TRANSCRIPTIONAL AND EPIGENETIC REGULATION OF HUMAN N-CADHERIN GENE EXPRESSION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 为子项目列出的总成本可能 表示子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 钙粘蛋白是一类组织完整性所需的粘附蛋白。 它们是跨膜蛋白，与相邻细胞上的钙粘蛋白形成同源二聚体，并直接或间接附着于细胞骨架。 由于其在上皮组织维护中的作用，E-钙粘蛋白是研究最彻底的钙粘蛋白。 大多数人类癌症发生在上皮组织中，并且在肿瘤进展期间，肿瘤细胞可能开始不适当地表达N-钙粘蛋白。 N-钙粘蛋白因其在神经突生长中的作用而命名，在更多的间充质组织中发现，并且开始表达N-钙粘蛋白的肿瘤细胞变得更具运动性和侵袭性。 N-钙粘蛋白基因只是最近才成为研究的主题，启动子表征，以了解转录的调控一直是有限的。 癌细胞中N-钙粘蛋白的错误表达似乎与正常发育上皮细胞向间充质细胞转化（EMT）期间发生的N-钙粘蛋白表达的增加非常相似，这表明可以从EMT模型细胞培养系统的研究中收集对这种转移性转化的见解。 本研究的总体目标是确定特定的转录因子结合位点，转录因子，和表观遗传的启动子修饰参与调节人类N-钙粘蛋白基因的转录。 具体目的1是使用荧光素酶报告基因详细评估N-钙粘蛋白启动子-462 bp和-1896 bp之间的推定阻遏位点。 具体目标2是也使用荧光素酶报告基因来评估第一外显子的60 bp 3'的LEF-1共有结合位点的拟定转录作用。 具体目标3涉及使用染色质免疫沉淀（ChIP）来检测转录因子（包括AP-1、LEF-1）、任何鉴定的阻遏物和潜在的表观遗传修饰（包括甲基化）的体内结合。 如果ChIP试验表明AP-1、LEF-1或其他相关转录因子与N-钙粘蛋白启动子片段结合，则具体目标4是评价N-钙粘蛋白启动子片段-荧光素酶报告基因在EMT模型细胞培养系统中的表达，其中EMT诱导信号传导途径的组分可能被中断。

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.