Epigenetic regulation of the FMR1 gene

 描述(申请人提供)：FMR1基因沉默的非编码CGG重复扩展超过200个重复会导致脆性X综合征，这是智力残疾的主要遗传形式，并导致自闭症的主要单基因形式。然而，尽管这种表观遗传现象非常重要，但导致沉默的机制(S)在很大程度上是由于没有工具来定位必要的甲基化事件，这些事件跨越单个等位基因中的启动子和CGG重复元件。我们现在已经开发和实施了一些方法，使我们能够第一次解决这个核心的表观遗传学问题。这项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，并解决了FMR1沉默的不同方面。特定目的1(“结构”)基于假设(假设1)，即FMR1启动子的甲基化是CGG重复内甲基化启动的结果。这一目标将通过单分子实时测序来实现，这将使我们能够完整地定义跨启动子(包括CpG岛)的个体甲基化模式(S)和大范围CGG重复长度的个体等位基因的CGG重复元件。特定目的2(“功能”)假设(假设2)特定的表观类型(mCpG和组蛋白修饰)将与不同水平的FMR1mRNA的表达有关。第二个目标是利用我们的能力，从复杂的马赛克个体中产生多个成纤维细胞亚克隆，使每个亚克隆包含一个可与特定表达水平匹配的单一表观类型。具体目标3(“机械论”)将解决如何触发甲基化的问题，并将澄清其作用 在这个过程中CGG重复的可能性。我们提出(假设3)在CGG重复上形成共转录R环的频率、长度和停留时间随着CGG重复长度的增加而增加。这反过来又被认为导致双链DNA断裂(DSB)的形成，双链DNA断裂的修复通过断裂相关的染色质重塑触发基因沉默。为了验证这一点，我们将分析R-环的形成模式、频率和动态以及DSB的形成，使用(I)稳定的、非整合的附体系统，该系统在可诱导启动子或自然FMR1启动子的控制下包含扩展的CGG重复；以及(Ii)在其天然染色体环境中携带各种扩展的FMR1等位基因的亚克隆。我们预计，实现这三个目标将带来对FMR1基因沉默的一致机制的理解，这反过来将促进以基因特异性方式靶向沉默机制元件的治疗方法的发展。

 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.