Nishio Yasuhiko, Dong Yufeng, Paris Mark, O'Keefe Regis J, Schwarz Edward M, Drissi Hicham
Center for Musculoskeletal Research, Department of Orthopaedics, Box 665, University of Rochester Medical Center, Rochester, NY 14642, USA.
Gene. 2006 May 10;372:62-70. doi: 10.1016/j.gene.2005.12.022. Epub 2006 Mar 29.
The zinc finger transcription factor Osterix (Osx) regulates bone formation and osteoblast differentiation in vitro and in vivo. We investigated the transcriptional mechanisms underlying the mouse Osx expression by isolating and characterizing its 5' upstream region. We performed 5' RACE on mRNA isolated from murine chondroprogenitor cells and determined a cap site of Osx approximately -99 nucleotides upstream of the initiation codon. Sequence analysis of this TATA-less promoter shows several putative response elements for Sox9, VDRE, Runx and Sp1. Transfection of the Osx promoter driving the luciferase reporter gene into C3H10T1/2 and ATDC5 cells shows a strong basal promoter activity between 565 bp and 2 kb. Deletion mutant analyses show that the most proximal 852 kb of the Osx promoter contains the highest activating domains, while strong repressive domains were identified between 1.8 and 2 kb. Over-expression experiments indicate that Runx2 significantly transactivates the Osx promoter by at least 2 fold indicating that Osx is downstream of Runx2 in mesenchymal cells. This up-regulation was abrogated when the Runx2 responsive element on the Osx promoter was mutated. Finally, we show that Runx2 specifically binds to this DNA element in the Osx promoter. Thus our results show for the first time Osx transcriptional regulation through the bone and cartilage related transcription factor Runx2.
锌指转录因子osterix（Osx）在体外和体内调节骨形成和成骨细胞分化。我们通过分离和鉴定其5'上游区域来研究小鼠Osx表达的转录机制。我们对从小鼠软骨祖细胞分离的mRNA进行了5'RACE，并确定了Osx的帽位点，其位于起始密码子上游约-99个核苷酸处。对这个无TATA框启动子的序列分析显示了几个针对Sox9、VDRE、Runx和Sp1的假定反应元件。将驱动荧光素酶报告基因的Osx启动子转染到C3H10T1/2和ATDC5细胞中，显示出在565 bp和2 kb之间有很强的基础启动子活性。缺失突变分析表明，Osx启动子最近端的852 kb包含最高的激活结构域，而在1.8至2 kb之间鉴定出强抑制结构域。过表达实验表明，Runx2显著激活Osx启动子至少2倍，表明在间充质细胞中Osx位于Runx2下游。当Osx启动子上的Runx2反应元件发生突变时，这种上调被消除。最后，我们表明Runx2特异性结合Osx启动子中的这个DNA元件。因此，我们的结果首次显示了通过骨和软骨相关转录因子Runx2对Osx的转录调控。
