STAG2 mutations and 3D genome organization in glioblastoma multiforme

项目摘要 多形性胶质母细胞瘤（GBM）是最常见的原发性脑肿瘤。黏连蛋白是一种染色质结合环 复杂的三维基因组组织，姐妹染色单体凝聚力，基因表达和DNA修复。 在GBM中，编码粘着蛋白复合物组分的基因的突变失活是常见的， STAG 2亚基的突变占所有粘着蛋白突变的>50%。然而， STAG 2肿瘤抑制仍然未知。粘着蛋白基础生物学的最新突破性研究 已经表明，粘着蛋白在产生和维持染色质环中起着关键作用， 大部分的3D基因组组织和连接增强子的启动子调节。但 粘附素的这些功能与GBM发病机制的关系尚不清楚。这项资助将验证 肿瘤源性STAG 2突变导致3D基因组组织改变并增强Polycomb 组（PcG）介导的转录抑制，以驱动GBM中的肿瘤转化。这种假设是 基于在Waldman（多PI）和Jin（多PI）实验室进行的实验，这些实验利用基因编辑来 用匹配的校正和未校正的细胞校正GBM细胞中STAG 2的内源性突变等位基因 通过Hi-C和RNA-seq分析。使用Jin开发的新生物信息学管道分析数据 名为“HiCorr”和“DeepLoop”的技术，可以从低于10亿的读数中清楚地识别染色质环， 以最高的灵敏度对测序深度。这些实验表明，尽管STAG 2 STAG 2是维持拓扑相关结构域（TADs）的关键，STAG 2是维持拓扑相关结构域（TADs）的关键。 调节单个CTCF和H3 K27 me 3锚定的染色质环的大小和强度，导致 相邻基因表达的改变。初步研究还表明，STAG 2突变GBM 细胞中染色质结合的H3 K27 me 3水平显著增加，Polycomb的抑制增强， 组（PcG）调节的基因，和敏感性抑制剂的PcG信号在体外。根据这些数据，2 提出了目标。在目标#1中，我们将研究STAG 2调节的染色质环与 GBM细胞和肿瘤中的基因表达。在目标#2中，我们将定义STAG 2在PcG介导的染色质中的作用。 成环和转录抑制。完成本补助金中提出的研究将确定 肿瘤来源的STAG 2基因突变在3D基因组组织和PcG介导的转录中的作用 抑制GBM细胞和肿瘤。这些发现将提供一个长期寻求的分子机制，为cohesin- 介导的肿瘤抑制，为如何靶向粘附素突变提供了重要线索， GBM的治疗目的。

PROJECT SUMMARY Glioblastoma multiforme (GBM) is the most common primary brain tumor. Cohesin is a chromatin-bound ring complex involved in 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Mutational inactivation of genes encoding components of the cohesin complex is common in GBM, and mutations of the STAG2 subunit account for >50% of all cohesin mutations. However, the mechanism(s) of STAG2 tumor suppression remain unknown. Recent ground-breaking studies in the basic biology of cohesin have shown that cohesin plays a critical role in generating and maintaining the chromatin loops that underly much of 3D genome organization and that link enhancers to the promoters the regulate. However, the relationship of these functions of cohesin to GBM pathogenesis is undefined. This grant will test the hypothesis that tumor-derived STAG2 mutations result in alterations to 3D genome organization and enhanced Polycomb Group (PcG)-mediated transcriptional repression to drive neoplastic transformation in GBM. This hypothesis is based on experiments performed in the Waldman (multi-PI) and Jin (multi-PI) labs that utilized gene editing to correct the endogenous mutant allele of STAG2 in GBM cells, with matched corrected and uncorrected cells analyzed by Hi-C and RNA-seq. The data were analyzed using a new bioinformatics pipeline Jin developed called “HiCorr” and “DeepLoop” that makes it possible to clearly identify chromatin loops from sub-billion read- pair sequencing depth with the highest possible sensitivity. These experiments showed that whereas STAG2 was dispensable for maintenance of Topologically Associating Domains (TADs), STAG2 was essential for regulating the size and strength of individual CTCF and H3K27me3-anchored chromatin loops, leading to alterations in the expression of adjacent genes. The preliminary studies also showed that STAG2-mutant GBM cells have dramatically increased levels of chromatin-bound H3K27me3, enhanced repression of Polycomb Group (PcG)-regulated genes, and sensitivity to inhibitors of PcG signaling in vitro. Based on these data, two aims are proposed. In Aim #1 we will examine the relationship between STAG2-regulated chromatin loops and gene expression in GBM cells and tumors. In Aim #2 we will define the role of STAG2 in PcG-mediated chromatin looping and transcriptional repression in GBM. Completion of the research proposed in this grant will define the role of tumor-derived STAG2 gene mutations in 3D genome organization and PcG-mediated transcriptional repression in GBM cells and tumors. These findings will provide a long-sought molecular mechanism for cohesin- mediated tumor suppression, providing important clues for how cohesin mutations can be targeted for therapeutic purposes in GBM.