INITIATION OF SILENCING BY METHYL BINDING PROTEINS

通过甲基结合蛋白引发沉默

• 批准号: 6514911
• 负责人: PHILLIP A YATES
• 金额: $ 4.81万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6514911
• 关键词: CpG islands; DNA binding protein; DNA methylation; adenine phosphoribosyltransferase; chimeric proteins; chromatin; complementary DNA; enzyme induction /repression; genetic promoter element; nucleic acid sequence; tissue /cell culture; transcription factor; tumor suppressor genes

Methylation-associated silencing of tumor suppressor genes is a common event implicated in the formation and progression of cancer. Although the causes of methylation-associated tumor suppressor gene silencing are unknown, it is thought that stable repression of methylated tumor suppressor gene promoters is mediated by one or more methyl-binding proteins (MBPs). These proteins bind specifically to methylated CpG (mCpG) dinucleotides. This proposal addresses the possibility that MBPs play a causal role in tumor suppressor gene inactivation prior to promoter methylation. Specifically, I propose that altered methylation patterns in tumor cells cause the mistargeting of MBPs to methylated regions flanking unmethylated CpG islands. Promoters that are normally protected from inactivation are unable to resist the increased repressor activity from MBPs bound nearby, become silenced and subsequently methylated. The experiments described here will directly test the predictions of this model using the well-characterized mouse adenine phosphoribosyltransferase gene (Aprt) as a model. The objectives of this proposal are: 1) to determine if mistargeting of MBPs via overexpression can cause the aberrant silencing of the endogenous Aprt gene; 2) to determine if unmethylated promoters that are resistant to methylation- associated silencing are also resistant to silencing by MBPs bound upstream; 3) to determine if silencing of an unmethylated promoter by MBPs leads to its methylation. The proposed studies will provide important insights into the nature of aberrant gene silencing in cancer and will reveal functional differences between MBPs.

甲基化相关的肿瘤抑制基因的沉默是一个常见事件，涉及癌症的形成和进展。尽管甲基化相关肿瘤抑制基因沉默的原因尚不清楚，但人们认为稳定抑制甲基化肿瘤抑制基因启动子是由一种或多种甲基结合蛋白（MBP）介导的。这些蛋白质特异性与甲基化的CpG（MCPG）二核苷酸结合。该提议解决了Mbps在启动子甲基化之前灭活肿瘤基因失活中起因果作用的可能性。具体而言，我建议改变肿瘤细胞中的甲基化模式会导致Mbps的误解到甲基化区域，该区域是未甲基化的CPG岛的甲基化区域。通常受到保护免于失活的启动子无法抵抗附近Mbps的抑制剂活性增加，沉默并随后甲基化。此处描述的实验将直接使用特征良好的小鼠腺嘌呤磷酸糖基转移酶基因（APRT）作为模型来测试该模型的预测。该提案的目标是：1）确定Mbps是否通过过表达对Mbps进行误解会导致内源性APRT基因的异常沉默； 2）确定抗甲基化相关的沉默的未甲基化启动子是否也抵抗了上游MBPS的沉默； 3）确定MBPS对未甲基化启动子的沉默是否导致其甲基化。拟议的研究将提供对癌症异常基因沉默的性质的重要见解，并揭示Mbps之间的功能差异。