Mechanisms of Gene Silencing in Friedreich's Ataxia

弗里德赖希共济失调的基因沉默机制

• 批准号: 7781576
• 负责人: JOEL M. GOTTESFELD
• 金额: $ 41.54万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7781576
• 关键词: Acetylation; Affinity Chromatography; Alleles; Binding Sites; Biochemical; Biotin; Cell Differentiation process; Cell Line; Cell model; Cells; Chromatin; Chromatin Structure; Code; Communities; DNA; DNA Sequence; Development; Disease; Down-Regulation; Environment; Enzymes; Event; Fibroblasts; Fluorescent Dyes; Friedreich Ataxia; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Heterochromatin; Histone Deacetylase; Histone Deacetylase Inhibitor; Histone H3; Histones; Human; Hybrids; In Vitro; Inherited; Introns; Knowledge; Lead; Lymphoid Cell; Lysine; Mass Spectrum Analysis; Mediating; Methods; Mitochondrial Proteins; Modeling; Modification; Molecular; Neurodegenerative Disorders; Neurons; Nuclear; Outcome Study; Pathogenesis; Patients; Principal Investigator; Process; Protein Binding; Proteins; Proteomics; RNA; Recruitment Activity; Repression; Research; Role; Signal Transduction; Small Interfering RNA; Structure; Testing; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Trinucleotide Repeats; Up-Regulation; Western Blotting; Yeasts; base; chromatin immunoprecipitation; frataxin; gene repression; histone methyltransferase; improved; induced pluripotent stem cell; inhibitor/antagonist; mouse model; nervous system disorder; novel; novel strategies; novel therapeutics; public health relevance; research study; therapeutic development

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.

描述（由主要研究者提供）：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由编码必需线粒体蛋白共济蛋白的核FXN基因的转录抑制引起。基因阻遏是由于FXN内含子中GAA7TTC三重重复序列的扩增，这导致异染色质形成。基于组蛋白的乙酰化状态负责基因沉默的假设，我们鉴定了一类新型HDAC抑制剂，其在源自FRDA患者的淋巴细胞和该疾病的小鼠模型中缓解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的可用性的限制，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。我们已经采取了一种新的方法来产生神经元细胞和细胞系，用于我们对FXN基因三联体重复序列介导的沉默机制的研究。我们已经从FRDA患者成纤维细胞中产生了诱导多能干细胞（iPS），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，并可作为探索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质的结合位点的假设，我们将使用遗传学和生化方法来鉴定结合GAA 7 TTC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。我们将研究组蛋白合成后修饰状态和异染色质蛋白在正常和FRDA FXN等位基因的FXN基因调控。将确定HDAC抑制剂在基因活化中的作用机制。基于这些研究的结果，可以确定治疗干预和治疗剂的新靶点。 公共卫生关系：这个应用程序的目的是了解遗传性神经系统疾病弗里德赖希共济失调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的重复扩增引起的，该基因编码一种称为共济失调蛋白的蛋白质。这些DNA重复序列可能通过将共济失调蛋白基因包装在非活性染色体环境中来沉默该基因。通过研究这些重复序列沉默frataxin基因表达的机制，新的治疗策略将来自这些研究。