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

• 批准号: 10207788
• 负责人: Marek Napierala
• 金额: $ 7.84万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10207788
• 关键词: Address; Affect; Ataxia; Binding; CRISPR/Cas technology; Cardiac; Cell Differentiation process; Cell model; Cells; ChIP-seq; Chromatin; Code; Complex; DNA replication fork; Data; Dedications; Defect; Development; Disease; Down-Regulation; Elements; Engineering; Epigenetic Process; Event; Fibroblasts; Friedreich Ataxia; Funding; Gene Silencing; Genes; Genetic Transcription; Head; Histone Deacetylase Inhibitor; Impairment; Inherited; Introns; Kinetics; Laboratories; Length; Measures; Mitochondrial Proteins; Modeling; Molecular; Neurodegenerative Disorders; Neurons; Nuclear; Nylons; Oligonucleotides; Pathogenesis; Patients; Pharmacology; Replication Origin; Research; Resolution; Risk; Role; Running; Techniques; Testing; Therapeutic; Therapeutic Intervention; Time; Trans-Activators; Transcription Elongation; Transcription Process; Trinucleotide Repeats; Work; base; cohort; effective therapy; epigenetic silencing; experimental study; frataxin; gallium arsenide; gene repression; genome editing; induced pluripotent stem cell; new therapeutic target; novel therapeutic intervention; overexpression; programs; promoter; replication stress; targeted treatment; therapy development; transcription factor S-II; transcriptome

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

Friedreich‘s共济失调(FRDA)是最常见的常染色体隐性共济失调。这是由于位于FXN基因第一内含子的GAA三核苷酸重复序列的大量扩张导致线粒体蛋白Frataxin(FXN)水平降低所致。虽然FRDA患者的FXN编码序列没有改变，但由于GAA的大规模扩增，该基因的转录受到抑制。FXN表达下调与GaAs周围染色质从激活状态到抑制状态的转变有关，然而FXN沉默的潜在分子机制在很大程度上仍不清楚。目前，FRDA尚无有效的治疗方法，FXN的转录沉默是治疗干预的主要靶点之一。因此，了解GAA诱导沉默的机制对于治疗的发展至关重要。根据我们的初步研究，我们假设，长的，扩大的GAA重复诱导复制压力，导致内源FXN基因座的复制程序的变化。由此产生的转录和复制之间的碰撞抑制了转录延伸，并刺激了GAA重复序列的扩张。由于缺乏特定的转录辅助因子，转录延伸缺陷在反式转录中被进一步放大。为了解决这一假说，我们将集中讨论关于Friedreich‘s共济失调分子发病机制的三个基本问题：1)内源性FXN基因座转录和复制之间的相互作用如何影响基因沉默和GAA重复序列的扩张？2)FRDA细胞中GAA重复序列扩增影响转录过程的哪一步？3)反式因子对FRDA转录缺陷有何贡献？首先，我们将使用一组CRISPR/Cas9工程FRDA细胞来剖析内源性FXN基因座转录和复制之间的分子相互作用机制。此外，我们将使用精确的核连续测序(PRO-SEQ)技术来确定FXN基因座新生转录的轮廓，同时还定义了受扩展的GAA重复序列影响的确切转录步骤。此外，我们将定义重新激活FXN转录对GaAs渐进扩展的影响，以评估与长期重新激活FXN表达相关的潜在风险。最后，我们对一大群FRDA和对照细胞的转录组图谱的初步数据表明，FRDA细胞中的一组转录延伸辅助因子显著下调。我们将阐明这些反式因子影响FXN基因转录过程的机制，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建立的一系列FRDA细胞模型，包括FRDA患者成纤维细胞、诱导的多潜能干细胞、从多潜能细胞分化而来的神经细胞和心脏细胞。总而言之，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，并确定对重复诱导的FXN沉默和GaAs扩展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发展