Instability of Triplet Repeats in Mammalian Cells

哺乳动物细胞中三联体重复的不稳定性

• 批准号: 7904472
• 负责人: JOHN H WILSON
• 金额: $ 16.88万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-31 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904472
• 关键词: Affect; Age; Alleles; Biological Assay; Biological Models; CAG repeat; Candidate Disease Gene; Cells; Clinical; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA repair protein; Disease; Elements; Embryonic Development; Epigenetic Process; Event; Genes; Genetic Transcription; Genomics; Goals; Grant; Human; Huntington Disease; Lead; Length; Libraries; Link; Maintenance; Mammalian Cell; Mammals; Methylation; Methyltransferase; Mismatch Repair; Molecular; Mus; Myotonic Dystrophy; Neurons; Nucleotide Excision Repair; Pathologic; Pathology; Pathway interactions; Patients; Play; Process; Proteins; Research Personnel; Role; Sea; Small Interfering RNA; Spinocerebellar Ataxias; Structure; System; Testing; Tetanus Helper Peptide; Tissues; Trinucleotide Repeats; age related; base; demethylation; disease phenotype; genome-wide; homologous recombination; human disease; innovation; mouse model; nervous system disorder; novel; programs; recombinational repair; repaired; small molecule libraries; vector

Expansions of CTG/CAG repeats in specific human genes cause numerous neurological diseases, including myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. These diseases arise when the number of triplet repeats increases beyond a threshold of about 25-35 repeats to a length that has pathologic consequences. This application focuses on the basis for CTG/CAG repeat instability. Studies in model systems have shown that processes that expose single strands of DMA¿replication, recombination, repair, transcription¿are capable of destabilizing triplet repeats. CTG and CAG repeats in single strands form hairpins and slipped-strand structures, which are the key intermediates in instability. These structures either interfere with normal repair or trigger aberrant repair, which changes the length of the repeat tract. In no case, however, has the mechanism for triplet repeat instability in humans been defined. Using a novel selection assay for CAG repeat contraction, we have identified two processes that greatly destabilize triplet repeats in mammalian cells: genome-wide demethylation and transcription. The two documented periods of repeat instability in development¿early embryogenesis and germline differentiation¿correspond to the two waves of epigenetic reprogramming that occur in mammals. Thus, demethylation-induced repeat instability is likely to be directly relevant to the germline events that lead to the progressive worsening of the disease phenotype in subsequent generations¿the clinical phenomenon of anticipation. Ongoing, age-dependent repeat instability occurs in affected patient tissues such as neurons, which do not divide, and may exacerbate the disease pathology. Because the disease genes are transcribed in these tissues, transcription-induced instability may be a major contributor to this ongoing, non-replication-dependent instability. We propose to define the specific DNA repair proteins responsible for demethylation- and transcription-induced instability, and in that way define the molecular mechanisms underlying CTG/CAG repeat instability. We will use siRNA knockdowns in selection assays in human cells to identify specific components required for repeat instability. We will extend our studies to mice to examine the effects of altered genomic methylation and transcription on repeat stability in germline and somatic tissues. Finally, we propose to use our selection assay to screen insertion vector libraries, siRNA libraries, and chemical libraries to identify genes that alter repeat stability. Our goal is to delineate those processes that are responsible for both the germline and somatic CTG/CAG repeat instability that characterizes myotonic dystrophy and other neurological diseases.

CTG/CAG重复序列在特定人类基因中的扩增导致许多神经系统疾病，包括 强直性肌营养不良、亨廷顿病和几种脊髓小脑共济失调。这些疾病是在 三联体重复序列的数目增加超过约25-35个重复序列的阈值， 病理后果。本申请集中在CTG/CAG重复不稳定性的基础上。研究 模型系统已经表明，暴露DNA单链的过程可以复制，重组， 修复、转录都能破坏三联体重复序列的稳定性。CTG和CAG重复单链 形成发夹和滑移链结构，这是不稳定性的关键中间体。这些结构 干扰正常修复或触发异常修复，从而改变重复序列的长度。在 然而，还没有人确定三联体重复不稳定性的机制。使用一种新 CAG重复收缩的选择性测定，我们已经确定了两个过程，极大地破坏了三联体 哺乳动物细胞中的重复：全基因组去甲基化和转录。两个有记录的时期 发育中的重复不稳定性<$早期胚胎发生和生殖系分化<$<$对应于两个 发生在哺乳动物身上的表观遗传重编程浪潮。因此，去甲基化诱导的重复不稳定性是 可能与导致疾病进行性恶化的生殖系事件直接相关 表型在随后的几代中的预测的临床现象。正在进行，年龄依赖性 重复不稳定性发生在受影响的患者组织中，例如不分裂的神经元，并且可能 加重了疾病的病理。因为疾病基因在这些组织中转录， 转录诱导的不稳定性可能是这种持续的，非复制依赖性的 不稳定我们建议定义负责去甲基化的特定DNA修复蛋白， 转录诱导的不稳定性，并以这种方式定义CTG/CAG的分子机制 重复不稳定。我们将在人类细胞的选择试验中使用siRNA敲低来鉴定特异性的 重复不稳定性所需的组件。我们将把我们的研究扩展到老鼠身上， 在生殖系和体细胞组织中改变基因组甲基化和转录对重复稳定性的影响。最后我们 我建议使用我们的选择试验来筛选插入载体文库、siRNA文库和化学文库 来鉴定改变重复稳定性的基因。我们的目标是描述那些负责 生殖细胞和体细胞CTG/CAG重复不稳定性都是强直性肌营养不良的特征， 神经系统疾病