STAG2 mutations and 3D genome organization in glioblastoma multiforme

项目总结 多形性胶质母细胞瘤(GBM)是最常见的原发性脑肿瘤。粘附素是染色质结合环 参与3D基因组组织、姐妹染色单体聚集、基因表达和DNA修复的复合体。 编码粘附素复合体成分的基因突变失活在GBM中很常见，并且 STAG2亚单位的突变占所有凝集素突变的50%。但是，这一机制(S) STAG2对肿瘤的抑制作用仍不清楚。粘附素基础生物学的最新开创性研究 已经证明，粘附素在产生和维持不充分的染色质环中起着关键作用 大部分3D基因组组织和将增强子连接到启动子的启动子是受调控的。然而， 粘附素的这些功能与GBM发病机制的关系尚不明确。这笔拨款将检验这一假说 肿瘤来源的STAG2突变导致3D基因组组织和增强的Polycomb改变 组(PcG)介导的转录抑制，以驱动GBM的肿瘤转化。这一假设是 基于Waldman(多PI)和JIN(多PI)实验室进行的实验，这些实验室利用基因编辑来 纠正GBM细胞中STAG2的内源性突变等位基因，与匹配的校正和未校正细胞 经Hi-C和RNA-seq分析。使用金开发的新的生物信息学管道对数据进行分析 这两种技术被称为“HiCorr”和“DeepLoop”，使得从几十亿的读数中清晰地识别染色质循环成为可能。 具有最高灵敏度的配对测序深度。这些实验表明，尽管STAG2 对于拓扑关联结构域(TADS)的维护是必不可少的，STAG2对于 调节单个CTCF和H3K27me3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，STAG2-突变体GBM 细胞显著增加了染色质结合的H3K27me3水平，增强了Polycomb的抑制作用 PcG组(PcG)调节的基因，以及体外对PcG信号抑制剂的敏感性。根据这些数据，有两个 提出了目标。在目标1中，我们将研究STAG2调节的染色质环和 基底膜细胞和肿瘤中的基因表达。在目标2中，我们将定义STAG2在PcG介导的染色质中的作用 GBM中的环路和转录抑制。完成这项拨款建议的研究将界定 肿瘤来源STAG2基因突变在3D基因组组织和PcG介导的转录中的作用 在基底膜细胞和肿瘤中的抑制。这些发现将为凝集素提供一个长期寻求的分子机制。 介导的肿瘤抑制，为如何靶向粘附素突变提供了重要线索 基底膜的治疗目的。

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.