GATA4 represses RANKL in osteoblasts via multiple long-range enhancers to regulate osteoclast differentiation

Abstract

GATA4 is a transcription factor that is responsible for tissue-specific gene regulation in many tissues, and more recent studies showed that it is necessary for osteoblast differentiation. Previously, we showed that in vivo deletion of Gata4 using Cre-recombinase under the control of the Col1a1 2.3 kb promoter, showed significantly reduced trabecular bone properties. To understand the role of GATA4 in more differentiated cells, GATA4fl/fl mice were crossed with mice expressing Cre-recombinase under the control of the osteocalcin promoter. MicroCT analysis of trabecular bone properties of the femur and tibia from 14-week-old female osteocalcin-Cre/ GATA4fl/fl (OCN-cKO) mice showed a significant reduction in percentage bone volume, a decrease in trabecular number and an increase in trabecular spacing. In vivo, histomorphometric analysis revealed a decrease in the number of osteoblasts and an increase in the number of osteoclasts in the tibiae of OCN-cKO mice. In vivo and in vitro systems correlated a decrease in Gata4 mRNA with increased RANKL gene expression. To determine if RANKL is a direct target of GATA4, chromatin immunoprecipitation (ChIP)-sequencing was performed, and it demonstrated that GATA4 is recruited to seven enhancers near RANKL. Furthermore, when Gata4 is knocked down, the chromatin at the RANKL region is further opened, as detected by a reduction in histone 3 lysine 27 trimethylation (H3K27me3) and an increase in histone 3 lysine 4 dimethylation (H3K4me2) in the RANKL locus. In vitro, TRAP staining of cells from bone marrow cultures from Gata4 knockout cells show that the increased levels of RANKL are sufficient for osteoclast formation. Together, the data suggest that GATA4 directly represses RANKL expression via seven cis-regulatory regions and plays an important role in maintaining proper bone development and osteoclast formation.

Osteocyte RANKL is required for cortical bone loss with age and is induced by senescence

“…Here, we show that an increase in Gata4 is sufficient to stimulate production of both IL-1α and RANKL. Khalid and coworkers have suggested that Gata4 binds to the Tnfsf11 promoter and represses Tnfsf11 expression (32). A reason for the difference between this previous study and the results presented here is not clear.…”

The cis-regulatory effects of modern human-specific variants

“…We also included 100 negative control fragments, created by randomly picking 100 of the designed DNA fragments and scrambling their sequence ( Supplementary file 1e ). Lastly, we incorporated 299 positive control fragments ( Ryu et al, 2018 ; Prabhakar et al, 2008 ; Visel et al, 2007 ; Inoue et al, 2019 ; Hojo et al, 2016 ; Meyer et al, 2016 ; Khalid et al, 2018a ; Khalid et al, 2018b ; Loots et al, 2005 ; Fukami et al, 2006 ; Kawane et al, 2018 ) (i.e., expected to drive expression; Supplementary file 1d ). As the library was infected into three cell types (see later), we designed positive controls for each of the cell types.…”

GATA4 regulates osteogenic differentiation by targeting miR‑144‑3p

“…Song et al demonstrated that GATA4 could negatively regulate osteogenic differentiation in BMSCs (17). Other previous studies have reported that GATA4 could not only inhibit the expression of RANKL in the process by which BMSCs differentiate into osteoblasts but also regulate the differentiation of osteoclasts (32)(33)(34)(35). In addition, GATA4 has been revealed to participate in the regulation of tooth development by affecting the expression of osteogenesis regulatory factors and promoting the proliferation of tooth mesenchymal cells (36).…”