Investigating cohesin regulators to target Cornelia de Lange Syndrome

项目总结 Cornelia de Lange综合征(CDLS)是由基因突变引起的多系统、显性遗传性疾病。 粘附素复合体的结构或调节亚基。粘附素调节染色质的3D组织， 特别是在染色质环和拓扑相关结构域(TAD)的水平上。我们最近展示了 粘附素耗竭破坏了TAD边界，CDLS患者细胞中差异表达的基因是 在这些边界上丰富，表明3D染色质组织是疾病发病机制的关键。我们 有更多的初步证据表明，共同耗尽WAPL，一种凝集素的负调控因子，能够拯救 粘附素丢失对基因组组织和基因表达的影响。目前还没有已知的靶向凝集素的药物 然而，用目前的方法，发现新的粘附素调节器仍然很困难。至 发现在TAD边界上调节凝集素作用的基因，我开发了TAD高通量 荧光原位杂交(TAD Hi-FISH)TAD Hi-Fish使用齐聚颜料和高含量成像技术 定量鉴定以粘附素或WAPL类方式调节TAD边界的基因。要确定 针对CDLS的潜在药物靶点，我将TAD Hi-FISH应用于人类的“可药物基因组”，即3,083个基因 是已知药物的靶标。我的主屏幕显示了70个类似粘附素的点击和57个类似WAPL的点击。GSK3A，a 多功能激酶已被初步证实为一种可抑制凝集素丢失的先导WAPL样HIT。 我建议应用TAD Hi-FISH和其他分析方法来进一步表征GSK3A和我所有的热门药物的效果 在TAD边界和粘附素调节上。在目标1中，我将策划一组凝集素的可用药调节器 染色质的结构功能。我将首先使用二级TAD Hi-Fish屏幕来确定哪些点击可以调节 TAD边界以区域非特异性和粘附素依赖的方式进行。在这些排名靠前的第二大热门歌曲中，我会 通过染色质分离和蛋白质印迹分析评价影响粘附素结合的因素 实验。最后，我将使用CHIP-SEQ评估最强的粘附素调节剂对全基因组的影响 用来凝聚。在目标2中，我将研究GSK3A与基因组组织、粘附素和 抄写。为了研究GSK3A蛋白及其催化功能，我将建立一个可诱导降解的细胞系 对于GSK3A，还优化了该细胞系中最近开发的GSK3A化学抑制剂的条件。这就做 然后在GSK3A耗尽和抑制后进行Hi-C以研究GSK3A对全基因组的影响 染色质结构。接下来，我将使用PRO-SEQ应用新生转录组学来确定GSK3A 共耗竭或抑制可以挽救粘附素丢失的基因表达变化。这些目标加在一起，将 策划一组新的凝集素调节因子，包括GSK3A，这将促进我们对3D基因组的理解 组织和潜在的CDLS治疗发展。

PROJECT SUMMARY Cornelia de Lange Syndrome (CdLS) is multi-system, dominant genetic disorder caused by mutations in the structural or regulatory subunits of the cohesin complex. Cohesin regulates the 3D organization of chromatin, especially at the level of chromatin looping and topologically associated domains (TAD). We recently showed that cohesin depletion disrupts TAD boundaries and that differentially expressed genes in CdLS patient cells are enriched at these boundaries, suggesting that 3D chromatin organization is key to disease pathogenesis. We have additional preliminary evidence that co-depletion of WAPL, a negative regulator of cohesin, is able to rescue the effects of cohesin loss of genome organization and gene expression. No drugs are known to target cohesin or WAPL, however, and discovering novel cohesin regulators remains difficult with current approaches. To discover genes that modulate the role of cohesin on TAD boundaries, I developed TAD high-throughput fluorescence in situ hybridization (TAD Hi-FISH). TAD Hi-FISH uses Oligopaints and high-content imaging to quantitatively identify genes that regulate TAD boundaries in a cohesin- or WAPL-like manner. To identify potential drug targets for CdLS, I applied TAD Hi-FISH to the human “druggable genome,” i.e. 3,083 genes targeted by known drugs. My primary screen uncovered 70 cohesin-like and 57 WAPL-like hits. GSK3A, a multifunctional kinase, has been preliminarily validated as a lead WAPL-like hit that can suppress cohesin loss. I propose to apply TAD Hi-FISH and other assays to further characterize the effects of GSK3A and all of my hits on TAD boundaries and cohesin regulation. In aim 1, I will curate a set of druggable regulators of cohesin’s chromatin architectural function. I will first use secondary TAD Hi-FISH screens to determine which hits regulate TAD boundares in both a region non-specific and cohesin-dependent manner. Of these top secondary hits, I will evaluate which affect cohesin chromatin binding by performing chromatin fractionation and western blot experiments. Finally, I will evaluate the genome-wide effects of the strongest cohesin regulators using ChIP-seq for cohesin. In aim 2, I will investigate the relationship of GSK3A to genome organization, cohesin, and transcription. To study both GSK3A protein and its catalytic function, I will develop an inducible degron cell line for GSK3A, and also optimize conditions in this cell line for a recently developed GSK3A chemical inhibitor. I will then perform Hi-C after GSK3A depletion and inhibition to investigate the genome-wide effects of GSK3A on chromatin architecture. Next, I will apply nascent transcriptomics using PRO-seq to determine whether GSK3A co-depletion or inhibition can rescue the gene expression changes of cohesin loss. Together, these aims will curate a novel set of cohesin regulators, including GSK3A, that will advance our understanding of 3D genome organization and potential CdLS therapeutic development.