权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

GAA REPEATS INDUCED EPIGENETIC SILENCING IN FRIEDREICH'S ATAXIA

GAA 在 FRIEDREICH 共济失调中重复诱导表观遗传沉默

基本信息

批准号：
10579665
负责人：
Marek Napierala
金额：
$ 33.86万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10579665
关键词：
GAA REPEATS INDUCED EPIGENETIC SILENCING

项目摘要

Friedreich’s ataxia (FRDA) is the most common autosomal recessive ataxia. It is caused by reduced levels of the mitochondrial protein frataxin (FXN) as a result of large expansions of GAA trinucleotide repeats located in the first intron of the FXN gene. Although the FXN coding sequence in FRDA patients is unaltered, transcription of the gene is suppressed as a consequence of the large GAA expansions. Downregulation of FXN expression is associated with a transition of chromatin surrounding the GAAs from an active to a repressed state, however the underlying molecular mechanism of FXN silencing remains largely unknown. At the present time there is no effective treatment for FRDA and transcriptional silencing of FXN is one of the primary targets for therapeutic intervention. Therefore, understanding the mechanism governing GAA-induced silencing is of critical importance for therapy development. Based on our preliminary studies we hypothesize that long, expanded GAA repeats induce replication stress leading to changes of the replication program at the endogenous FXN locus. A resulting collision between transcription and replication suppresses transcription elongation and stimulates expansions of GAA repeats. The transcription elongation defect is further amplified in trans by deficiency of specific transcriptional co-factors. To address this hypothesis, we will focus on three fundamental questions regarding the molecular pathogenesis of Friedreich’s ataxia: 1) How does interplay between transcription and replication at the endogenous FXN locus affect gene silencing and expansions of GAA repeats? 2) Which step of the transcription process is affected by expanded GAA repeats in FRDA cells? 3) What is the contribution of trans-factors to the transcriptional defect in FRDA? First, we will dissect mechanisms of molecular interplay between transcription and replication in the endogenous FXN locus using a set of CRISPR/Cas9 engineered FRDA cells. Furthermore, we will employ the precision nuclear run-on sequencing (PRO-seq) technique to determine the profile of nascent transcription at the FXN locus, while also defining the exact step of transcription affected by expanded GAA repeats. Additionally, we will define the influence of reactivation of FXN transcription on progressive expansions of the GAAs to evaluate potential risks associated with long-term reactivation of FXN expression. Lastly, our preliminary data from transcriptome profiling of a large cohort of FRDA and control cells demonstrated a profound downregulation of a set of transcription elongation co-factors in FRDA cells. We will elucidate the mechanism whereby these trans-factors affect transcriptional processivity of the FXN gene to identify new therapeutic targets for FRDA. To answer these questions, we will use a battery of FRDA cell models generated in our laboratory, including FRDA patient fibroblasts, induced pluripotent stem cells, neuronal and cardiac cells differentiated from the pluripotent cells. Collectively, successful completion of this project will uncover the molecular events occurring at the FXN locus in FRDA cells and define cis- elements as well as trans-factors critical for repeat-induced FXN silencing and GAAs expansion. Combined approaches of genome editing, pharmacological modulation, and high resolution transcriptome analyses performed in a spectrum of thoughtfully chosen FRDA models will fuel development of new therapeutic approaches

弗里德赖希共济失调（FRDA）是最常见的常染色体隐性共济失调。它是由线粒体蛋白 frataxin (FXN) 水平降低引起的，这是由于位于 FXN 基因第一个内含子中的 GAA 三核苷酸重复序列的大量扩展所致。尽管 FRDA 患者的 FXN 编码序列未改变，但由于 GAA 大量扩增，该基因的转录受到抑制。 FXN 表达的下调与 GAA 周围的染色质从活跃状态转变为抑制状态有关，然而 FXN 沉默的潜在分子机制仍然很大程度上未知。目前 FRDA 尚无有效治疗方法，FXN 的转录沉默是治疗干预的主要目标之一。因此，了解 GAA 诱导沉默的机制对于治疗开发至关重要。根据我们的初步研究，我们假设长的、扩展的 GAA 重复序列会诱导复制应激，从而导致内源 FXN 基因座的复制程序发生变化。由此产生的转录和复制之间的碰撞会抑制转录延伸并刺激 GAA 重复序列的扩展。由于特定转录辅助因子的缺乏，转录延伸缺陷在反式中进一步放大。为了解决这一假设，我们将重点关注有关弗里德赖希共济失调分子发病机制的三个基本问题：1）内源 FXN 位点的转录和复制之间的相互作用如何影响基因沉默和 GAA 重复序列的扩展？ 2) FRDA 细胞中扩增的 GAA 重复序列会影响转录过程的哪一步？ 3）反式因子对FRDA转录缺陷的贡献是什么？首先，我们将使用一组 CRISPR/Cas9 工程 FRDA 细胞剖析内源 FXN 位点转录和复制之间分子相互作用的机制。此外，我们将采用精确核连续测序 (PRO-seq) 技术来确定 FXN 基因座的新生转录谱，同时还定义受扩展 GAA 重复序列影响的准确转录步骤。此外，我们将定义 FXN 转录重新激活对 GAA 渐进扩张的影响，以评估与 FXN 表达长期重新激活相关的潜在风险。最后，我们对大量 FRDA 和对照细胞的转录组分析的初步数据表明，FRDA 细胞中一组转录延伸辅因子的深度下调。我们将阐明这些反式因子影响 FXN 基因转录持续性的机制，以确定 FRDA 的新治疗靶点。为了回答这些问题，我们将使用我们实验室生成的一系列 FRDA 细胞模型，包括 FRDA 患者成纤维细胞、诱导多能干细胞、从多能细胞分化的神经元和心脏细胞。总的来说，该项目的成功完成将揭示 FRDA 细胞中 FXN 基因座发生的分子事件，并定义对重复诱导的 FXN 沉默和 GAA 扩增至关重要的顺式元件和反式因子。在一系列精心选择的 FRDA 模型中进行基因组编辑、药理学调节和高分辨率转录组分析的组合方法将推动新治疗方法的开发