Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation [RNAseq_2D]

Epigenetic regulator genes are key in controlling cell identity and are frequently disrupted in breast cancers. However, specific epigenetic drivers (epidrivers) of mammary cell plasticity and their mechanistic contributions to this phenotype are poorly characterized. Here, we employed a CRISPR/Cas9 loss-of-function screening strategy to identify potential epidrivers of the emergence of mesenchymal breast cancer stem cell-like phenotypes in non-tumorigenic mammary cells. We unveiled that loss of the histone deubiquitinase BAP1 led to increased expression of breast cancer stem cell and several epithelial-to-mesenchymal transition markers accompanied by disrupted cell organization in mammary spheroids. In-depth characterization of epigenome and transcriptome changes following BAP1 loss revealed less permissive chromatin and downregulation in gene expression impacting programs that control cellular glycosylation, leading to decreased glycan abundance and complexity. BAP1 rescue restored the expression of several deregulated genes in a catalytic activity-dependent manner, suggesting that BAP1-mediated cell identity and glycosylation regulation are dependent on its histone deubiquitinase activity. Our results point to BAP1 as an epidriver of mammary cell plasticity and glycosylation aberrations, through the disruption of its histone modifying and gene expression regulation roles. Overall design: To assess changes in gene expression after BAP1 loss, we have generated BAP1 knockouts in MCF10A Cas9-expressing cells, and performed RNA-sequencing using two independent BAP1 knockout clones and Cas9-expressing control cells, in duplicates.

表观遗传调控基因（epigenetic regulator genes）是维持细胞身份的关键，且在乳腺癌中常发生异常。然而，目前对于乳腺细胞可塑性的特定表观遗传驱动因子（epidrivers），以及它们对该表型的机制性贡献，仍鲜有深入阐释。本研究采用CRISPR/Cas9功能缺失筛选策略，在非致瘤性乳腺细胞中鉴定介导间质样乳腺癌干细胞表型出现的潜在表观遗传驱动因子。本研究发现，组蛋白去泛素化酶BAP1的缺失会上调乳腺癌干细胞及多种上皮间质转化标志物的表达，并伴随乳腺球体的细胞结构紊乱。对BAP1缺失后表观基因组与转录组变化的深度解析显示，染色质开放程度降低，且控制细胞糖基化的基因表达调控程序出现下调，最终导致聚糖丰度与复杂度下降。恢复BAP1表达后，多个异常表达基因的表达得以以依赖其催化活性的方式恢复，这表明BAP1介导的细胞身份维持与糖基化调控依赖于其组蛋白去泛素化酶活性。本研究结果表明，BAP1通过破坏其组蛋白修饰与基因表达调控功能，成为乳腺细胞可塑性与糖基化异常的表观遗传驱动因子。实验整体设计：为评估BAP1缺失后的基因表达变化，本研究在表达Cas9的MCF10A细胞中构建了BAP1基因敲除模型，并分别以两个独立的BAP1敲除克隆与表达Cas9的对照细胞为材料，进行双重复的RNA测序（RNA-sequencing）。