title: Osteoblast-specific transcription factor Osterix (Osx) is an upstream regulator of Satb2 during bone formation.
abstract: Osterix (Osx) is an osteoblast-specific transcription factor essential for osteoblast differentiation and bone formation. Osx knock-out mice lack bone completely. Satb2 is critical for osteoblast differentiation as a special AT-rich binding transcription factor. It is not known how Satb2 is transcriptionally regulated during bone formation. In this study, quantitative real-time RT-PCR results demonstrated that Satb2 was down-regulated in Osx-null calvaria. In stable C2C12 mesenchymal cells using the tetracycline (Tet)-Off system, overexpression of Osx stimulated Satb2 expression. Moreover, inhibition of Osx by siRNA led to repression of Satb2 expression in osteoblasts. These results suggest that Osx controls Satb2 expression. Transient transfection assay showed that Osx activated 1kb Satb2 promoter reporter activity in a dose-dependent manner. To define the region of Satb2 promoter responsive to Osx activation, a series of deletion mutants of Satb2 constructs were made, and the minimal region was narrowed down to the proximal 130 bp of the Satb2 promoter. Further point mutation studies found that two GC-rich region mutations disrupted the Satb2 130bp promoter activation by Osx, suggesting that these GC-rich binding sites were responsible for Satb2 activation by Osx. Gel shift assay showed that Osx bound to the Satb2 promoter sequence directly. ChIP assays indicated that endogenous Osx associated with the native Satb2 promoter in osteoblasts. Importantly, Satb2 siRNA significantly inhibited Osx-induced osteoblast marker gene expressions. Taken together, our findings indicate that Osx is an upstream regulator of Satb2 during bone formation. This reveals a new additional link of the transcriptional regulation mechanism that Osx controls bone formation.
authors.affiliation: Bone Research Laboratory, Texas Scottish Rite Hospital for Children, Dallas, Texas 75390, USA.
authors.forename: Wanjin
authors.initials: W
authors.lastname: Tang
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: Lindsey
authors.initials: L
authors.lastname: Osimiri
authors.forename: Chi
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001665:Binding Sites; D015536:Down-Regulation / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D036961:Matrix Attachment Region Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009928:Organ Specificity / Q000235:genetics; D010006:Osteoblasts / Q000378:metabolism*; D010012:Osteogenesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D036961:Matrix Attachment Region Binding Proteins; D034741:RNA, Small Interfering; C501473:SATB2 protein, mouse; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D014157:Transcription Factors