Testing the role of small RNAs in FMR1 promoter silencing in Fragile X Syndrome

描述(由申请人提供)：拟议研究的目标是确定导致脆性X综合征(FXS)的异常表观遗传重编程的分子基础。FXS是由脆性X智力低下蛋白(FMRP)基因FMR1的5？非翻译区(5？UTR)上的一个扩大的CGG三核苷酸重复引起的。含有&gt；200个CGG重复拷贝的等位基因与FMR1启动子的异染色质化以及随后的FMR1转录和FMRP表达的丧失有关。FMR1沉默发生在胎儿发育的早期，这阻碍了研究FMR1基因沉默所涉及的分子通路的体外范例的发展。最近的一项研究表明，FXS人胚胎干细胞(HESC)含有未甲基化的FMR1启动子，在分化过程中会发生甲基化，这提供了第一个FMR1沉默的体外培养模型。这一建议测试了小RNA在FXS中FMR1沉默中的作用。在体外，CGG重复RNA被RNA干扰酶Disher加工成小RNA。最近的研究表明，针对mRNA转录本或基因启动子的小RNA可以导致哺乳动物细胞中启动子的沉默。小RNA也在介导哺乳动物反转录转座子基因沉默方面发挥关键作用，与CGG重复序列一样，这些基因通常由重复的核苷酸元件组成。这些研究提出了小RNA通路可能参与FXS中FMR1沉默的可能性。这项研究的目的是利用这一新的模型系统首次确定在FXS中介导FMR1启动子沉默的途径，并确定小RNA在FMR1沉默中的作用。该建议的具体目的是：(1)确定RNA干扰途径在FXS中FMR1沉默中的作用。使用野生型和FXS HESC系，我们将确定FMR1启动子是否获得与RNA指导的启动子沉默相关的特征染色质标记。我们还将确定MicroRNA和PIWI RNA处理所需的途径是否对于FMR1沉默是必要的；以及(2)我们将确定FMR1转录本在FXS HESC中是如何处理的。我们将监测内源FMR1全重复转录本以及异源表达的含有CGG重复转录本的处理过程，以确定CGG重复RNA在FMR1基因沉默过程中是否产生小RNA。FXS干细胞的出现为从分子上剖析导致异常表观遗传沉默的信号通路提供了第一次机会，而这种异常的表观遗传沉默导致了这种高度普遍的智力低下综合征。干细胞在这个高风险/高回报的R21项目中的使用将为FMR1沉默所涉及的分子成分提供新的见解，并将使我们能够解决FXS、三核苷酸重复障碍和新兴的小RNA定向基因沉默之间的联系。

DESCRIPTION (provided by applicant): The objective of the proposed research is to identify the molecular basis for the aberrant epigenetic reprogramming that leads to fragile X syndrome (FXS). FXS is caused by an expanded CGG trinucleotide repeat in the 5¿ untranslated region (5¿UTR) of the fragile X mental retardation protein (FMRP) gene, FMR1. Alleles containing >200 copies of the CGG repeat are associated with heterochromatinization of the FMR1 promoter and the subsequent loss of FMR1 transcription and FMRP expression. FMR1 silencing occurs early in fetal development, which has prevented the development of an in vitro paradigm for dissecting the molecular pathways involved in FMR1 gene silencing. Recently, a study showed that FXS human embryonic stem cells (HESC) contain unmethylated FMR1 promoters, which become methylated upon differentiation, providing the first in vitro culture model of FMR1 silencing. This proposal tests the role of small RNAs in FMR1 silencing in FXS. CGG repeat RNAs are processed into small RNAs in vitro by the RNA interference enzyme Dicer. Recent studies have shown that small RNAs directed against mRNA transcripts or gene promoters can result in the silencing of promoters in mammalian cells. Small RNAs also have critical roles in mediating silencing of mammalian retrotransposon genes, which, like CGG repeat sequences, are often characterized by repeated nucleotide elements. These studies raise the possibility that small RNA pathways may be involved in FMR1 silencing in FXS. The goal of the research described in this proposal is to use this new model system to identify, for the first time, the pathways that mediate FMR1 promoter silencing in FXS and to determine the role of small RNAs in FMR1 silencing. The specific aims of this proposal are: (1) To determine the role of RNA interference pathways in FMR1 silencing in FXS. Using wild-type and FXS HESC lines, we will determine if the FMR1 promoter acquires characteristic chromatin marks that are associated with RNA-directed promoter silencing. We will also determine if the pathways required for microRNA and PIWI RNA processing are necessary for FMR1 silencing; and (2) We will determine how the FMR1 transcript is processed in FXS HESC. We will monitor the processing of the endogenous FMR1 full repeat transcript as well as heterologously expressed CGG repeat-containing transcripts, to determine whether CGG repeat RNAs generate small RNAs during FMR1 gene silencing. The advent of FXS stem cells provides the first opportunity to molecularly dissect the signaling pathways that lead to the aberrant epigenetic silencing that causes this highly prevalent mental retardation syndrome. The use of stem cells in this high risk/high reward R21 project will provide new insight into the molecular components involved in FMR1 silencing and will allow us to address a link between FXS, trinucleotide repeat disorders, and the emerging area of small RNA-directed gene silencing.