TRANSCRIPTIONAL AND EPIGENETIC REGULATION OF HUMAN N-CADHERIN GENE EXPRESSION

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 钙粘附素是一类组织完整性所必需的黏附蛋白。它们是跨膜蛋白，与相邻细胞上的钙粘附素形成同源二聚体，并直接或间接地附着在细胞骨架上。由于其在上皮组织维持中的作用，E-钙粘素是研究最深入的钙粘附素。大多数人类癌症发生在上皮组织中，在肿瘤进展过程中，肿瘤细胞可能开始不适当地表达N-钙粘素。N-钙粘蛋白因其在轴突生长中的作用而得名，在更多的间质组织中发现，开始表达N-钙粘蛋白的肿瘤细胞变得更具运动性和侵袭性。N-钙粘蛋白基因最近才成为研究的对象，而为了了解转录调控而进行的启动子鉴定一直是有限的。癌细胞中N-钙粘附素的错误表达似乎与正常发育的上皮向间充质转化(EMT)过程中N-钙粘附素的表达增加非常相似，这表明可以通过对EMT模型细胞培养系统的研究来了解这种转移转化。这项研究的总体目标是确定参与调控人N-钙粘蛋白基因转录的特定转录因子结合部位、转录因子和表观遗传启动子修饰。具体目的1是利用荧光素酶报告基因详细评估N-钙粘蛋白启动子-462bp至-1896bp之间的一个可能的抑制子位点。具体目的2是使用荧光素酶报告评估第一外显子60bp3‘的Lef-1共识结合位点的转录作用。具体目的3涉及使用染色质免疫沉淀(ChIP)来检测体内转录因子的结合，包括AP-1，Lef-1，任何已识别的阻遏因子，以及潜在的表观遗传修饰，包括甲基化。如果芯片分析表明AP-1、Lef-1或其他相关转录因子与N-钙粘素启动子片段结合，则特异性目的4是评估N-钙粘素启动子片段-荧光素酶报告基因在EMT模型细胞培养系统中的表达，在该系统中，EMT诱导信号通路的组成部分可以被中断。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cadherins are a class of adhesion proteins required for tissue integrity. They are transmembrane proteins that form homodimers with cadherins on adjacent cells and attach either directly or indirectly to the cytoskeleton. E-cadherin is the most thoroughly studied cadherin due to its role in epithelial tissue maintenance. Most human cancers arise in epithelial tissues, and, during tumor progression, tumor cells may begin expressing N-cadherin inappropriately. N-cadherin is named for its role in neurite outgrowth, is found in more mesenchymal tissues, and tumor cells that begin expressing N-cadherin become more motile and invasive. The N-cadherin gene has only recently become the subject of study and promoter characterization in order to understand the regulation of transcription has been limited. The misexpression of N-cadherin in cancer cells appears to be very similar to the increase in N-cadherin expression that occurs during the normal developmental epithelial-to-mesenchymal transition (EMT), suggesting that insight into this metastatic transition can be gleaned from studies in EMT model cell culture systems. The overall goal of this research is to identify specific transcription factor binding sites, transcription factors, and epigenetic promoter modifications involved in regulating transcription of the human N-cadherin gene. Specific Aim 1 is to evaluate a putative repressor site between -462bp and -1896bp of the N-cadherin promoter in detail using luciferase reporters. Specific Aim 2 is to evaluate the proposed trancriptional role of a LEF-1 consensus binding site 60bp 3' of the first exon also using luciferase reporters. Specific Aim 3 involves using chromatin immuno-precipitation (ChIP) to detect in vivo binding of transcription factors including AP-1, LEF-1, any identified repressor, and potential epigenetic modifications including methylation. If ChIP assays indicate that AP-1, LEF-1 or other relevant transcription factors are binding to N-cadherin promoter fragments, Specific Aim 4 is to evaluate expression of N-cadherin promoter fragment-luciferase reporters in EMT model cell culture systems in which components of the EMT inducing signaling pathways can be interrupted.