Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis.

Most BRCA1-associated breast tumors are basal-like yet originate from luminal progenitor cells. BRCA1 is best known for its functions in DNA repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions of BRCA1 fully explains the cell lineage-specific increase in breast tumor development. Cell culture-based studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcriptional regulation and genetic instability. We found that BRCA1 mutation-associated R-loop accumulates preferentially in luminal epithelial cells of cancer-free human breast tissue, and at the 5' end of those genes that experience promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of mouse NELF-B/COBRA1, a Pol II-pausing factor and BRCA1-binding protein, in Brca1 knockout mouse mammary epithelium ameliorates R-loop accumulation and reduces mammary tumorigenesis. Our studies show that Pol II pausing is a previously unappreciated contributor to BRCA1-associated R-loop accumulation and breast cancer development. Normal breast tissue samples from four BRCA1 mutation carriers (B1) and four non-carriers (NC) were procured, digested into single cells, and sorted by flow cytometry using established cell surface markers (EpCAM and CD49f). Four distinct cell populations were acquired: stromal cells, basal epithelial cells, luminal progenitor cells (LP), and mature luminal epithelial cells (ML). Global R-loops were examined in each population. Global Pol II and NELF were examined in non-carrier patient unsorted normal breast tissue.