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

• 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
• 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

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.

项目摘要 Cornelia de Lange综合征（CDLS）是多系统的，主要是由突变引起的主要遗传疾病 粘蛋白复合物的结构或调节亚基。粘着素调节染色质的3D组织， 特别是在染色质环和拓扑相关的域（TAD）的水平上。我们最近显示 凝聚素的耗竭会破坏TAD边界，并且CDLS患者细胞中表达的基因不同 在这些边界上富集，这表明3D染色质组织是疾病发病机理的关键。我们 还有其他初步证据表明，WAPL的共抑制（粘蛋白的负调节剂）能够营救 基因组组织和基因表达的粘着蛋白丧失的影响。不知道靶向粘着蛋白的药物 但是，或WAPL，发现新型的粘着蛋白调节剂在当前的方法中仍然很困难。到 发现调节粘蛋白在TAD边界中的作用的基因，我开发了TAD高通量 荧光原位杂交（TAD HI-FISH）。 tad hi-fish使用寡头和高含量成像 定量地识别以粘蛋白或WAPL样方式调节TAD边界的基因。识别 我对CDL的潜在药物靶标，我将Hi-Fish应用于人的“可药基因组”，即3,083个基因 由已知药物靶向。我的主要屏幕发现了70种粘蛋白样和57个WAPL样命中。 GSK3A，a 多功能激酶已被初步验证为铅WAPL样命中，可以抑制粘蛋白损失。 我建议使用TAD HI-FISH和其他Assas进一步描述GSK3A和我所有的命中的影响 在TAD边界和粘着蛋白调节上。在AIM 1中，我将策划一组粘蛋白的可吸毒调节剂 染色质体系结构功能。我将首先使用次级TAD HI-FISH屏幕来确定哪些命中率调节 TAD边界在非特异性和粘蛋白依赖性方式中均以边界为单位。在这些顶级次要的热门歌曲中，我会 评估哪些通过执行染色质分馏和蛋白质印迹影响粘蛋白染色质结合 实验。最后，我将使用CHIP-SEQ评估强粘着蛋白调节剂的全基因组效应 用于粘着蛋白。在AIM 2中，我将调查GSK3A与基因组组织，粘蛋白和 转录。为了同时研究GSK3A蛋白及其催化功能，我将开发可诱导的Degron细胞系 对于GSK3A，还可以优化该细胞系中最近开发的GSK3A化学抑制剂的条件。我会 然后在GSK3A部署和抑制后执行HI-C，以研究GSK3A的全基因组影响 染色质体系结构。接下来，我将使用Pro-Seq应用新生的转录组学来确定GSK3A是否是否 共灭症或抑制可以挽救粘着蛋白丧失的基因表达变化。这些目标将在一起 策划了包括GSK3A在内的一组新型的粘着蛋白调节剂，这将提高我们对3D基因组的理解 组织和潜在的CDL治疗开发。