GAA Repeats Induced Epigenetic Silencing in Friedreich's Ataxia

描述（由申请人提供）：弗里德赖希共济失调（FRDA）由FXN基因的表观遗传沉默引起，导致共济失调蛋白（铁硫簇合成途径的关键组分）缺乏。FXN的转录抑制是由内含子GAA重复序列的大量扩增引起的，并且可以通过调节表观遗传环境来部分逆转。在弗里德赖希共济失调中发生的转录抑制和表观遗传变化的机制的确切触发因素仍然未知。本申请的目的是确定由扩展的GAA重复序列诱导的表观遗传沉默的机制，其可用作治疗弗里德赖希共济失调的潜在靶点。我们将讨论Friedreich共济失调的分子发病机制的三个基本方面：1）是什么触发了突变FXN基因座的表观遗传变化？2)什么是参与沉默的主要染色质修饰途径？3)控制WT FXN基因表达的哪些因素受到FRDA中GAA扩增的影响？为了回答这些问题并确定疾病相关细胞模型中表观遗传沉默的机制，我们产生了FRDA和对照诱导多能干细胞系（iPSC），并将其分化为神经元细胞。我们将使用这些新的FRDA模型来检验我们的假设，即GAA重复序列的扩增启动了一系列事件，这些事件始于重复序列内的DNA构象变化，随后是GAA侧翼序列的表观遗传变化，从而导致FXN表达的失调。首先，我们将确定在生理条件下控制FXN基因表达的机制。基于我们的初步研究结果，我们将确定GCN 5组蛋白乙酰转移酶和c-MYC转录因子在调节FXN表达中的作用。接下来，我们将通过定义扩展的GAA重复序列在FXN基因的天然背景下的构象来确定GAA重复序列诱导转录的触发因素。我们还将确定非典型构象的形成、表观遗传沉默的程度和GAA重复序列的长度之间的联系。此外，我们将在各种表观遗传调节剂的存在下采用体细胞重编程为iPSC，以辨别染色质修饰途径对GAA重复介导的沉默的贡献。总的来说，这些实验将定义FXN表达的表观遗传控制，以及导致其在Friedreich共济失调中发生的失调和沉默的分子机制。我们在体细胞重编程期间对表观基因组进行基因组编辑和药理学调节的组合方法将推动FRDA新治疗方法的开发。

DESCRIPTION (provided by applicant): Friedreich's ataxia (FRDA) is caused by the epigenetic silencing of the FXN gene resulting in the deficiency of frataxin, a critical component of the iron-sulfur clusters synthesis pathway. Transcriptional repression of FXN results from large expansions of the intronic GAA repeats and can be partially reversed by modulating the epigenetic environment. The exact trigger of transcriptional inhibition and the mechanisms of epigenetic changes that occur in Friedreich's ataxia remain unknown. The aim of this application is to define mechanisms of epigenetic silencing induced by expanded GAA repeats, which could be used as potential targets for therapy of Friedreich's ataxia. We will address three fundamental aspects of the molecular pathogenesis responsible for Friedreich's ataxia: 1) What triggers the epigenetic changes in the mutant FXN locus? 2) What is the primary chromatin modification pathway involved in silencing? 3) What factors controlling the expression of the WT FXN gene are affected by the GAA expansion in FRDA? In order to answer these questions and to define the mechanism of the epigenetic silencing in the disease-relevant cellular models we generated FRDA and control induced pluripotent stem cell lines (iPSCs) and differentiated them to neuronal cells. We will use these novel FRDA models to test our hypothesis that the expansion of GAA repeats initiates a cascade of events that begins with DNA conformational changes within the repeats, followed by epigenetic changes in the sequences flanking the GAAs, which consequently leads to deregulation of FXN expression. First, we will identify mechanisms controlling expression of the FXN gene in physiological conditions. Based on our preliminary findings, we will define the roles of GCN5 histone acetyltransferase and c-MYC transcription factor in regulating FXN expression. Next, we will identify the trigger for GAA repeats-induced transcription by defining the conformation of the expanded GAA repeats in their natural context of the FXN gene. We will also determine a link between formation of the non canonical conformations, extent of epigenetic silencing and length of the GAA repeats. Furthermore, we will employ somatic cell reprogramming to iPSCs in the presence of various epigenetic modulators to discern the contributions of chromatin modification pathways to the GAA repeats-mediated silencing. Collectively, these experiments will define the epigenetic control of FXN expression, as well as the molecular mechanisms leading to its deregulation and silencing that occur in Friedreich's ataxia. Our combined approach of genome editing and pharmacological modulation of the epigenome during somatic cell reprogramming will fuel development of new therapeutic approaches for FRDA.