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

项目摘要 科尔内利亚德兰格综合征（CdLS）是一种多系统遗传性疾病，由调节基因突变引起。 或者是粘着蛋白复合物的结构成分。有丝分裂过程中粘附素介导姐妹染色单体的粘附 并且理论上在整个间期挤出DNA以形成增强子-启动子染色质环， 维持称为拓扑关联结构域（TADs）的DNA的自相互作用簇。染色体 在患者中很少观察到不稳定。相反，广泛的转录变化已被确定， 在CdLS患者样本中，暗示了粘附素在疾病发病机制中的结构作用。我 假设在缺乏粘着蛋白的情况下，介导增强子-启动子相互作用的染色质环被 破坏，而是在基因和近端调控元件之间形成接触。我进一步预计， 增强子重组将在转录爆发水平上影响基因表达。可视化 为了研究单细胞中的染色质组织，我设计了Oligopaints来探测人类中邻近的TADs 荧光原位杂交（FISH）。我的初步数据表明，这种检测方法对 检测常规和免疫抑制剂急性粘着蛋白耗竭后局部染色体拓扑结构的变化， 超分辨率显微镜因此，我将应用这个和其他分析来回答有关 粘着蛋白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 确定粘连蛋白丢失对启动子拓扑结构和转录调控的影响。研究的作用 在这些过程中，我将在细胞培养模型中进行这些实验， 黏连蛋白降解我将应用一种高通量RNA FISH技术，seq-FISH，来评估 基因组范围内的爆发频率。然后，我将绘制增强子-启动子接触的差异， 使用高分辨率Capture-C方法解释改变的基因表达。在aim 2中，我将探测到 CdLS中染色质解体和转录爆发变化的特征。我会调查 组织和新生基因的表达。然后，我将把表型与 每个患者的突变类型、临床严重程度和粘附素丰度。总之，这些目标将阐明 转录调节与局部基因组结构之间的关系，同时提供新的 机制来解释染色体错误折叠如何导致发育障碍，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.