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（适用提供）：非编码CGG重复扩展的FMR1基因的沉默超过200重复会导致脆弱的X综合症，X综合症是智力残疾的领先遗传形式，以及主要的单基因自闭症形式。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并不理解，这在很大程度上是由于无法绘制跨越启动子和单个等位基因中CGG重复元件的必要甲基化事件的工具所不可用的。现在，我们已经开发并实施了方法，使我们能够首次解决这一中央表观遗传问题。拟议的研究包括三个相互关联的特定目的，每个目标都基于一个工作假设，并解决了FMR1沉默的不同方面。具体的目标1（“结构”）是基于假设（假设1），即FMR1启动子的甲基化是CGG重复中甲基化的起源。该目标将使用单分子，实时（SMRT）测序来解决，这将使我们能够在启动子（包括CPG岛）和CGG重复元件上完全定义单个甲基化模式，以获得一系列CGG重复长度长度的单个等位基因。特定的目标2（“功能”）假定（假设2），特定表观型（MCPG和组蛋白修饰）将与FMR1 mRNA的不同水平相关。第二个目标将利用我们从复杂的镶嵌个体中生成多个成纤维细胞子clones的能力，使每个子字符具有单一的表观型，可以匹配特定的表达水平。具体目标3（“机械”）将解决甲基化如何触发的问题，并将阐明角色 我们提出（假设3）表明，在CGG重复次数上，频率，长度和停留时间增加了CGG重复长度的频率，长度和停留时间。反过来，这又提出导致形成双链DNA断裂（DSB），其修复的修复会通过断裂相关的染色质复制物触发基因沉默。为了测试这一点，我们将使用（i）使用（i）稳定的，非集成的偶发系统分析R环的形成模式，频率和动力学，并在诱导的启动子或天然FMR1启动子的控制下使用（i）构成稳定的，非整合的偶发性偶发系统； （ii）在本机染色体环境中携带各种扩展FMR1等位基因的亚克隆。我们预计实现这三个目标将对FMR1-GENE沉默产生连贯的机械理解，这反过来又应以基因特异性的方式制定了靶向治疗方法的发展，这是沉默机制的元素。