"R-loop formation protects CpG islands against epigenetic silencing".

描述（由申请人提供）：项目概述：CpG岛（CGI）作为超过60%的人类基因的启动子。重要的是，尽管基因组中的绝大多数CpG位点都是甲基化的，但这些元件仍然大多没有CpG甲基化--一种与稳定转录沉默相关的表观遗传标记。虽然CGIs对表观遗传沉默的相对免疫力已经被注意到多年，并且对其功能至关重要，但其潜在机制仍然难以捉摸。在这里，我们报告了与该过程相关的三个新的和关键的观察结果：（i）大部分CGI启动子，虽然总体上富含GC，但在G和C残基的分布中显示出显著的链不对称或偏斜;（ii）通过这些高GC偏斜区域的转录导致长R环结构的形成，其中新转录的富G RNA保持与模板富C DNA链杂交，迫使非模板DNA链进入大部分单链构象;和（iii）R环形成保护潜在的DNA序列免受DNA甲基转移酶（DNMT）的作用。基于这些知识，我们假设，在哺乳动物CGI启动子的R-环的形成，以保护这些地区对表观遗传沉默。我们建议通过三个特定目标结合计算，基因组学，生物化学和分子遗传学方法在人类和小鼠细胞中进一步验证这一假设。具体目的1：检验R环形成是哺乳动物CGI启动子的广泛和保守性质的假设。具体目标2：检验R环形成防止DNA甲基化的假设。具体目标3：检验R环形成改变导致异常DNA甲基化模式的假设。 公共卫生相关性：项目叙述：该提案提供了一个新的框架，用于理解CGI启动子功能，并解决CGI的保护失调如何导致人类疾病，包括癌症，印记，最重要的是，自身免疫疾病，如Aicardi-Goutieres综合征。

DESCRIPTION (provided by applicant): Project Summary: CpG islands (CGIs) function as promoters for greater than 60% of human genes. Importantly, these elements remain mostly free of CpG methylation - an epigenetic mark associated with stable transcriptional silencing - despite the fact that the vast majority of CpG sites in the genome are methylated. While the relative immunity of CGIs against epigenetic silencing has been noted for years and is critical to their function, its underlying mechanism has remained elusive. Here, we report three novel and key observations relevant to this process: (i) a large fraction of CGI promoters, while GC-rich overall, display marked strand asymmetry, or skew, in the distribution of G and C residues; (ii) transcription through such regions of high GC-skew leads to the formation of long R-loop structures in which the newly transcribed G-rich RNA remains hybridized to the template C-rich DNA strand, forcing the non-template DNA strand into a largely single-stranded conformation; and (iii) R-loop formation protects the underlying DNA sequence from the action of DNA methyltransferases (DNMTs). Based on this knowledge, we hypothesize that R-loop formation at mammalian CGI promoters serves to protect these regions against epigenetic silencing. We propose to further test this hypothesis through three Specific Aims combining computational, genomics, biochemical, and molecular genetics approaches in human and mouse cells. Specific Aim 1: To test the hypothesis that R-loop formation is a widespread and conserved property of mammalian CGI promoters. Specific Aim 2: To test the hypothesis that R-loop formation protects against DNA methylation. Specific Aim 3: To test the hypothesis that altered R-loop formation leads to aberrant DNA methylation patterns. PUBLIC HEALTH RELEVANCE: Project Narrative: This proposal provides a novel framework for understanding CGI promoter function and for addressing how deregulated protection of CGIs often leads to human diseases, including cancer, imprinting, and most importantly, auto-immune disorders such as Aicardi-Goutieres Syndrome.