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.

肿瘤抑制基因的甲基化相关沉默是涉及癌症形成和进展的常见事件。虽然甲基化相关的肿瘤抑制基因沉默的原因是未知的，它被认为是由一个或多个甲基结合蛋白（MBP）介导的甲基化肿瘤抑制基因启动子的稳定抑制。这些蛋白质特异性结合甲基化CpG（mCpG）二核苷酸。该建议解决了MBPs在启动子甲基化之前在肿瘤抑制基因失活中发挥因果作用的可能性。具体来说，我建议，改变肿瘤细胞中的甲基化模式，导致错误的MBP甲基化区域侧翼未甲基化的CpG岛。通常被保护免于失活的启动子不能抵抗来自附近结合的MBP的增加的阻遏物活性，变得沉默并随后甲基化。这里描述的实验将直接测试该模型的预测，使用充分表征的小鼠腺嘌呤磷酸核糖基转移酶基因（Aprt）作为模型。该提议的目的是：1）确定通过过表达的MBP的错误启动是否可以引起内源Aprt基因的异常沉默; 2）确定对甲基化相关沉默具有抗性的未甲基化启动子是否也对上游结合的MBP的沉默具有抗性; 3）确定MBP对未甲基化启动子的沉默是否导致其甲基化。拟议的研究将为癌症中异常基因沉默的性质提供重要的见解，并将揭示MBP之间的功能差异。