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: 8525464
• 负责人: JOEL M. GOTTESFELD
• 金额: $ 39.29万
• 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/8525464
• 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; DNA Structure; 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; 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; Acetylation; Affinity Chromatography; Alleles; Binding Sites; Biochemical; Biotin; Cell Differentiation process; Cell Line; Cell model; Cells; Chromatin; Chromatin Structure; Code; Communities; DNA; DNA Sequence; DNA Structure; 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; 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（由首席研究者提供）：此应用旨在进一步了解我们对神经退行性疾病弗里德里希共济失调（FRDA）的分子基础的理解，以期，这种知识能够改善该疾病的治疗剂。 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三胞胎重复的扩展，从而导致异染色质形成。基于以下假设：组蛋白蛋白的乙酰化状态负责基因沉默，我们确定了一种新型的HDAC抑制剂，该抑制剂可以缓解源自FRDA患者的淋巴样细胞中FXN基因的抑制作用，并在小鼠模型中缓解了该疾病的淋巴样细胞。 HDAC抑制剂直接作用于与FXN基因相关的组蛋白，从而增加了组蛋白H3和H4的特定赖氨酸残基的乙酰化，从而直接证明了染色质结构在基因沉默中的作用。尽管这些结果令人鼓舞，但FRDA发病机理和治疗发育的研究受到适当的神经元细胞模型的可用性的限制，在该模型中，研究导致FXN基因沉默并测试可能的新治疗剂的分子事件。我们采用了一种新的方法来产生神经元细胞和细胞系，以研究三胞胎重复介导的FXN基因的机理。我们已经从FRDA患者成纤维细胞产生了诱导的多能茎（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。基于以下假设：膨胀重复的DNA序列或结构形成启动基因沉默的细胞蛋白的结合位点，我们将使用遗传和生化方法来识别结合GaA7TTC三重序重复序列的蛋白质。染色质免疫沉淀方法将用于验证这些蛋白确实确实与来自FRDA患者的细胞系中的沉默FXN基因相互作用，并且将使用siRNA方法来测试这些蛋白质在FXN基因沉默中的作用。我们将确定与非活性FXN等位基因相关的组蛋白脱乙酰基酶（S），并类似地使用siRNA方法来验证该酶在基因抑制中的作用。我们将在正常和FRDA FXN等位基因中的FXN基因调节中检查组蛋白后修饰态和异染色质蛋白。将确定HDAC抑制剂在基因激活中的作用机理。可以根据这些研究的结果确定治疗干预和治疗剂的新靶标。 公共卫生相关性：该应用旨在理解遗传神经疾病弗里德里希共济失调中基因沉默的分子基础。该疾病是由简单DNA序列GAA重复序列扩展而引起的，该基本基因编码称为Frataxin的蛋白质。这些DNA可以通过在不活跃的染色体环境中包装Frataxin基因包装来使基因沉默。通过研究这些重复静音Frataxin基因表达的机制，这些研究将来自新的治疗策略。