Epigenetic regulation of the FMR1 gene

FMR1基因的表观遗传调控

• 批准号: 8857166
• 负责人: Frederic Louis Chedin
• 金额: $ 38.31万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-21 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8857166
• 关键词: Address; Alleles; Autistic Disorder; CGG repeat; CGG repeat expansion; Cells; Complex; CpG Islands; DNA Double Strand Break; DNA Modification Process; Development; Elements; Epigenetic Process; Event; FMR1; FMR1 Gene; Fibroblasts; Fragile X Gene; Fragile X Syndrome; Frequencies; Gene Silencing; Genes; Genotype; Individual; Inherited; Intellectual functioning disability; Lead; Length; Maps; Messenger RNA; Methods; Methylation; Mosaicism; Mutation; Pattern; Pattern Formation; Process; Relative (related person); Research; Role; System; Testing; Time; Transcriptional Silencer Elements; Untranslated RNA; Work; base; chromatin modification; chromatin protein; chromatin remodeling; deep sequencing; epigenetic regulation; histone modification; methylation pattern; promoter; public health relevance; repaired; residence; single molecule; therapeutic development; tool

 DESCRIPTION (provided by applicant): Silencing of the FMR1 gene for non-coding CGG-repeat expansions in excess of 200 repeats gives rise to fragile X syndrome, the leading inherited form of intellectual disability, and to a principal single-gene form of autism. However, despite the critical importance of this epigenetic phenomenon, the mechanism(s) leading to silencing are not understood in large part due to the unavailability of tools for mapping the necessary methylation events that span both the promoter and CGG-repeat element within single alleles. We have now developed and implemented methods that will allow us to address this central epigenetic issue for the first time. The proposed research comprises three interrelated specific aims, each based on a working hypothesis and addressing a different aspect of FMR1 silencing. Specific Aim 1 ("structural") is based on the hypothesis (Hypothesis 1) that methylation of the FMR1 promoter is a consequence of the initiation of methylation within the CGG repeat. This aim will be addressed using single molecule, real-time (SMRT) sequencing, which will enable us to completely define individual methylation pattern(s) across the promoter (inclusive of the CpG island) and the CGG-repeat element for a broad range of CGG-repeat-length individual alleles. Specific Aim 2 ("functional") posits (Hypothesis 2) that specific epigenotypes (both mCpG and histone modifications) will be associated with differing levels of expression of FMR1 mRNA. This second aim will utilize our ability to generate multiple fibroblast sub-clones from complex mosaic individuals such that each sub-clone harbors a single epigenotype that can be matched to a specific expression level. Specific Aim 3 ("mechanistic") will address the question of how methylation is triggered and will clarify the role of the CGG repeat in this process. We propose (Hypothesis 3) that co-transcriptional R-loop formation at the CGG repeat increases in frequency, length, and residence time with increasing CGG-repeat length. This in turn is proposed to lead to the formation of double-strand DNA breaks (DSBs), the repair of which triggers gene silencing through break-associated chromatin-remodeling. To test this, we will analyze R-loop formation patterns, frequency, and dynamics together with the formation of DSBs using (i) a stable, non-integrating episomal system harboring expanded CGG repeats under the control of either an inducible promoter or the native FMR1 promoter; and (ii) subclones carrying various expanded FMR1 alleles in their native chromosomal context. We expect that achieving these three aims will bring about a coherent mechanistic understanding of FMR1-gene silencing, which should in turn facilitate the development of therapeutic approaches to target, in a gene-specific fashion, elements of the silencing mechanism.

 描述（由申请人提供）：FMR1 基因因非编码 CGG 重复扩展超过 200 个重复而沉默会引起脆性 X 综合征，这是智力障碍的主要遗传形式，也是自闭症的主要单基因形式然而，尽管这种表观遗传现象至关重要，但导致沉默的机制在很大程度上仍不清楚，因为缺乏绘制跨越两个基因组的必要甲基化事件的工具。我们现在已经开发并实施了一些方法，使我们能够首次解决这一核心表观遗传问题。拟议的研究包括三个相互关联的具体目标，每个目标都基于一个工作假设并解决一个问题。 FMR1 沉默的不同方面。 具体目标 1（“结构性”）基于以下假设（假设 1）：FMR1 启动子的甲基化是 CGG 重复序列内甲基化起始的结果。这一目标将通过单分子实时 (SMRT) 测序来实现，这将使我们能够完全定义跨启动子（包括 CpG 岛）和广泛范围的 CGG 重复元件的个体甲基化模式。与特定表观基因型（mCpG 和组蛋白修饰）相关的特定目标 2（“功能”）位置（假设 2）。第二个目标将利用我们从复杂嵌合体个体中生成多个成纤维细胞亚克隆的能力，以便每个亚克隆都具有可与特定表达水平相匹配的单一表观基因型。 “机制”）将解决甲基化是如何触发的问题并阐明其作用 我们提出（假设 3），随着 CGG 重复长度的增加，CGG 重复处的共转录 R 环形成的频率、长度和停留时间也会增加。双链 DNA 断裂 (DSB) 的形成，其修复通过断裂相关的染色质重塑触发基因沉默。为了测试这一点，我们将分析 R 环形成模式、频率和影响。使用（i）稳定的非整合附加型系统，在诱导型启动子或天然 FMR1 启动子的控制下包含扩展的 CGG 重复序列，以及（ii）在其天然中携带扩展的各种 FMR1 等位基因的亚克隆我们预计，实现这三个目标将为 FMR1 基因沉默带来连贯的机制理解，从而促进靶向治疗方法的开发。基因特异性时尚，沉默机制的要素。