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

“R 环的形成可保护 CpG 岛免受表观遗传沉默”。

• 批准号: 8512740
• 负责人: Frederic Louis Chedin
• 金额: $ 26.81万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8512740
• 关键词: Aberrant DNA Methylation; Address; Affect; Algorithms; Base Pairing; Binding; Biochemical; Biological Assay; Cell Culture System; Cells; Characteristics; Complex; CpG Islands; DNA; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA Sequence; Elements; Enzymes; Epigenetic Process; Evolution; Exoribonuclease II; Family; Genes; Genetic Transcription; Genome; Genomics; Histones; Human; Human Genome; Immune System Diseases; Immunity; In Vitro; Knowledge; Lead; Malignant Neoplasms; Maps; Measures; Mediating; Methods; Methylation; Molecular Conformation; Molecular Genetics; Mus; Mutation; Patients; Pattern; Process; Property; Proteins; RNA; Recruitment Activity; Relative (related person); Reporting; Single-Stranded DNA; Site; Structure; Syndrome; System; Testing; Tetanus Helper Peptide; base; chromatin immunoprecipitation; embryonic stem cell; histone methyltransferase; human disease; imprint; novel; prevent; promoter; public health relevance; ribonuclease H1; ribonuclease HII; scale up; small hairpin RNA; vertebrate genome

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.

描述（由申请人提供）： 项目摘要：CpG 岛 (CGI) 充当超过 60% 的人类基因的启动子。重要的是，这些元件基本上没有 CpG 甲基化（一种与稳定转录沉默相关的表观遗传标记），尽管基因组中的绝大多数 CpG 位点都是甲基化的。虽然 CGI 对表观遗传沉默的相对免疫力多年来一直受到关注，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颖且关键的观察结果：（i）大部分 CGI 启动子虽然总体上富含 GC，但在 G 和 C 残基的分布中表现出明显的链不对称或倾斜； (ii) 通过此类高 GC 偏度区域的转录导致形成长 R 环结构，其中新转录的富含 G 的 RNA 仍与模板富含 C 的 DNA 链杂交，迫使非模板 DNA 链进入大部分为单链构象； (iii) R 环的形成保护底层 DNA 序列免受 DNA 甲基转移酶 (DNMT) 的作用。基于这些知识，我们假设哺乳动物 CGI 启动子处的 R 环形成有助于保护这些区域免受表观遗传沉默。我们建议通过结合计算、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的三个具体目标来进一步检验这一假设。具体目标 1：检验 R 环形成是哺乳动物 CGI 启动子的广泛且保守的特性这一假设。具体目标 2：检验 R 环形成可防止 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。 公共健康相关性：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 保护放松管制如何导致人类疾病，包括癌症、印记，以及最重要的 Aicardi-Goutieres 等自身免疫性疾病综合症。