Regulation of DMP1 Gene Expression in Bone and Tooth

描述（申请人提供）：牙本质基质蛋白（DMP 1）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成分。DMP 1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基础问题。目前，关于骨骼和牙齿发育过程中DMP 1表达的调控知之甚少。本R 03提案的目的是通过体外启动子研究来研究DMP 1基因在骨和牙齿的高度特化矿化细胞与其他非表达、非矿化细胞类型中表达的调控机制。我们的初步研究已经确定了一个新的元素在DMP 1启动子，可能控制DMP 1基因表达的矿化与非矿化细胞。DNA数据库分析显示，在许多其他牙本质/骨基因的启动子中发现了类似的DNA共有序列。这些体外研究将为进一步确定这种新型顺式元件及其基因调控功能提供初步信息。我们的假设是，在矿化与非矿化细胞的DMP 1基因表达的调节部分由一种新的保守的顺式元件通过离散的相互作用与相应的转录因子。为了实现这一目标，本研究提出了两个具体的目标：1）通过电泳迁移率变动分析，利用选择性扩增的结合和突变方法，确定DNA-蛋白质结合位点的共有序列; 2）通过使用体外启动子检测顺式/反式相互作用，确定以细胞特异性方式调节DMP 1基因表达的共有序列的生物学作用。记者分析。我们提出的工作是创新的，因为我们已经确定了一个新的DNA-蛋白质结合元件在DMP 1基因启动子，是保守的其他牙本质/牙齿胶原和非胶原基因的启动子。这些研究的结果有望为研究DMP 1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基础。这些实验也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.