Elucidating the role of chromatin architecture in the pathophysiology of Cornelia de Lange Syndrome

项目总结 Cornelia de Lange综合征(CDLS)是一种由调节性基因突变引起的多系统遗传性疾病 或粘附素复合体的结构成分。粘附素在有丝分裂过程中介导姐妹染色单体凝聚 其理论上是在整个间期挤出DNA以形成增强子-启动子染色质环和 维持被称为拓扑相关结构域(TADS)的DNA自我相互作用的簇。染色体 在患者中很少观察到不稳定。相反，已经确定了广泛的转录变化。 跨CDLS患者样本，暗示粘附素在该疾病的发病机制中的结构作用。我 假设在没有粘附素的情况下，介导增强子-启动子相互作用的染色质环是 相反，基因和邻近的调控元件之间形成了联系。我进一步期待这一点 增强子重组将在转录爆发水平上影响基因表达。形象化 单细胞中染色质的组织，我设计了寡色剂来探测人类中邻近的TADS 基因组的荧光原位杂交(FISH)。我的初步数据显示这项检测对 检测急性粘附素耗竭后局部染色体拓扑的变化 超分辨率显微镜。因此，我将应用这一分析和其他分析来回答关于 粘附素折叠染色质以促进单细胞水平转录的机制。在《目标1》中，我将 确定粘附素丢失对启动子拓扑和转录调控的影响。研究……的作用 在这些过程中，我将在一个细胞培养模型中进行这些实验，该模型有助于 粘附素降解。我将应用高通量RNA FISH技术seq-Fish来评估 全基因组范围内的突发频率。然后，我将绘制增强子-启动子接触的差异图，以努力 用高分辨率的Capture-C方法解释改变的基因表达。在《目标2》中，我将检测到 CDL中染色质解体和转录突发性变化的特征。我会调查TAD的 CDLS患者来源细胞的组织和新生基因表达。然后我将把表型与 每个患者的突变类型、临床严重性和粘附素丰度。这些目标加在一起，将照亮 转录调控与局部基因组结构之间的关系 解释染色体错误折叠如何导致发育障碍的机制，如CDLS。

PROJECT SUMMARY Cornelia de Lange Syndrome (CdLS) is a multi-system genetic disorder caused by mutations in the regulatory or structural components of the cohesin complex. Cohesin mediates sister chromatid cohesion during mitosis and is theorized to extrude DNA throughout interphase to form enhancer-promoter chromatin loops and maintain self-interacting clusters of DNA called topologically associating domains (TADs). Chromosome instability is rarely observed in patients. Rather, widespread transcriptional changes have been identified across CdLS patient samples, implicating the structural role of cohesin in the pathogenesis of the disorder. I hypothesize that in the absence of cohesin, chromatin loops that mediate enhancer-promoter interactions are disrupted and instead contacts form between genes and proximal regulatory elements. I further expect that this enhancer reorganization will affect gene expression at the level of transcriptional bursting. To visualize chromatin organization in single cells, I have designed Oligopaints to probe neighboring TADs in the human genome by fluorescence in situ hybridization (FISH). My preliminary data indicate this assay is sensitive to detect changes in local chromosome topology following acute cohesin depletion by both conventional and super resolution microscopy. Therefore, I will apply this and other assays to answer questions regarding the mechanisms by which cohesin folds chromatin to facilitate transcription at the single cell level. In aim 1, I will determine the effect of cohesin loss on promoter topology and transcriptional regulation. To study the role of cohesin in these processes, I will perform these experiments in a cell culture model that facilitates acute cohesin degradation. I will apply a high-throughput RNA FISH technique, seq-FISH, to assess changes in bursting frequencies genome-wide. Then, I will map differences in enhancer-promoter contacts in effort to explain the altered gene expression using a high-resolution Capture-C method. In aim 2, I will detect signatures of chromatin disorganization and transcriptional bursting changes in CdLS. I will probe TAD organization and nascent gene expression in cells derived from CdLS patients. I will then relate phenotypes to the mutation type, clinical severity, and cohesin abundance in each patient. Together, these aims will illuminate the relationship between transcriptional regulation and local genomic architecture while providing new mechanisms to explain how chromosome misfolding may lead to developmental disorders such as CdLS.