Regulation of DMP1 Gene Expression in Bone and Tooth

描述(申请人提供)：牙本质基质蛋白(DMP1)是一种酸性磷酸化蛋白，是牙齿和骨骼矿化细胞外基质的重要组成部分。DMP1的时空表达模式高度局限于矿化组织。了解调控组织特异性基因表达的过程是发育生物学中的一个重要的基本问题。目前，对DMP1在骨和牙齿发育过程中的表达调控知之甚少。本R03建议的目的是通过体外启动子研究DMP1基因在高度特化的骨骼和牙齿矿化细胞中表达的调控机制，以比较其他不表达、非矿化细胞类型。我们的初步研究已经在DMP1启动子中发现了一个新的元件，它可能控制矿化细胞和非矿化细胞中DMP1基因的表达。DNA数据库分析表明，在其他一些牙本质/骨基因的启动子中也发现了类似的DNA共有序列。这些体外研究将为进一步确定这一新的顺式元件及其与基因调控相关的功能提供初步信息。我们的假设是，DMP1基因在矿化细胞和非矿化细胞中的表达部分是由一个新的保守顺式元件通过与相应转录因子的离散相互作用来调节的。为了实现这一目标，提出了两个具体的目标：1)通过选择和扩增结合和突变的方法，通过凝胶迁移率改变分析确定DNA-蛋白质结合位点的一致序列(S)；2)通过体外启动子-报告分析，通过检测顺式/反式相互作用，确定一致序列(S)以细胞特异性的方式调节DMP1基因表达的生物学作用。我们的工作具有创新性，因为我们在DMP1基因启动子中发现了一个新的DNA-蛋白质结合元件，该元件在其他牙本质/牙齿胶原性和非胶原性基因的启动子中保守。这些研究结果有望为研究DMP1在骨骼和牙齿以及矿化和非矿化组织中时空表达模式的分子机制提供体内研究的基础。这些实验还将为矿化组织基因调控的基本机制提供重要的新知识。因此，这些研究将对全面了解骨/牙齿特异性基因表达的分子基础做出重要贡献。

DESCRIPTION (provided by applicant): Dentin matrix protein (DMP1) is an acidic phosphorylated protein that is an important component of the mineralized extracellular matrix of tooth and bone. Spatial-temporal expression patterns of DMP1 are highly restricted to mineralized tissues. Understanding the processes that regulate tissue-specific gene expression is an important fundamental issue in developmental biology. Currently, very little is known regarding the regulation of DMP1 expression during bone and tooth development. The purpose of this R03 proposal is to investigate through in vitro promoter studies the regulatory mechanisms of DMP1 gene expression in the highly specialized mineralized cells of bones and teeth versus other non-expressing, non-mineralizing cell types. Our preliminary studies have identified a novel element in the DMP1 promoter that potentially controls DMP1 gene expression in mineralizing versus non-mineralizing cells. DNA database analysis reveals that similar DNA consensus sequences are found in the promoters of a number of other dentin/bone genes. These in vitro studies will provide initial information to further define this novel cis-element and its function related to gene regulation. Our hypothesis is that the DMP1 gene expression in mineralizing versus non-mineralizing cells is regulated in part by a novel conserved cis-element through discrete interactions with corresponding transcription factors. In order to achieve this goal, two Specific Aims are proposed: 1) To determine consensus sequence(s) of the DNA-protein binding site by using selected and amplified binding and mutagenesis methods by electrophoretic mobility shift assay; 2) To determine the biological role of the consensus sequence(s) regulating DMP1 gene expression in the cell-specific manner by examining the cis/trans interactions using in vitro promoter-reporter analysis. Our proposed work is innovative because we have identified a novel DNA-protein binding element in DMP1 gene promoter that is conserved in the promoters of other dentin/tooth collagenous and non-collagenous genes. Results of these studies are expected to provide the foundation for in vivo studies investigating the molecular mechanisms of spatial-temporal expression patterns of DMP1 in bones and teeth as well as mineralizing and non-mineralizing tissues. These experiments will also provide significant new knowledge concerning the basic mechanism of mineralized tissue gene regulation. These studies will therefore make an important contribution toward an overall understanding of the molecular basis of bone/tooth specific gene expression.