Mechanisms of Gene Silencing in Friedreich's Ataxia

描述（由首席研究员提供）：本申请旨在进一步了解神经退行性疾病弗里德赖希共济失调（FRDA）的分子基础，希望这些知识将导致改善该疾病的治疗方法。FRDA是由编码线粒体必需蛋白frataxin的核FXN基因转录抑制引起的。基因抑制是由于FXN内含子中的GAA7TTC三重重复扩增，导致异染色质形成。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，可以缓解来自FRDA患者的淋巴样细胞中FXN基因的抑制，以及该疾病的小鼠模型。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发病机制和治疗发展的研究受到适当的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治疗方法。为了研究FXN基因三联体重复沉默的机制，我们采用了一种新的方法来产生神经细胞和细胞系。我们从FRDA患者的成纤维细胞中产生了诱导多能干细胞（iPS），并证明这些细胞保留了FXN基因的抑制。这些细胞可在体外分化为神经细胞，并可作为FXN基因沉默机制的研究模型。基于扩展重复序列的DNA序列或结构形成启动基因沉默的细胞蛋白的结合位点的假设，我们将使用遗传和生化方法来鉴定结合GAA7TTC三重重复序列的蛋白。染色质免疫沉淀方法将用于验证这些蛋白确实与来自FRDA患者的细胞系中沉默的FXN基因相互作用，siRNA方法将用于测试这些蛋白在FXN基因沉默中的作用。我们将鉴定与失活FXN等位基因相关的组蛋白去乙酰化酶(s)，并类似地使用siRNA方法验证该酶在基因抑制中的作用。我们将在正常和FRDA FXN等位基因中检测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抑制剂在基因活化中的作用机制将被确定。根据这些研究的结果，可能会确定新的治疗干预靶点和治疗剂。

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.