Transcriptional Regulation in hMLH1-Silenced Colon Cells

描述（由申请方提供）：本提案中的研究将剖析在用2-脱氧-5-氮杂胞苷[AzadC]处理细胞后，导致位于结肠癌主要亚组的细胞系中3号染色体上的一组基因逃避异常甲基化的条件。我们实验室对错配修复基因hMLH 1表达异常沉默的人结肠肿瘤细胞系进行了详细的微阵列分析，确定了3号染色体上与hMLH 1基因共诱导表达的一组基因。拟议的实验将利用滚环探针定位这些基因在间期核。拟议的研究将确定这些基因在3号染色体上相互分离的共表达是否存在结构基础，并将探讨它们的调控与其他AzadC反应基因的差异。进一步的研究将解剖这些基因中的每一个的启动子区域的精细结构，以确定它们在沉默细胞中的CpG甲基化模式，以及在短暂的AzadC诱导的这种抑制释放后，并利用AzadC暴露后分离的一组组成型表达亚克隆。这些研究将检验这样的假设，即这些基因是异常甲基化过程导致的共调节基因组的代表。他们将进一步对比导致该基因亚组中调控机制废除的条件与AzadC诱导的其他基因位点调节所需的条件。导致基因沉默和逃避这些基因抑制过程的机制将通过分析改变表达的基因在用AzadC处理的细胞中与针对三种主要的人DNA甲基转移酶DNMT 1、3a和3b的反义RNA相比的交叉来进一步检查。甲基化相关基因表达调控的机制分化也将进一步解剖采用反义RNA特定的组蛋白去乙酰化酶。作为拟议研究重点的细胞系代表了15-20%的表现出微卫星不稳定性[MSI]的人类结肠癌，这几乎普遍与DNA MMR缺陷同义。这些MSI癌症中的2/3被归类为散发性起源的癌症，因为它们的发生与已知的家族缺陷无关。

DESCRIPTION (provided by applicant): The studies in this proposal will dissect conditions leading to escape from aberrant methylation in a cohort of genes localized on chromosome 3 in a cell line representative of a major subgroup of colon cancers following treatment of cells with 2-deoxy-5- azacytidine [AzadC]. Detailed microarray analyses in our laboratory of a human colon tumor cell line in which the mismatch repair gene, hMLH1 is aberrantly silenced for expression have defined a set of genes on chromosome 3 that are co-induced for expression with the hMLH1 gene. Proposed experiments will utilize rolling circle probes to localize these genes in interphase nuclei. Proposed studies will determine if there is a structural basis for co-expression of these genes that are widely separated from each other on chromosome 3 and will probe how their regulation differs from other AzadC responsive genes. Further studies will dissect the fine structure of the promoter regions of each of these genes for their CpG methylation patterns in gone silenced cells and after transient AzadC-induced release from this repression, and exploit a group of constitutively expressing subclones isolated after AzadC exposure. These studies will test the hypothesis that these genes are representative of a co-regulated gene cohort resulting from the aberrant methylation process. They will further contrast conditions leading to abrogation of regulatory mechanisms in this subset of genes with those required for AzadC-induced modulation of other gene sites. The mechanisms leading to gene silencing and escape from these gene repression processes will be further examined by analyzing the intersection of genes altered for expression in cells treated with AzadC versus antisense RNA to the three major human DNA methyltransferase enzymes, DNMT1, 3a and 3b. Mechanistic differentiation of methylation-related gene expression regulation will also be further dissected employing antisense RNAs to specific histone deacetylases. The cell lines that are a focus of the proposed studies are representative of 15-20% of human colon cancers that exhibit microsatellite instability [MSI], which is almost universally synonymous with defects in DNA MMR. 2/3 of these MSI cancers are classified as cancers of sporadic origin because their occurrence does not correlate with a known familial defect.