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

弗里德里希（Friedreich）的共济失调（FRDA）是最常见的常染色体隐性共济失调。它是由于位于FXN基因的第一个内含子中的GAA三核苷酸重复序列大量扩展而导致的线粒体蛋白frataxin（FXN）的水平降低。尽管FRDA患者的FXN编码序列未经改变，但由于大型GAA扩张而抑制了基因的转录。 FXN表达的下调与GAA周围的染色质从活性状态到反射状态有关，但是FXN沉默的潜在分子机制仍然在很大程度上未知。目前，尚无有效的FRDA治疗方法，FXN的转录沉默是治疗干预的主要目标之一。因此，了解控制GAA引起的沉默的机制对于治疗发展至关重要。基于我们的初步研究，我们假设长期扩大的GAA重复诱导复制应力，从而导致内源性FXN基因座的复制程序发生变化。转录和复制之间的碰撞抑制了转录扩展并刺激GAA重复的扩展。通过特定转录副因素的缺乏，转录脱毛缺陷进一步扩大。为了解决这一假设，我们将重点关注有关弗里德里希共济失调的分子发病机理的三个基本问题：1）在内源性FXN基因座的转录与复制之间的相互作用如何影响GAA重复的基因沉默和扩张？ 2）转录过程的哪一步受到FRDA细胞中GAA重复的扩展影响？ 3）跨因素对FRDA中转录缺陷的贡献是什么？首先，我们将使用一组CRISPR/CAS9工程FRDA细胞在内源性FXN基因座中的转录和复制之间进行分子相互作用的机制。此外，我们将采用精确的核跑步测序（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GAA重复序列影响的转录的确切步骤。此外，我们将定义FXN转录重新激活对GAA的进行性扩张的影响，以评估与FXN表达的长期重新激活相关的潜在风险。最后，我们来自大量FRDA和对照细胞的转录组分析的初步数据表明，FRDA细胞中一组转录伸长的副因素的深度下调。我们将阐明这些跨因素会影响FXN基因转录加工以鉴定FRDA的新治疗靶标的机制。为了回答这些问题，我们将使用实验室中产生的一系列FRDA细胞模型，包括FRDA患者成纤维细胞，诱导多能干细胞，神经元和心脏细胞与多能细胞区分开的。总的来说，该项目的成功完成将发现FRDA细胞中FXN基因座发生的分子事件，并定义顺式元素以及对重复诱导的FXN沉默和GAAS扩展至关重要的跨因素。基因组编辑，药物调制和高分辨率转录组分析的合并方法在经过精心选择的FRDA模型中进行