GATA4 represses RANKL in osteoblasts via multiple long-range enhancers to regulate osteoclast differentiation.
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.
['Khalid|Aysha B|AB|', 'Slayden|Alexandria V|AV|', 'Kumpati|Jerusha|J|', 'Perry|Chanel D|CD|', 'Berryhill|Stuart B|SB|', 'Crawford|Julie A|JA|', 'Fatima|Iram|I|', 'Morselli|Marco|M|', 'Pellegrini|Matteo|M|', 'Miranda-Carboni|Gustavo A|GA|', 'Krum|Susan A|SA|']
D000818:Animals D001862:Bone Resorption D000071556:Cancellous Bone D002454:Cell Differentiation D045744:Cell Line, Tumor D004742:Enhancer Elements, Genetic D050991:GATA4 Transcription Factor D006801:Humans D018345:Mice, Knockout D010006:Osteoblasts D010010:Osteoclasts D010012:Osteogenesis D053245:RANK Ligand D012333:RNA, Messenger D012097:Repressor Proteins
GATA4 RANKL Osteoblast Osteoclast Bone