Trinucleotide repeat disorders and the 3D genome

项目摘要 被称为短串联重复序列(STR)的重复DNA序列的不稳定扩展是 机械学基础上有超过25种遗传性人类疾病。不稳定重复扩张的患者 疾病有一系列复杂的症状，包括：心脏缺陷、白内障、焦虑、多动、 低智商、社交缺陷、呼吸缺陷和癫痫。在某些疾病中，如脆性X综合征和 Freidreich‘s共济失调，下游的表型在很大程度上是通过基因表达减少来调节的。在所有的 这些疾病的持续重复扩增与疾病的严重程度有关。治疗三核苷酸重复 因此，疾病是复杂的，因为疾病的主要影响因素包括 重复序列以及包含重复序列的基因表达中断。因此，增加了 STR不稳定性和扩张相关基因调控的分子机制研究 调节失调将有助于开发预防和治疗重复扩张性疾病的治疗方法。 在我们的前期工作中，我们引入了高阶染色质结构作为一个新的维度 了解重复扩张性疾病的这些特征。我们的数据显示：(1)绝大多数疾病 相关的STR精确地位于划分3D基因组折叠结构域的边界上，称为 拓扑相关结构域(TADs)和亚TADs以及(2)FMR1基因的重复扩增，遗传 脆性X综合征的驱动因素，导致CTCF占位消融和大规模TAD/亚TAD重组 以一种与STR束长度、疾病严重性和FMR1转录中断相关的方式。 鉴于3D基因组的重要性日益增加，迫切需要将我们的初步数据扩展到 了解3D基因组如何被三核苷酸重复扩增所干扰，以及这是否 扰动可能导致起源于因果基因本身的疾病的主要影响因素：重复 不稳定和基因表达失调。这项提案概述了这样做的下一步。在第一个目标中，我 将进行基因组工程实验，以确定我们观察到的结构域重组 在FMR1附近，基因表达下降。在第二个目标中，我将创建高分辨率 FXN基因是Freidreich‘s共济失调的遗传驱动因素，其周围的拓扑图决定了FXN基因是否 边界破坏出现在另一种三核苷酸重复紊乱中。在第三个目标中，我将执行 额外的基因组编辑实验，以阐明结构域边界中断是否会影响重复序列 FXN基因的不稳定性和基因表达。总之，这些目标的实现将证明 3D基因组可以在重复扩增障碍中受到干扰，并且这种扰动可以调节 重复不稳定和基因表达中断。最终，我们可以使用这些结果来确定 操纵3D基因组可能成为治疗这些疾病的潜在治疗目标。

Project Summary Unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs) serves as the mechanistic basis for more than 25 inherited human disorders. Patients with unstable repeat expansion diseases suffer from a complex array of symptoms, including: cardiac defects, cataracts, anxiety, hyperactivity, low IQ, social deficits, respiratory defects and seizures. In some diseases, such as Fragile X Syndrome and Freidreich’s Ataxia, the downstream phenotype is mediated in large part by reduced gene expression. In all of these diseases, continuous repeat expansion is associated with disease severity. Treating trinucleotide repeat disorders is thus complex because the primary effectors of disease include both the continuous expansion of repetitive sequences as well as disrupted expression of the gene containing the repeat. Thus, an increased understanding of the molecular mechanisms governing STR instability and expansion related gene dysregulation would facilitate efforts to develop therapies to prevent and treat repeat expansion disorders. In our preliminary work, we introduce the higher order chromatin architecture as a new dimension in understanding these features in repeat expansion disorders. Our data shows that (1) the large majority of disease associated STRs are located precisely at boundaries demarcating 3D genome folding domains termed topologically associating domains (TADs) and subTADs and (2) repeat expansion in the FMR1 gene, the genetic driver for Fragile X Syndrome, results in CTCF occupancy ablation and large-scale TAD/subTAD reorganization in a manner that correlates with STR tract length, disease severity, and transcriptional disruption of FMR1. Given the increasing importance of the 3D genome, there is a critical need to extend our preliminary data to understand how the 3D genome may be perturbed by trinucleotide repeat expansion and whether this perturbation could contribute to the primary effectors of disease originating from the causal gene itself: repeat instability and dysregulated gene expression. This proposal outlines the next steps doing so. In the first aim, I will perform genome engineering experiments to determine if the domain reorganization we have observed around FMR1 contributes to decreased gene expression. In the second aim, I will create high resolution topological maps around the FXN gene, the genetic driver for Freidreich’s ataxia, the determine whether boundary disruption is present in an additional trinucleotide repeat disorder. In the third aim, I will perform additional genome editing experiments to elucidate whether domain boundary disruptions can influence repeat instability and gene expression of the FXN gene. In sum, the accomplishment of these aims would demonstrate that the 3D genome can be perturbed in repeat expansion disorders and that this perturbation can mediate repeat instability and disrupted gene expression. Ultimately, we could use these results to determine whether manipulating the 3D genome could be a potential therapeutic target for treating these diseases.