GATA4 Directly Regulates Runx2 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 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.

Keywords: BONE; GATA4; OSTEOBLASTS; RUNX2.

Figure 1

Trabecular bone volume is decreased in GATA4 cKO mice. (A, B) μCT reconstruction of trabecular bone in the distal tibia metaphysis of 14‐week‐old female mice. (C–F) Mean percentage bone volume (BV/TV), trabecular number (Tb.N), separation (Tb.Sp), and thickness (Tb.Th). Black bars indicate wild‐type (WT) mice; white bars indicate GATA4 conditional knockout (cKO) mice. Data are mean ± standard deviation from 12 WT and 12 cKO mice. Student's t test: *p < 0.05, **p < 0.01 compared with WT. (G–J) Section of femur stained with H&E and (K, L) RUNX2. Pr = proliferating chondrocytes. Arrows indicate binucleate chondrocytes. Ch = chondrocytes. Tr = trabecular bone. Scale bars = 1000 μm (G, H); 200 μm (K, L).

Figure 2

GATA4 regulates mineralization in vitro. (A) Calvarial osteoblasts were transduced with lentivirus expressing shC or shGATA4. RNA was obtained and qPCR was performed for Gata4 and normalized to actb (β‐actin) mRNA. (B) Calvarial osteoblasts transduced with lentivirus directed toward shC or shGATA4 and were cultured in osteogenic media for 2 weeks. Cells were then fixed and stained using Alizarin Red. (C) Alizarin Red from B was eluted and the mineral content measured at an OB of 570 nm. Silencing was performed in three wells and the average OD is displayed. (D) Bone marrow from WT and cKO mice were cultured in osteogenic media for 2 weeks. RNA was obtained and qPCR was performed for Gata4 and normalized to Actb (β‐actin) mRNA. (E) Cells were then fixed and stained using Alizarin Red. Three different mice were tested and a representative image from one mouse is presented in triplicate. (F) Alizarin Red from E was eluted and the mineral content measured at an OB of 570 nm. The average OD from all wells is displayed. Student's t test: *p < 0.05, ***p < 0.001 compared with wild type. OD = optical density.

Figure 3

GATA4 regulates Runx2 in both primary calvarial cells and in bone marrow MSCs. (A) Calvarial osteoblasts were transduced with lentivirus expressing shC or shGATA4. Cells were then differentiated for 2 weeks in osteogenic media and RNA was obtained. qPCR was performed for Runx2 and normalized to Actb (β‐actin) mRNA. (B) Bone marrow was isolated from tibia and femurs of wild‐type and conditional GATA4 knockout mice. Cells were then differentiated for two weeks in osteogenic media and RNA was obtained. qPCR was performed for Runx2 and normalized to Actb (β‐actin) mRNA. (C) Calvarial osteoblasts were transduced with lentivirus expressing either shC or shGATA4. Cells were then differentiated for 2 weeks in osteogenic media and protein was obtained and immunoblots for RUNX2 and β‐actin were performed. (D) The Runx2‐promoter luciferase construct was transfected into U2OS cells, along with pcDNA3‐GATA4, where indicated. The Runx2‐promoter mutant has two point mutations in the sequence “GATA.” Luciferase values were normalized to Renilla luciferase. n = 3, Student's t test: *p < 0.05, ***p < 0.001.

Figure 4

GATA4 regulates Runx2 at two promoters and an enhancer. (A) Schematic diagram of Runx2 genomic locus. The arrow indicates the direction of gene transcription. The GATA4 binding sites are Runx2 promoter 1, promoter 2, and an enhancer site, which are represented by solid squares and triangle. (B) Streptavidin‐coated beads were used to immunoprecipitate either wild‐type or Flag‐biotin‐GATA4. qPCR was performed with primers to the indicated regions. Calvarial osteoblasts were transduced with either shC or shGATA4 lentivirus. ChIP was performed with antibodies to (C) H3K4me2, (D) H3K27ac (open chromatin), (E) H3K27me3 (closed chromatin), or IgG. qPCR was performed to the indicated regions. Each PCR was normalized to input and represented as fold enrichment over a negative genomic locus (H3K4me2 and H3K27ac: mo. −ve, and H3K27me3: actin). n = 3, Student's t test: *p < 0.05, ***p < 0.001 compared with shC.

Figure 5

DNase assays demonstrate GATA4 is necessary for open chromatin at the Runx2 locus. (A–D) Gata4 was knocked down in primary calvarial osteoblast cells with lentivirus and then DNase assays were performed at the indicated Runx2 cis regulatory regions. n = 3, Student's t test: *p < 0.05.

Figure 6

GATA4 regulates Runx2 gene transcription early in differentiation. Calvarial osteoblasts were infected with lentivirus expressing shC or shGATA4. Cells were either collected 48 hours after infection (day 0) or differentiated for 2 weeks in osteogenic media. RNA was obtained from undifferentiated and differentiated cells and qPCR was performed for (A) Gata4 and (B) Runx2 and normalized to Actb (β‐actin) mRNA. (C) ChIP was performed with streptavidin beads on undifferentiated (day 0) or differentiated (day 14) osteoblasts from GATA4‐Flag‐Biotin mice. ChIP was performed with an antibody to (D) H3K4me2 (open chromatin), (E) H3K27ac, or (F) H3k27me3 and qPCR was performed to detect GATA4 binding sites at Runx2 promoters and enhancer region. Each qPCR was normalized to input and represented as fold enrichment over a negative genomic locus (mo. −ve) or the actin (Actb) promoter. n = 3, Student's t test: *p < 0.05, **p < 0.01, ***p < 0.001.