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Osterix acetylation at K307 and K312 enhances its transcriptional activity and is required for osteoblast differentiation.

ABSTRACT: Osterix (Osx) is an essential transcription factor involved in osteoblast differentiation and bone formation. The precise molecular mechanisms of the regulation of Osx expression are not fully understood. In the present study, we found that in cells, both endogenous and exogenous Osx protein increased after treatment with histone deacetylase inhibitors Trichostatin A and hydroxamic acid. Meanwhile, the results of immunoprecipitation indicated that Osx was an acetylated protein and that the CREB binding protein (CBP), and less efficiently p300, acetylated Osx. The interaction and colocalization of CBP and Osx were demonstrated by Co-immunoprecipitation and immunofluorescence, respectively. In addition, K307 and K312 were identified as the acetylated sites of Osx. By contrast, HDAC4, a histone deacetylase (HDAC), was observed to interact and co-localize with Osx. HDAC4 was demonstrated to mediate the deacetylation of Osx. Moreover, we found that acetylation of Osx enhanced its stability, DNA binding ability and transcriptional activity. Finally, we demonstrated that acetylation of Osx was required for the osteogenic differentiation of C2C12 cells. Taken together, our results provide evidence that CBP-mediated acetylation and HDAC4-mediated deacetylation have critical roles in the modification of Osx, and thus are important in osteoblast differentiation.

SUBMITTER: Lu J

PROVIDER: S-EPMC5122325 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Osterix acetylation at K307 and K312 enhances its transcriptional activity and is required for osteoblast differentiation.

Lu Jianlei J Qu Shuang S Yao Bing B Xu Yuexin Y Jin Yucui Y Shi Kaikai K Shui Yifang Y Pan Shiyang S Chen Li L Ma Changyan C

Oncotarget 20160601 25

Osterix (Osx) is an essential transcription factor involved in osteoblast differentiation and bone formation. The precise molecular mechanisms of the regulation of Osx expression are not fully understood. In the present study, we found that in cells, both endogenous and exogenous Osx protein increased after treatment with histone deacetylase inhibitors Trichostatin A and hydroxamic acid. Meanwhile, the results of immunoprecipitation indicated that Osx was an acetylated protein and that the CREB ...[more]

PMID: 27250035

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Project description:Commitment of Runx2-expressing precursor osteoblasts to functional osteoblasts and then to osteocytes is triggered by Osterix (Osx), which activates its target genes in those cells during bone formation. It is not yet known whether Osx has a role in remodeling the chromatin architecture of its target genes during the transition from preosteoblast to osteoblast. In testing the hypothesis that Osx is indispensable for active chromatin architecture, we first showed that in Osx-null calvarial cells occupancy of the transcriptional activators, including lysine 4 methyl transferase (Wdr5), c-Myc, and H2A.Z, at the Osx target gene Bsp was very markedly decreased. The levels of methylation of lysines 4 and 36 and acetylation of histone H3, markers for active chromatin, were also reduced at the Bsp gene in these cells. In contrast, occupancy of the transcriptional repressors HP1 and the nucleolar protein 66 (NO66), a histone demethylase previously identified as an Osx-interacting protein, was increased at the Bsp gene in Osx-null calvarial cells. Furthermore, the Bsp promoter was hypermethylated in embryonic stem (ES) cells and in embryonic day 9.5 (E9.5) embryos but was markedly hypomethylated in the calvaria of E18.5 embryos, coinciding with robust Bsp expression. In contrast, CpG methylation in the Bsp promoter remained high in Osx-null calvaria compared to Osx-wild-type calvaria. Our data also revealed that NO66 interacted with DNA Methyltransferase 1A (DNMT1A), histone deacetylase 1A (HDAC1A), and HP1, which are known to control histone and DNA methylation. In addition, HP1 stimulated the demethylase activity of NO66 for its substrates "trimethylation of histone H3 at lysine 4" (H3K4me3) and "trimethylation of histone H3 at lysine 36" (H3K36me3). Our findings strongly suggest that in the absence of Osx, the chromatin of Osx target genes is transcriptionally inactive. We propose that Osx is a molecular switch for the formation of an active chromatin state during osteoblast differentiation, whereas NO66 helps gene repression through histone demethylation and/or formation of a repressor complex, resulting in multilayered control of the chromatin architecture of specific osteoblast genes.

Project description:Bone formation via intramembranous and endochondral ossification is necessary for successful healing after a wide range of bone injuries. The pleiotropic cytokine, vascular endothelial growth factor A (VEGFA) has been shown, via nonspecific pharmacologic inhibition, to be indispensable for angiogenesis and ossification following bone fracture and cortical defect repair. However, the importance of VEGFA expression by different cell types during bone healing is not well understood. We sought to determine the role of VEGFA from different osteoblast cell subsets following clinically relevant models of bone fracture and cortical defect. Ubiquitin C (UBC), Osterix (Osx), or Dentin matrix protein 1 (Dmp1) Cre-ERT2 mice (male and female) containing floxed VEGFA alleles (VEGFAfl/fl ) were either given a femur full fracture, ulna stress fracture, or tibia cortical defect at 12 weeks of age. All mice received tamoxifen continuously starting 2 weeks before bone injury and throughout healing. UBC Cre-ERT2 VEGFAfl/fl (UBC cKO) mice, which were used to mimic nonspecific inhibition, had minimal bone formation and impaired angiogenesis across all bone injury models. UBC cKO mice also exhibited impaired periosteal cell proliferation during full fracture, but not stress fracture repair. Osx Cre-ERT2 VEGFAfl/fl (Osx cKO) mice, but not Dmp1 Cre-ERT2 VEGFAfl/fl (Dmp1 cKO) mice, showed impaired periosteal bone formation and angiogenesis in models of full fracture and stress fracture. Neither Osx cKO nor Dmp1 cKO mice demonstrated significant impairments in intramedullary bone formation and angiogenesis following cortical defect. These data suggest that VEGFA from early osteolineage cells (Osx+), but not mature osteoblasts/osteocytes (Dmp1+), is critical at the time of bone injury for rapid periosteal angiogenesis and woven bone formation during fracture repair. Whereas VEGFA from another cell source, not from the osteoblast cell lineage, is necessary at the time of injury for maximum cortical defect intramedullary angiogenesis and osteogenesis. © 2019 American Society for Bone and Mineral Research.

Dataset Information

Osterix acetylation at K307 and K312 enhances its transcriptional activity and is required for osteoblast differentiation.

Publications

Osterix acetylation at K307 and K312 enhances its transcriptional activity and is required for osteoblast differentiation.

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