ARID2 promotes clear cell renal cell carcinoma in the absence of functional PBRM1 [microarray]. Mus musculus

Subunits of SWI/SNF chromatin remodeling complexes are frequently mutated in human malignancies. The PBAF complex is composed of multiple subunits, including the putative tumor suppressor proteins PBRM1 (BAF180) and ARID2 (BAF200) that are unique to this SWI/SNF complex. PBRM1 is mutated in various cancers, with a high mutation frequency in clear cell renal cell carcinoma (ccRCC). Here, we integrate RNA-seq, ARID2 and histone mark ChIP-seq, and ATAC-seq data to show that PBAF acts to enhance or repress gene expression depending on the genomic context. At baseline, ARID2 binds to areas of open chromatin at both active enhancers and promoters. Depletion of PBRM1 leads to attenuated and redistributed ARID2 chromatin binding that correlates significantly with gene expression changes. At enhancers, ARID2 binding loss leads to diminishment of the histone mark H3K4me1 and gene downregulation. Alternatively, at a subset of promoters, ARID2 binding loss derepresses gene expression. Interestingly, ARID2, which remains bound to other PBAF subunits after loss of PBRM1, is essential for many of the pro-tumorigenic transcriptional changes observed after loss of PBRM1, whereas other core SWI/SNF components are dispensable. Upon loss of PBRM1, ARID2 positively regulates cancer-related genes and pathways, including the cancer stem cell marker ALDH1A1 and ­­­EG­F signaling, to stimulate tumor cell growth. Therefore, ARID2 is crucial for maintaining the transformed state of PBRM1-deficient ccRCC cells. In total, this study suggests a novel mechanism of transcriptional control by PBRM1, whereby its loss alters the chromatin distribution of the residual PBAF complex leading to altered transcription that promotes tumorigenesis. Overall design: We isolated murine embryonic fibroblasts (MEFs) from conditional Pbrm1 mice we had generated, where Cre-mediated recombination caused a frame-shift mutation of the Pbrm1 allele and loss of Pbrm1 expression. We then performed microarray analysis on 3 matcheds of MEFs with or without Pbrm1 expression to investigate gene expression differences.SWI/SNF染色质重塑复合物（SWI/SNF chromatin remodeling complexes）的亚基在人类恶性肿瘤中频发突变。PBAF复合物（PBAF complex）由多个亚基组成，其中包含该SWI/SNF复合物特有的推定肿瘤抑制蛋白PBRM1（BAF180）与ARID2（BAF200）。PBRM1在多种癌症中发生突变，其中透明细胞肾细胞癌（ccRCC）的突变频率较高。本研究整合RNA测序（RNA-seq）、ARID2与组蛋白标记染色质免疫共沉淀测序（ChIP-seq）以及转座酶可及性测序（ATAC-seq）数据，证实PBAF复合物可根据基因组背景增强或抑制基因表达。在基线状态下，ARID2结合于活跃增强子与启动子区域的开放染色质。敲除PBRM1会导致ARID2的染色质结合能力减弱且分布重排，这与基因表达变化显著相关。在增强子区域，ARID2结合缺失会导致组蛋白H3赖氨酸4单甲基化（H3K4me1）水平降低以及基因表达下调；而在部分启动子区域，ARID2结合缺失则会解除基因的表达抑制。值得注意的是，在PBRM1缺失后仍与其他PBAF亚基结合的ARID2，对PBRM1缺失后观察到的诸多促肿瘤转录变化至关重要，而其他核心SWI/SNF组分则并非必需。在PBRM1缺失后，ARID2可正向调控癌症相关基因与通路，包括癌症干细胞标记物乙醛脱氢酶1A1（ALDH1A1）以及表皮生长因子（EGF）信号通路，从而促进肿瘤细胞增殖。因此，ARID2对于维持PBRM1缺陷型ccRCC细胞的转化状态至关重要。综上，本研究揭示了PBRM1调控转录的全新机制：PBRM1缺失会改变残留PBAF复合物的染色质分布，进而引发转录变化并促进肿瘤发生。实验设计：我们从自行构建的条件性Pbrm1小鼠中分离小鼠胚胎成纤维细胞（MEFs），通过Cre重组酶介导的重组使Pbrm1等位基因产生移码突变并导致Pbrm1表达缺失。随后我们对3对分别表达或缺失Pbrm1的MEFs进行微阵列分析，以探究基因表达差异。