Abstract
ABSTRACTGATA4 is a zinc‐finger transcription factor that is a pioneer factor in various tissues and regulates tissue‐specific gene regulation. In vivo deletion of Gata4 using Cre‐recombinase under the control of the Col1a1 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 Gata4 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 Gata4 correlates with reduced Runx2 gene expression, along with reduced osteoblast mineralization. To determine if Runx2 is a direct target of GATA4, chromatin immunoprecipitation (ChIP) was performed, and it demonstrated that GATA4 is recruited to the two Runx2 promoters and an enhancer region. Furthermore, when Gata4 is knocked down, the chromatin at the Runx2 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 Runx2 promoter and enhancer and helps maintain open chromatin to regulate Runx2 expression leading to bone mineralization. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.
Highlights
Osteoblast differentiation is controlled by directly inducing the expression of, or regulating the activities of, the “master” osteoblast transcription factor RUNX2
Gata4 knockout mice die early in embryogenesis due to the heart and intestinal defects.[3,4] We have recently demonstrated that GATA4 is a critical transcription factor for osteoblast differentiation, and GATA4 controls osteoblast function by regulating the balance between TGFb and BMP pathways.[5]. Conditional Gata4 knockout mice with Cre expression driven by the 2.3-kb promoter of Col1a1 showed reduced trabecular bone mass and exhibited both appendicular and cranial defects in newborn mice
We found that newborn Conditional Gata4 knockout mice (cKO) (Gata4fl/fl/Creþ) mice were not born at the expected Mendelian ratio, and microcomputed tomography (mCT) analysis of the newborn femur and vertebra showed reduced trabecular bone properties and skeletal defects.[5]. After an expansion of the breeding colony, 429 mice were since bred to obtain 7.9% cKO adult mice (25% was expected based on breeding Gata4fl/fl female mice with Gata4fl/þ/Creþ male mice)
Summary
Osteoblast differentiation is controlled by directly inducing the expression of, or regulating the activities of, the “master” osteoblast transcription factor RUNX2. Gata knockout mice die early in embryogenesis due to the heart and intestinal defects.[3,4] We have recently demonstrated that GATA4 is a critical transcription factor for osteoblast differentiation, and GATA4 controls osteoblast function by regulating the balance between TGFb and BMP pathways.[5] Conditional Gata knockout mice (cKO) with Cre expression driven by the 2.3-kb promoter of Col1a1 showed reduced trabecular bone mass and exhibited both appendicular and cranial defects in newborn mice. GATA4 has been shown to act as a regulator of osteoblasts by controlling estrogen receptor recruitment to the chromatin in a tissue-specific manner.[5,6]. We present evidence that GATA4 maintains open chromatin at both the Runx promoters and an enhancer to upregulate Runx, the master regulator of osteoblast differentiation, and to control proper adult bone formation in vivo.
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