TRANSCRIPTIONAL CONTROL OF THE TISSUE-SPECIFIC, DEVELOPMENTALLY-REGULATED OSTEOCALCIN GENE REQUIRES A BINDING MOTIF FOR THE MSX FAMILY OF HOMEODOMAIN PROTEINS
TRANSCRIPTIONAL CONTROL OF THE TISSUE-SPECIFIC, DEVELOPMENTALLY-REGULATED OSTEOCALCIN GENE REQUIRES A BINDING MOTIF FOR THE MSX FAMILY OF HOMEODOMAIN PROTEINS
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DOI：
10.1073/pnas.91.26.12887
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发表时间：
1994-12-20
影响因子：
11.1
通讯作者：
STEIN, JL
中科院分区：
文献类型：
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作者：
HOFFMANN, HM;CATRON, KM;STEIN, JL
大鼠骨钙素启动子的OC盒(NT-99至-76)是组织特异性和发育调控骨钙素基因表达的主要近端调控元件。OC盒的中心基序包括某些同源结构域蛋白的完美共识DNA结合位点。同源结构域蛋白是一种转录因子，通过调节基因表达的特定时间和空间模式来指导适当的发育。因此，我们讨论了同源结构域结合基序在OC启动子活性中的作用。在本研究中，通过结合突变和定点蛋白识别分析，我们检测了ROS 17/2.8骨肉瘤细胞核蛋白和纯化的MSX-1同源结构域蛋白与OC盒的相互作用。我们检测到一系列相关的特定蛋白质-DNA相互作用，其中一部分被针对MSX-1同源结构域的抗体抑制，但也识别MSX-2同源结构域。我们的结果表明，与MSX-2同源结构域的MSX-1结合所需的序列与体内骨钙素基因启动子活性所需的序列非常相似。这种功能关系通过在ROS17/2.8骨肉瘤细胞中瞬时表达一系列骨钙素启动子(NT-1097到+24)报告基因构建体来证明，该报告基因构建体包含在OC盒的同源结构域结合位点内和两侧的突变。对几种骨相关细胞的Northern印迹分析表明，所有细胞都表达MSX-1，而MSX-2仅限于转录骨钙素的细胞。综上所述，我们的结果提示MSX-1和MSX-2或相关的同源结构域蛋白在骨钙素基因的转录中起作用。
The OC box of the rat osteocalcin promoter (nt -99 to -76) is the principal proximal regulatory element contributing to both tissue-specific and developmental control of osteocalcin gene expression. The central motif of the OC box includes a perfect consensus DNA binding site for certain homeodomain proteins. Homeodomain proteins are transcription factors that direct proper development by regulating specific temporal and spatial patterns of gene expression. We therefore addressed the role of the homeodomain binding motif in the activity of the OC promoter. In this study, by the combined application of mutagenesis and site-specific protein recognition analysis, we examined interactions of ROS 17/2.8 osteosarcoma cell nuclear proteins and purified Msx-1 homeodomain protein with the OC box. We detected a series of related specific protein-DNA interactions, a subset of which were inhibited by antibodies directed against the Msx-1 homeodomain but which also recognize the Msx-2 homeodomain. Our results show that the sequence requirements for binding the Msx-1 of Msx-2 homeodomain closely parallel those necessary for osteocalcin gene promoter activity in vivo. This functional relationship was demonstrated by transient expression in ROS 17/2.8 osteosarcoma cells of a series of osteocalcin promoter (nt -1097 to +24)-reporter gene constructs containing mutations within and flanking the homeodomain binding site of the OC box. Northern blot analysis of several bone related cell types showed that all of the cells expressed msx-1, whereas msx-2 expression was restricted to cells transcribing osteocalcin. Taken together, our results suggest a role for Msx-1 and -2 or related homeodomain proteins in transcription of the osteocalcin gene.