Tamiya Hiroyuki, Ikeda Toshiyuki, Jeong Jae-Hwan, Saito Taku, Yano Fumiko, Jung Youn-Kwan, Ohba Shinsuke, Kawaguchi Hiroshi, Chung Ung-Il, Choi Je-Yong
Center for Disease Biology and Integrative Medicine, Faculty of Medicine, the University of Tokyo, Tokyo, 113-8655, Japan.
Gene. 2008 Jun 15;416(1-2):53-60. doi: 10.1016/j.gene.2008.03.003. Epub 2008 Mar 15.
Little is known about the upstream regulator of Runx2, a master regulator of osteoblast differentiation in bone tissues. To elucidate the molecular mechanism of Runx2 gene expression, we analyzed Runx2 promoter activity in osseous (MC3T3-E1, KS483, Kusa) and non-osseous (NIH3T3, C3H10T1/2, mouse embryonic fibroblasts) cells and also identified Runx2 upstream regulator using a Runx2 promoter-derived luciferase reporter system. After cloning 15 serial deletion constructs from -6832 bp/+390 bp to -37 bp/+390 bp of the Runx2-P1 promoter, we performed a transient transfection assay in osseous and non-osseous cells. A reduction in Runx2 promoter activity was observed in two regions; one was between -3 kb and -1 kb, and the other was between -155 bp and -75 bp. The step-down pattern in promoter activity between -3 kb and -1 kb was observed only in osseous cells. Interestingly, the step-down pattern between -155 bp and -75 bp was revealed in both cell types. Consistently, beta-galactosidase staining in axial skeleton of -3 kb-Runx2-P1-LacZ transgenic mice was positive, but that of all skeletal tissues of -1 kb-Runx2-P1-LacZ transgenic mice was negative. To identify upstream regulators of the Runx2-P1 promoter, we screened 100 transcription factors using Runx2-P1-luciferase reporter constructs in NIH3T3 fibroblasts and HeLa cells. Among them, HIF2A was identified as the strongest activator of Runx2-P1 promoter activity. A HIF2A-responsive site on the Runx2 promoter was identified between -106 bp and -104 bp by mutation analysis. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding of HIF2A to the Runx2-P1 promoter in vitro and in vivo, respectively. Knock-down using siRNA against HIF2A confirmed that HIF2A is an important regulator of Runx2 gene expression. Collectively, these results suggest that the region between -3 kb and -1 kb is required for the minimal skeletal tissue-specific expression of Runx2, and that the region between -155 bp and -75 bp is important for its basal transcription, which may be in part mediated by HIF2A in bone tissues.
关于骨组织中骨细胞分化的主要调节因子Runx2的上游调节因子，人们了解甚少。为了阐明Runx2基因表达的分子机制，我们分析了骨细胞（MC3T3-E1、KS483、Kusa）和非骨细胞（NIH3T3、C3H10T1/2、小鼠胚胎成纤维细胞）中Runx2启动子的活性，并使用Runx2启动子衍生的荧光素酶报告系统鉴定了Runx2上游调节因子。从Runx2-P1启动子的-6832 bp/+390 bp至-37 bp/+390 bp克隆了15个连续缺失构建体后，我们在骨细胞和非骨细胞中进行了瞬时转染实验。在两个区域观察到Runx2启动子活性降低；一个在-3 kb和-1 kb之间，另一个在-155 bp和-75 bp之间。仅在骨细胞中观察到-3 kb和-1 kb之间启动子活性的逐步下降模式。有趣的是，-155 bp和-75 bp之间的逐步下降模式在两种细胞类型中均有显示。一致地，-3 kb-Runx2-P1-LacZ转基因小鼠轴向骨骼中的β-半乳糖苷酶染色呈阳性，但-1 kb-Runx2-P1-LacZ转基因小鼠所有骨骼组织中的染色均为阴性。为了鉴定Runx2-P1启动子的上游调节因子，我们在NIH3T3成纤维细胞和HeLa细胞中使用Runx2-P1-荧光素酶报告构建体筛选了100种转录因子。其中，HIF2A被鉴定为Runx2-P1启动子活性最强的激活剂。通过突变分析在Runx2启动子上-106 bp和-104 bp之间鉴定出一个HIF2A反应位点。电泳迁移率变动分析和染色质免疫沉淀分析分别在体外和体内证实了HIF2A与Runx2-P1启动子的结合。使用针对HIF2A的siRNA进行敲低证实HIF2A是Runx2基因表达的重要调节因子。总体而言，这些结果表明，-3 kb和-1 kb之间的区域是Runx2最小骨骼组织特异性表达所必需的，而-155 bp和-75 bp之间的区域对其基础转录很重要，这可能部分由骨组织中的HIF2A介导。
