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

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 identify epigenetic regulators with potential driver roles in the acquistion of mesenchymal breast cancer stem cell markers, we have transduced two independent clones of MCF10A Cas9-expressing cells with a lentiviral gRNA library targeting 426 epigenetic regulator genes. Mesenchymal breast cancer stem cell-like populations were isolated by FACS based on CD24, CD44 and EpCAM marker expression. gRNAs were sequenced through MiSeq, and the enriched and depleted gRNAs were identified by comparing the mesenchymal breast cancer stem cell-like population with the bulk of cells on the day of sorting.