx2</i> expression leading to bone mineralization.