Analysis of the Runx2 promoter in osseous and non-osseous cells and identification of HIF2A as a potent transcription activator
Analysis of the Runx2 promoter in osseous and non-osseous cells and identification of HIF2A as a potent transcription activator
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DOI：
10.1016/j.gene.2008.03.003
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发表时间：
2008-06-15
期刊：
影响因子：
3.5
通讯作者：
Choi, Je-Yong
中科院分区：
文献类型：
--
作者：
Tamiya, Hiroyuki;Ikeda, Toshiyuki;Choi, Je-Yong
Runx 2是骨组织中成骨细胞分化的主要调控因子，但其上游调控因子知之甚少。为了阐明Runx 2基因表达的分子机制，我们分析了Runx 2启动子在骨细胞（MC 3 T3-E1，KS 483，Kusa）和非骨细胞（NIH 3 T3，C3 H10 T1/2，小鼠胚胎成纤维细胞）中的活性，并使用Runx 2启动子衍生的荧光素酶报告系统鉴定了Runx 2上游调控因子。在克隆Runx 2-P1启动子的-6832 bp/+390 bp至-37 bp/+390 bp的15个连续缺失构建体后，我们在骨和非骨细胞中进行瞬时转染测定。在两个区域观察到Runx 2启动子活性降低;一个区域在-3 kb和-1 kb之间，另一个区域在-155 bp和-75 bp之间。仅在骨细胞中观察到启动子活性在-3 kb和-1 kb之间的逐步下降模式。有趣的是，在两种细胞类型中揭示了-155 bp和-75 bp之间的逐步下降模式。β-半乳糖苷酶在-3 kb-Runx 2-P1 - LacZ转基因小鼠中轴骨组织中呈阳性染色，而在-1 kb-Runx 2-P1-LacZ转基因小鼠的所有骨骼组织中均呈阴性染色。为了鉴定Runx 2-P1启动子的上游调控因子，我们在NIH 3 T3成纤维细胞和HeLa细胞中使用Runx 2-P1-荧光素酶报告基因构建体筛选了100个转录因子。其中，HIF 2A被鉴定为Runx 2-P1启动子活性的最强激活剂。通过突变分析在Runx 2启动子上的-106 bp和-104 bp之间鉴定了HIF 2A应答位点。电泳迁移率变动试验和染色质免疫沉淀试验证实了HIF 2A的结合Runx 2-P1启动子在体外和体内，分别。HIF 2A是Runx 2基因表达的重要调控因子，提示-3 kb ~-1 kb的区域是Runx 2在骨组织特异性表达所必需的，而-155 bp ~-75 bp的区域是Runx 2基础转录的重要区域，这可能是HIF 2A在骨组织中介导的。(C)2008 Elsevier B. V.保留所有权利。
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. (C) 2008 Elsevier B.V. All rights reserved.