GAA Repeats Induced Epigenetic Silencing in Friedreich's Ataxia

描述（由申请人提供）：friedrich 's ataxia （FRDA）是由FXN基因的表观遗传沉默导致frataxin缺乏引起的，frataxin是铁硫簇合成途径的关键成分。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。弗里德赖希共济失调中发生的转录抑制的确切触发因素和表观遗传变化的机制尚不清楚。本应用的目的是确定扩大GAA重复序列诱导的表观遗传沉默机制，这可能作为治疗弗里德赖希共济失调的潜在靶点。我们将探讨弗里德赖希共济失调的分子发病机制的三个基本方面：1)是什么触发了突变体FXN位点的表观遗传变化？2)沉默中涉及的初级染色质修饰途径是什么？3) FRDA中GAA扩增影响了哪些控制WT FXN基因表达的因素？为了回答这些问题并确定表观遗传沉默在疾病相关细胞模型中的机制，我们生成了FRDA并控制诱导多能干细胞系（iPSCs）并将其分化为神经元细胞。我们将使用这些新的FRDA模型来验证我们的假设，即GAA重复序列的扩展引发了一系列事件，首先是重复序列内的DNA构象变化，然后是GAA两侧序列的表观遗传变化，最终导致FXN表达的解除。首先，我们将确定生理条件下控制FXN基因表达的机制。基于我们的初步研究结果，我们将确定GCN5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基因的自然背景下的构象来确定GAA重复序列诱导转录的触发因素。我们还将确定非规范构象的形成、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，我们将在多种表观遗传调节剂存在的情况下对iPSCs进行体细胞重编程，以辨别染色质修饰途径对GAA重复序列介导的沉默的贡献。总的来说，这些实验将定义FXN表达的表观遗传控制，以及导致其在弗里德赖希共济失调中解除管制和沉默的分子机制。我们的基因组编辑和体细胞重编程过程中表观基因组的药理调节相结合的方法将推动FRDA新治疗方法的发展。

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.