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，希望这些知识能够改进该疾病的治疗方法。 FRDA 是由编码必需线粒体蛋白 frataxin 的核 FXN 基因的转录抑制引起的。基因抑制是由于 FXN 内含子中 GAA7TTC 三联体重复序列的扩展导致异染色质形成。基于组蛋白的乙酰化状态导致基因沉默的假设，我们发现了一类新型 HDAC 抑制剂，可以缓解 FRDA 患者淋巴细胞和该疾病小鼠模型中 FXN 基因的抑制。 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元细胞模型可用性的限制，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。我们采用了一种新方法来生成神经元细胞和细胞系，用于研究三联体重复介导的 FXN 基因沉默机制。我们从 FRDA 患者成纤维细胞中产生了诱导多能干 (iPS) 细胞，并表明这些细胞保留了 FXN 基因的抑制。这些细胞可以在体外分化为神经元细胞，并用作探索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质的结合位点的假设，我们将使用遗传和生化方法来鉴定结合 GAA7TTC 三联体重复序列的蛋白质。染色质免疫沉淀方法将用于验证这些蛋白质确实与来自 FRDA 患者的细胞系中沉默的 FXN 基因相互作用，并且 siRNA 方法将用于测试这些蛋白质在 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脱乙酰酶，并类似地使用 siRNA 方法来验证该酶在基因抑制中的作用。我们将检查正常和 FRDA FXN 等位基因中 FXN 基因调控中的组蛋白合成后修饰状态和异染色质蛋白。将确定 HDAC 抑制剂在基因激活中的作用机制。可以根据这些研究的结果确定治疗干预和治疗药物的新目标。 公共健康相关性：本申请旨在了解遗传性神经系统疾病弗里德赖希共济失调中基因沉默的分子基础。这种疾病是由编码一种名为 frataxin 的蛋白质的人类必需基因中简单 DNA 序列 GAA 的重复扩展引起的。这些 DNA 重复序列可能通过将 frataxin 基因包装在不活跃的染色体环境中来沉默该基因。通过研究这些重复沉默 frataxin 基因表达的机制，新的治疗策略将来自这些研究。

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.