INITIATION OF SILENCING BY METHYL BINDING PROTEINS

肿瘤抑制基因的甲基化相关沉默是与癌症的形成和发展有关的常见事件。虽然甲基化相关的抑癌基因沉默的原因尚不清楚，但人们认为甲基化的抑癌基因启动子的稳定抑制是由一个或多个甲基结合蛋白(MBP)介导的。这些蛋白质与甲基化的CpG(MCpG)二核苷酸特异结合。这一建议解决了MBPS在启动子甲基化之前的肿瘤抑制基因失活中起因果作用的可能性。具体地说，我认为，肿瘤细胞中甲基化模式的改变导致MBP错误地定位于未甲基化的CpG岛两侧的甲基化区域。通常受到保护而不被失活的启动子无法抵抗附近结合的Mbps增加的阻遏活性，变得沉默，随后甲基化。这里描述的实验将直接使用特征明确的小鼠腺嘌呤磷酸核糖基转移酶基因(Aprt)作为模型来测试该模型的预测。该建议的目的是：1)确定过度表达导致的Mbps的错误定位是否会导致内源aprt基因的异常沉默；2)确定抵抗甲基化相关沉默的未甲基化启动子是否也抵抗上游结合的Mbps沉默；3)确定Mbps沉默非甲基化启动子是否导致其甲基化。这项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.