Mouse Col10a1 Gene Regulation and Skeletal Development

描述(由申请人提供)：这项建议的具体目的是表征在软骨细胞肥大过程中赋予X型胶原组织特异性表达的顺式元件和潜在的转录决定因素。其长期目标是了解软骨细胞成熟的分子调控。 软骨细胞肥大是长骨软骨内骨化过程中软骨细胞分化的终末阶段，X型胶原是唯一已知的肥大软骨细胞特异性分子标志物。在人类中，它的缺乏会导致施密德干骺端软骨发育不良症(SMCD)。小鼠遗传学研究表明，X胶原蛋白缺乏症具有部分类似人类SMCD的表型异常。到目前为止，指导其在体内肥大的软骨细胞特异性表达的顺式元件或转录因子尚未被描述。初步数据表明，10kb的小鼠COL10a1启动子/内含子元件可以引导报告基因在整个肥厚区的表达。体内转基因、体外转基因和电泳学分析表明，在COL10a1远端启动子和第二内含子中均含有保守元件。我们建议通过转基因小鼠的方法在体内和体外通过软骨细胞肥大的MCT细胞模型进一步剖析这个10kb的Col10a1启动子/内含子元件和可能的DNA结合蛋白，这些蛋白指导其组织特异性表达。这对于了解软骨内骨化的分子机制以及X型胶原等骨骼发育不良的分子发病机制是至关重要的。

DESCRIPTION (provided by applicant): The specific aim of this proposal is to characterize the cis elements and potential transcriptional determinants that confer the tissue-specific expression of type X collagen during chondrocyte hypertrophy. The long-term objective is to understand the molecular regulation of chondrocyte maturation. Chondrocyte hypertrophy is the terminal stage of chondrocyte differentiation during endochondral ossification of long bone growth and type X collagen is the only known hypertrophic chondrocyte-specific molecular marker. Its deficiency in humans causes Schmid metaphyseal chondrodysplasia (SMCD). Mouse genetic studies show that collagen X deficiency has phenotypic abnormalities partly resemble human SMCD. Until now, cis elements or transcription factors that direct its hypertrophic chondrocyte-specific expression in vivo have not been described. The preliminary data within this proposal show that the 10 kb murine Col10a1 promoter/intron element can direct reporter gene expression throughout the hypertrophic zone. In vivo transgenic, in vitro transfection and in silico analysis of Col10a1 suggests that there are conserved elements within both Col10a1 distal promoter and second intron. We propose to further dissect this 10 kb Col10a1 promoter/intron element and the putative DNA-binding proteins that direct its tissue-specific expression in vivo by using transgenic mice approaches and in vitro by using the MCT cell model of chondrocyte hypertrophy. This is essential for understanding the molecular mechanisms that specify endochondral ossification and that underlie the molecular pathogenesis of skeletal dysplasias like those involving type X collagen.