Multidimensional OMICs reveal ARID1A orchestrated control of DNA damage, splicing, and cell cycle in normal and malignant urothelial cells [RNA-seq]. Multidimensional OMICs reveal ARID1A orchestrated control of DNA damage, splicing, and cell cycle in normal and malignant urothelial cells [RNA-seq]

Epigenetic regulators, such as the SWI/SNF complex, play an important role in tissue development and homeostasis, and are frequently mutated in cancer. ARID1A, a subunit of the SWI/SNF complex, is mutated in approximately 20% of all bladder tumors, however, our understanding of the consequences thereof remains limited. Here, we generated various loss- and gain-of-function models to conduct Omni-ATAC-Seq, RNA-Seq, interactome analyses, and first functional studies to characterize ARID1A-affected pathways potentially suitable for the treatment of ARID1A-deficient bladder cancers. We observed decreased cell proliferation and deregulation of stress-regulated pathways including DNA repair in ARID1A-deficient cells. Furthermore, ARID1A was linked to alternative splicing and translational regulation on RNA and interactome levels. The absence of ARID1A drastically impacted the accessibility of chromatin, displaying significantly less accessible chromatin especially around distal, but not proximal, enhancers. Less accessible chromatin areas were mapped to pathways such as DNA damage response and cell proliferation. Indeed, the G2/M checkpoint appeared impaired after DNA damage in ARID1A-deficient cells. Together, this highlights the broad impact of ARID1A and the possibility of targeting proliferative and DNA repair pathways. Overall design: To investigate the impact of ARID1A-deficiency in four bladder cell lines, mRNA-Seq was performed. T24 and UROtsa with and without a CRISPR/Cas9-mediated ARID1A Knockout, HBLAK with or without siRNA against ARID1A, and JMSU-1 (carrying a homozygous ARID1A mutation) were transformed to re-express ARID1A, were tested.

表观遗传调控因子（epigenetic regulators）如SWI/SNF复合物（SWI/SNF complex）在组织发育与稳态维持中发挥关键作用，且在癌症中频发突变。作为SWI/SNF复合物的亚基，ARID1A（ARID1A）在约20%的膀胱肿瘤中存在突变，但目前学界对其致病后果的认知仍较为有限。本研究构建了多种功能缺失与功能获得性模型，开展Omni-ATAC测序（Omni-ATAC-Seq）、RNA测序（RNA-Seq）及相互作用组学分析，并首次通过功能实验表征了ARID1A调控的、可用于治疗ARID1A缺陷型膀胱癌的潜在通路。
我们在ARID1A缺陷细胞中观察到细胞增殖能力下降，以及包括DNA损伤修复在内的应激调控通路失调。此外，ARID1A在RNA层面与相互作用组层面均与可变剪接及翻译调控相关。ARID1A的缺失会显著影响染色质可及性，尤其在远端而非近端增强子区域，染色质开放程度显著降低。染色质可及性降低的区域富集于DNA损伤应答与细胞增殖等通路。进一步实验证实，ARID1A缺陷细胞在DNA损伤后，其G2/M检验点（G2/M checkpoint）功能出现受损。综上，本研究揭示了ARID1A的广泛调控作用，以及靶向细胞增殖与DNA损伤修复通路的治疗潜力。
实验设计概述：为探究ARID1A缺陷对四种膀胱细胞系的影响，本研究开展了mRNA测序（mRNA-Seq）。检测样本包括：经CRISPR/Cas9介导敲除ARID1A的T24、UROtsa细胞及其对应野生型对照；转染靶向ARID1A的siRNA或阴性对照的HBLAK细胞；以及携带纯合ARID1A突变的JMSU-1细胞及其经改造后重新表达ARID1A的细胞系。