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

描述(申请人提供)：项目摘要：CPG岛(CGI)作为启动子作用于超过60%的人类基因。重要的是，这些元件大部分没有CpG甲基化--一种与稳定转录沉默相关的表观遗传标记--尽管基因组中绝大多数CpG位点是甲基化的。虽然CGI对表观遗传沉默的相对免疫力已经被注意到多年，并且对它们的功能至关重要，但其潜在的机制仍然难以捉摸。在这里，我们报告了与这一过程相关的三个新的关键观察结果：(I)很大一部分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明显的链不对称或扭曲；(Ii)通过GC高扭曲的区域转录导致形成长R环结构，在该结构中，新转录的富含G的RNA仍然与富含模板C的DNA链杂交，迫使非模板DNA链变成基本上单链的构象；(Iii)R环的形成保护了潜在的DNA序列不受DNA甲基转移酶(DNMT)的作用。基于这一知识，我们假设哺乳动物CGI启动子上R环的形成有助于保护这些区域免受表观遗传沉默的影响。我们建议通过在人类和小鼠细胞中结合计算、基因组学、生化和分子遗传学方法的三个具体目标来进一步检验这一假说。具体目的1：验证R环形成是哺乳动物CGI启动子广泛而保守的特性的假设。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DNA甲基化模式异常的假设。 公共卫生相关性：项目叙述：这项建议为理解CGI启动子的功能和解决CGI的非管制保护通常如何导致人类疾病提供了一个新的框架，包括癌症、印记，最重要的是自身免疫性疾病，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.