Transcriptional Regulation in hMLH1-Silenced Colon Cells

hMLH1 沉默的结肠细胞中的转录调控

• 批准号: 6904650
• 负责人: W DAVID SEDWICK
• 金额: $ 30.24万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6904650
• 关键词: CpG islands; DNA methylation; DNA replication; amidohydrolases; antisense nucleic acid; azacitidine; cell line; chromosome aberrations; colon neoplasms; functional /structural genomics; gene expression; gene induction /repression; genetic promoter element; genetic regulation; methyltransferase; molecular cloning; neoplasm /cancer genetics; nucleic acid quantitation /detection; nucleic acid repetitive sequence; protein localization; serial analysis of gene expression

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.

描述（由申请人提供）：该提案中的研究将剖析条件，导致在一个细胞系中定位于3染色体3染色体的基因中的异常甲基化，代表了用2-脱氧5-偶氮替丁的细胞处理细胞后，代表了一个主要亚组结肠癌[azacytidine [azadc]。在我们的人类结肠肿瘤细胞系中，在我们的实验室中进行了详细的微阵列分析，其中不匹配修复基因HMLH1异常沉默以表达表达，已定义了3号染色体上的一组基因，该基因与HMLH1基因共同诱导以表达以表达。提出的实验将利用滚动圆探针将这些基因定位在相间核中。拟议的研究将确定是否存在这些基因共表达这些基因的结构基础，这些基因在3染色体上彼此广泛分离，并将探测它们的调节与其他Azadc反应性基因的不同。进一步的研究将剖析这些基因中每个基因的启动子区域的精细结构，以使其在消失的细胞中以及瞬时阿扎德诱导的这种抑制作用释放后，并利用一组构成性表达的亚克隆，该亚克隆在AzADC暴露后分离出来。这些研究将检验以下假设：这些基因代表了异常甲基化过程所产生的共同调节基因队列。它们将进一步对比条件，从而导致基因子集中的调节机制与AzADC诱导的其他基因位点的调节所需的条件。导致基因沉默和逃离这些基因抑制过程的机制将通过分析用AzADC与抗义RNA与反义RNA处理的细胞中的基因相交，以与三种主要人DNA甲基转移酶，DNMT1、3A和3B和3B。甲基化相关基因表达调节的机械分化也将进一步解剖，采用特定组蛋白脱乙酰基酶的反义RNA。拟议研究的重点的细胞系代表了15-20％表现出微卫星不稳定性的人类结肠癌[MSI]，这几乎是DNA MMR缺陷的普遍代名词。这些MSI癌症中有2/3被分类为零星来源的癌症，因为它们的发生与已知的家族缺陷无关。