Mechanisms of Gene Silencing in Friedreich's Ataxia

描述(由首席研究人员提供)：此应用程序旨在加深我们对神经退行性疾病Friedreich‘s共济失调(FRDA)的分子基础的了解，希望这一知识将导致对该疾病的治疗方法的改进。FRDA是由编码必需线粒体蛋白Frataxin的核FXN基因转录抑制引起的。基因抑制是由于FXN内含子中GAA7TTC三联体重复序列的扩张，导致异染色质的形成。基于组蛋白乙酰化状态导致基因沉默的假设，我们发现了一类新的HDAC抑制剂，它可以缓解FRDA患者来源的淋巴细胞中FXN基因的抑制，并在FRDA的小鼠模型中发挥作用。HDAC抑制剂直接作用于与FXN基因相关的组蛋白，增加组蛋白H3和H4上特定赖氨酸残基的乙酰化，为染色质结构在基因沉默中的作用提供了直接证据。虽然这些结果令人鼓舞，但FRDA发病机制和治疗开发的研究受到适当神经细胞模型的限制，在该模型中，研究导致FXN基因沉默的分子事件并测试可能的新疗法。我们采用了一种新的方法来产生神经细胞和细胞系，以研究三重重复介导的FXN基因沉默的机制。我们已经从FRDA患者的成纤维细胞中培养出诱导多能干细胞(IPS)，并表明这些细胞保留了对FXN基因的抑制。这些细胞可以在体外分化为神经细胞，并作为探索FXN基因沉默机制的模型。基于DNA序列或扩展重复序列的结构形成启动基因沉默的细胞蛋白的结合部位的假设，我们将使用遗传和生物化学方法来鉴定结合GAA7TTC三联体重复序列的蛋白质。染色质免疫沉淀方法将被用来验证这些蛋白质确实与FRDA患者细胞系中沉默的FXN基因相互作用，并将使用siRNA方法来测试这些蛋白质在FXN基因沉默中的作用。我们将鉴定与非活性FxN等位基因相关的组蛋白脱乙酰基酶(S)，并同样使用小干扰RNA方法来验证该酶(S)在基因抑制中的作用。我们将在正常和FRDA FXN等位基因中检验组蛋白合成后修饰状态和异染色质蛋白在FXN基因调控中的作用。HDAC抑制剂在基因激活中的作用机制将被确定。根据这些研究的结果，可以确定治疗干预和治疗剂的新靶点。 公共卫生相关性：这项应用旨在了解遗传性神经疾病Friedreich‘s共济失调中基因沉默的分子基础。这种疾病是由人类基本基因中简单DNA序列GAA重复序列的扩大引起的，该基因编码一种名为Frataxin的蛋白质。这些DNA重复使基因沉默，可能是通过将Frataxin基因包装在一个不活跃的染色体环境中。通过研究这些重复抑制Frataxin基因表达的机制，这些研究将产生新的治疗策略。

DESCRIPTION (provided by principal investigator): This application is aimed at furthering our understanding of the molecular basis for the neurodegenerative disease Friedreich's ataxia (FRDA), in the hope that this knowledge will lead to improved therapeutics for the disease. FRDA is caused by transcriptional repression of the nuclear FXN gene encoding the essential mitochondrial protein frataxin. Gene repression is due to expansion of a GAA7TTC triplet-repeat in an intron of FXN, which leads to heterochromatin formation. Based on the hypothesis that the acetylation state of the histone proteins is responsible for gene silencing, we identified a novel class of HDAC inhibitors that relieve repression of the FXN gene in lymphoid cells derived from FRDA patients, and in a mouse model for the disease. The HDAC inhibitors act directly on the histones associated with the FXN gene, increasing acetylation at particular lysine residues on histones H3 and H4, providing direct evidence for a role for chromatin structure in gene silencing. While these results are encouraging, studies in FRDA pathogenesis and therapeutic development are limited by the availability of an appropriate neuronal cell model in which to study the molecular events that lead to FXN gene silencing and to test possible new therapeutics. We have taken a novel approach to generate neuronal cells and cell lines for our studies on the mechanism of triplet repeat- mediated silencing of the FXN gene. We have generated induced pluripotent stem (iPS) cells from FRDA patient fibroblasts, and shown that these cells retain repression of the FXN gene. These cells can be differentiated into neuronal cells in vitro, and used as a model for exploring the mechanisms of FXN gene silencing. Based on the hypothesis that either the DNA sequence or structure of expanded repeats forms the binding site for cellular proteins that initiate gene silencing, we will use both genetic and biochemical methods to identify proteins that bind GAA7TTC triplet repeats. Chromatin immunoprecipitation methods will be used to verify that these proteins do indeed interact with silenced FXN genes in cell lines derived from FRDA patients, and siRNA approaches will be used to test the role of these proteins in FXN gene silencing. We will identify the histone deacetylase enzyme(s) associated with inactive FXN alleles, and similarly use siRNA methods to verify the role of this enzyme(s) in gene repression. We will examine histone postsynthetic modification states and heterochromatin proteins in FXN gene regulation in normal and FRDA FXN alleles. The mechanism of action of the HDAC inhibitors in gene activation will be determined. New targets for therapeutic intervention and therapeutic agents may be identified based on the outcome of these studies. PUBLIC HEALTH RELEVANCE: This application is aimed at understanding the molecular basis for gene silencing in the inherited neurological disease Friedreich's ataxia. This disease is caused by expansion of repeats of the simple DNA sequence GAA in an essential human gene that codes for a protein called frataxin. These DNA repeats silence the gene, possibly by packaging the frataxin gene in an inactive chromosomal environment. By studying the mechanisms whereby these repeats silence frataxin gene expression, new therapeutic strategies will come from these studies.