Instability of Triplet Repeats in Mammalian Cells

人类特定基因中CTG/CAG重复序列的扩展会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性共济失调。这些疾病在以下情况下出现 三联体重复的数量超过大约25-35个重复的阈值，增加到具有 病理性后果。本应用重点研究了CTG/CAG重复序列不稳定性的基础。研究项目： 模型系统已经表明，暴露单链DMA的过程？复制、重组、 修复、转录能够破坏三联体重复序列的稳定。CTG和CAG在单链中重复 形成发夹和滑链结构，这是不稳定的关键中间体。这些结构 要么干扰正常修复，要么触发异常修复，从而改变重复束的长度。在……里面 然而，还没有病例定义人类三联体重复不稳定的机制。用一本小说 CAG重复收缩的选择实验，我们发现了两个极大破坏三联体稳定的过程 哺乳动物细胞中的重复：全基因组去甲基化和转录。记录在案的两个时期 发育的重复不稳定性？早期胚胎发育和生殖系分化？与两者相对应 发生在哺乳动物身上的表观遗传重编程浪潮。因此，去甲基化导致的重复序列不稳定性是 可能与导致疾病进行性恶化的生殖系事件直接相关 后代的表型？期待的临床现象。持续的、取决于年龄的 重复不稳定性发生在受影响的患者组织中，如神经元，这些组织不会分裂，可能 加重了疾病的病理。因为疾病基因是在这些组织中转录的， 转录诱导的不稳定性可能是这种持续的、非复制依赖的主要原因 不稳定。我们建议定义负责去甲基化的特定DNA修复蛋白--和 转录诱导的不稳定性，并以这种方式定义了CTG/CAG的分子机制 重复不稳定。我们将在人类细胞的选择分析中使用siRNA敲除来识别特定的 重复不稳定所需的组件。我们将把我们的研究扩展到小鼠身上，以检验 生殖系和体细胞组织中基因组甲基化和转录对重复序列稳定性的影响。最后，我们 建议使用我们的选择实验来筛选插入载体文库、siRNA文库和化学文库 以确定改变重复序列稳定性的基因。我们的目标是描述那些负责 生殖系和躯体CTG/CAG重复不稳定是强直性肌营养不良和其他 神经系统疾病。

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.