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

描述（由申请人提供）：拟议研究的目的是确定导致脆性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FMR 1的5 <$UTR中CGG三核苷酸重复扩增引起的。 含有>200个CGG重复拷贝的等位基因与FMR 1启动子的异染色质化以及随后FMR 1转录和FMRP表达的丧失相关。 FMR 1沉默发生在胎儿发育的早期，这阻碍了用于解剖FMR 1基因沉默所涉及的分子途径的体外范例的发展。 最近，一项研究表明，FXS人胚胎干细胞（HESC）含有未甲基化的FMR 1启动子，其在分化时变得甲基化，提供了第一个FMR 1沉默的体外培养模型。 该提案测试了小RNA在FXS中FMR 1沉默中的作用。 CGG重复RNA在体外被RNA干扰酶Dicer加工成小RNA。 最近的研究表明，针对mRNA转录物或基因启动子的小RNA可以导致哺乳动物细胞中启动子的沉默。 小RNA在介导哺乳动物逆转录转座子基因沉默中也具有关键作用，其与CGG重复序列一样，通常以重复的核苷酸元件为特征。 这些研究提出了小RNA途径可能参与FXS中FMR 1沉默的可能性。 本提案中描述的研究目标是使用这种新的模型系统首次鉴定介导FXS中FMR 1启动子沉默的途径，并确定小RNA在FMR 1沉默中的作用。 本研究的具体目的是：（1）确定RNA干扰途径在FXS中FMR 1沉默中的作用。 使用野生型和FXS HESC系，我们将确定FMR 1启动子是否获得与RNA指导的启动子沉默相关的特征性染色质标记。 我们还将确定microRNA和PIWI RNA加工所需的途径是否是FMR 1沉默所必需的;以及（2）我们将确定FMR 1转录物在FXS HESC中如何加工。 我们将监测内源性FMR 1全重复转录本以及异源表达的CGG重复含有转录本的加工，以确定CGG重复RNA是否在FMR 1基因沉默期间产生小RNA。 FXS干细胞的出现提供了第一次机会，从分子上剖析导致异常表观遗传沉默的信号通路，这种异常表观遗传沉默导致这种高度流行的精神发育迟滞综合征。 在这个高风险/高回报的R21项目中使用干细胞将为FMR 1沉默所涉及的分子组分提供新的见解，并使我们能够解决FXS，三核苷酸重复序列疾病和小RNA指导的基因沉默的新兴领域之间的联系。

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.