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，它是由位于 FXN 基因第一个内含子的 GAA 三核苷酸重复序列大量扩增导致的线粒体蛋白 frataxin (FXN) 水平降低引起的。 FRDA 患者的编码序列未改变，由于 FXN 表达下调，导致该基因的转录受到抑制。随着 GAA 周围的染色质从活跃状态转变为抑制状态，然而 FXN 沉默的潜在分子机制仍然很大程度上未知。目前，还没有针对 FRDA 的有效治疗方法，FXN 的转录沉默是主要目标之一。因此，了解 GAA 诱导的沉默的机制对于治疗开发至关重要，根据我们的初步研究，我们发现长的、扩展的 GAA 重复会诱导复制应激，从而导致复制程序的变化。内源 FXN 基因座之间产生的碰撞会抑制转录延伸并刺激 GAA 重复序列的扩增，因为特定转录辅因子的缺乏会进一步放大转录延伸缺陷。关于 Friedreich 共济失调分子发病机制的基本问题：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 的扩增2) FRDA 细胞中扩增的 GAA 重复会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？此外，我们将采用精确核连续测序 (PRO-seq) 技术来确定新生转录的概况。此外，我们将定义 FXN 转录重新激活对 GAA 逐步扩展的影响，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助因子发生了深刻的下调。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。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顺式元件。以及对重复诱导的 FXN 沉默和 GAA 扩增至关重要的反式因子，在一系列精心选择的 FRDA 模型中进行的基因组编辑、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。