Epigenetic regulation of the FMR1 gene

 描述（由申请人提供）：FMR 1基因的非编码CGG重复扩增超过200个重复的沉默引起脆性X综合征，这是智力残疾的主要遗传形式，并引起孤独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还不清楚，这是由于无法获得用于定位跨越单个等位基因内的启动子和CGG重复元件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个核心的表观遗传问题。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FMR 1沉默的不同方面。具体目标1（“结构”）基于以下假设（假设1）：FMR 1启动子的甲基化是CGG重复序列内甲基化起始的结果。这一目标将使用单分子实时（SMRT）测序来解决，这将使我们能够完全定义跨启动子（包括CpG岛）和CGG重复元件的单个甲基化模式，用于广泛的CGG重复长度单个等位基因。特定目的2（“功能性”）假定（假设2）特定表观基因型（mCpG和组蛋白修饰）将与FMR 1 mRNA的不同表达水平相关。这第二个目标将利用我们的能力，从复杂的嵌合体个体产生多个成纤维细胞亚克隆，使得每个亚克隆具有可以与特定表达水平匹配的单个表观基因型。具体目标3（“机制”）将解决甲基化是如何触发的问题，并将阐明甲基化的作用。 在这个过程中，CGG重复。我们提出（假设3），在CGG重复的共转录R-环的形成增加的频率，长度和停留时间与CGG重复长度的增加。这反过来又被认为导致双链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、频率和动力学以及DSB的形成，使用（i）在诱导型启动子或天然FMR 1启动子的控制下携带扩增的CGG重复的稳定的非整合附加型系统;和（ii）在其天然染色体背景中携带各种扩增的FMR 1等位基因的亚克隆。我们希望实现这三个目标将带来一个连贯的机制理解FMR 1基因沉默，这反过来又会促进治疗方法的发展目标，在基因特异性的方式，沉默机制的元素。

 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.