title: Runx2 contributes to murine Col10a1 gene regulation through direct interaction with its cis-enhancer.
abstract: We have recently shown that a 150-bp Col10a1 distal promoter (-4296 to -4147 bp) is sufficient to direct hypertrophic chondrocyte-specific reporter (LacZ) expression in vivo. More recently, through detailed sequence analysis we identified two putative tandem-repeat Runx2 binding sites within the 3'-end of this 150-bp region (TGTGGG-TGTGGC, -4187 to -4176 bp). Candidate electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation, and transfection studies demonstrate that these putative Runx2 sites bind Runx2 and mediate upregulated Col10a1/reporter activity in vitro. Transgenic studies using the 5'-sequence without Runx2 sites were not able to drive the cell-specific LacZ reporter activity, suggesting the in vivo requirement of the Runx2 sites located in the 3'-end in mediating Col10a1/reporter expression. Indeed, mutating the Runx2 sites in the context of the 150-bp promoter abolishes its capacity to drive hypertrophic chondrocyte-specific reporter expression in transgenic mice. We have also generated multiple transgenic mouse lines using only the 3'-sequence containing the Runx2 sites to drive the LacZ gene. Interestingly, no hypertrophic chondrocyte-specific blue staining was observed in these transgenic mice. Together, our data support that Runx2 directly interacts with murine Col10a1 cis-enhancer. This interaction is required but not sufficient for cell-specific Col10a1 promoter activity in vivo. Additional cooperative/repressive elements within the 5'- or 3'-sequences of this 150-bp promoter are needed to work with Runx2 together to mediate cell-specific Col10a1 expression. Further delineation of these elements/factors has the potential to identify novel therapeutic targets for multiple skeletal disorders, including osteoarthritis, that show abnormal Col10a1 expression and altered chondrocyte maturation.
authors: Department of Anatomy and Cell Biology, Rush University Medical Center, Chicago, IL, USA.
authors: Feifei
authors: F
authors: Li
authors: Yaojuan
authors: Y
authors: Lu
authors: Ming
authors: M
authors: Ding
authors: Dobrawa
authors: D
authors: Napierala
authors: Sam
authors: S
authors: Abbassi
authors: Yuqing
authors: Y
authors: Chen
authors: Xiangyun
authors: X
authors: Duan
authors: Siying
authors: S
authors: Wang
authors: Brendan
authors: B
authors: Lee
authors: Qiping
authors: Q
authors: Zheng
mesh_terms: D000818:Animals; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D024121:Collagen Type X / Q000235:genetics* / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020080:Tandem Repeat Sequences / Q000235:genetics
chemical_list: C570620:Col10a1 protein, mouse; D024121:Collagen Type X; D050659:Core Binding Factor Alpha 1 Subunit; C493716:Runx2 protein, mouse; D004247:DNA