INITIATION OF SILENCING BY METHYL BINDING PROTEINS

甲基化相关的肿瘤抑制基因沉默是涉及癌症形成和进展的常见事件。虽然甲基化相关肿瘤抑制基因沉默的原因尚不清楚，但人们认为甲基化肿瘤抑制基因启动子的稳定抑制是由一种或多种甲基结合蛋白（MBPs）介导的。这些蛋白特异性结合甲基化CpG （mCpG）二核苷酸。该建议提出了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，我提出肿瘤细胞中甲基化模式的改变导致MBPs错误靶向未甲基化的CpG岛两侧的甲基化区域。正常情况下免于失活的启动子无法抵抗附近结合的MBPs增加的抑制活性，从而沉默并随后甲基化。本文描述的实验将直接测试该模型的预测，该模型使用具有良好特征的小鼠腺嘌呤磷酸核糖基转移酶基因（Aprt）作为模型。本研究的目的是：1)确定通过过表达错误靶向MBPs是否会导致内源性Aprt基因的异常沉默；2)确定对甲基化相关沉默具有抗性的非甲基化启动子是否也对上游结合的MBPs沉默具有抗性；3)确定MBPs沉默未甲基化启动子是否会导致其甲基化。拟议的研究将为癌症中异常基因沉默的本质提供重要的见解，并将揭示MBPs之间的功能差异。

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.