Investigating cohesin regulators to target Cornelia de Lange Syndrome

研究针对科妮莉亚·德·朗格综合症的粘连蛋白调节剂

基本信息

批准号：
10394802
负责人：
Daniel Sungjoo Park
金额：
$ 3.5万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10394802
关键词：
3-Dimensional Affect Architecture Binding Biochemistry Biological Assay Biology Bruck-de Lange syndrome Cell Line Cells ChIP-seq Chemicals Chromatin Chromatin Loop Classification Complex DNA Disease Dominant Genetic Conditions Drug Targeting Fluorescent in Situ Hybridization Fractionation Gene Expression Gene Targeting Genes Genetic Diseases Genetic Transcription Genome Genomics Goals Hi-C Hour Human Image Interphase Label Lead Length Live Birth Measures Methods Molecular Mutation Pathogenesis Patients Pharmaceutical Preparations Phenotype Phosphotransferases Proteins Regulation Resources Role Shapes Small Interfering RNA System Transcriptional Regulation Visual Western Blotting Work cohesin cost effective developmental disease differential expression experimental study follow-up gene correction gene discovery gene product genome-wide inhibitor knock-down new therapeutic target novel protein complex small molecule therapeutic development transcriptomics

项目摘要

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.

项目概要 Cornelia de Lange 综合征 (CdLS) 是一种由基因突变引起的多系统显性遗传性疾病。粘连蛋白复合物的结构或调节亚基调节染色质的 3D 组织，特别是在染色质循环和拓扑相关域（TAD）水平上。粘连蛋白消耗破坏了 TAD 边界，并且 CdLS 患者细胞中差异表达的基因在这些边界处富集，表明 3D 染色质组织是疾病发病机制的关键。有额外的初步证据表明，共同耗尽 WAPL（粘连蛋白的负调节因子）能够挽救粘连蛋白损失对基因组组织和基因表达的影响目前还没有已知的药物可以靶向粘连蛋白。然而，用目前的方法发现新的粘连蛋白调节剂仍然很困难。发现调节 TAD 边界上粘连蛋白作用的基因，我开发了 TAD 高通量荧光原位杂交 (TAD Hi-FISH) 使用 Oligopaints 和高内涵成像技术。定量识别以粘连蛋白或 WAPL 样方式调节 TAD 边界的基因。 CdLS 的潜在药物靶标，我将 TAD Hi-FISH 应用于人类“可成药基因组”，即 3,083 个基因我的主要筛选发现了 70 个类似粘连蛋白的药物和 57 个类似 GSK3A 的药物。多功能激酶，已被初步验证为一种主要的 WAPL 样命中，可以抑制粘连蛋白损失。我建议应用 TAD Hi-FISH 和其他检测来进一步表征 GSK3A 和我所有命中的效果关于 TAD 边界和粘连蛋白调节在目标 1 中，我将策划一套粘连蛋白的可药物调节剂。我将首先使用二级 TAD Hi-FISH 筛选来确定哪些命中调节。 TAD 边界以区域非特异性和粘连蛋白依赖性方式进行。通过染色质分级和蛋白质印迹评估哪些因素会影响粘连蛋白染色质结合最后，我将使用 ChIP-seq 评估最强的粘连蛋白调节剂的全基因组效应。对于粘连蛋白，我将研究 GSK3A 与基因组组织、粘连蛋白和为了研究 GSK3A 蛋白及其催化功能，我将开发一种诱导型降解决定子细胞系。对于 GSK3A，我还将优化该细胞系中最近开发的 GSK3A 化学抑制剂的条件。然后在 GSK3A 耗尽和抑制后进行 Hi-C，以研究 GSK3A 对全基因组的影响接下来，我将使用 PRO-seq 应用新生转录组学来确定 GSK3A 是否存在。共耗竭或抑制可以挽救粘连蛋白损失的基因表达变化，这些目标将共同实现。策划一套新颖的粘连蛋白调节因子，包括 GSK3A，这将增进我们对 3D 基因组的理解组织和潜在的 CdLS 治疗开发。