Abstract A61: BRCA1-associated R-loop accumulation at noncoding putative ERα enhancer area blocks luminal epithelial differentiation

Abstract

Abstract Most BRCA1-associated breast tumors are basal-like, yet originate from luminal progenitor cells. BRCA1 is best known for its functions in double-strand break (DSB) 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. By sorting and profiling lineage-specific cells from precancerous human breast tissue, we found that BRCA1 mutation carriers tend to have a luminal cell-specific accumulation of R-loop, a transcriptional byproduct of DNA-RNA hybrid structure important for transcriptional regulation and genomic instability. R-loops accumulate preferentially at enhancers and transcription start sites (TSS) of luminal fate-related genes. Using CRISPR-Cas9 system, we have deleted an ERα enhancer area with BRCA1-associated R-loops accumulation in MCF7 cells. The deletion of this noncoding putative ERα enhancer area reduced the gene expression level of the neighboring genes of the deleted region, including ERα, RMND1, and CCDC170. In addition, compare to the reduced expression level of ERα, RMND1, and CCDC170 in BRCA1 knockdown of the parental MCF7, knocking down BRCA1 in these deletion clones showed less reduction of these genes. On the other hand, overexpression of RNASEH1, an enzyme that specifically removes R-loop, had little effect in these genes after BRCA1 knockdown. These results indicate BRCA1-associated R-loop accumulation at least partially affects luminal gene transcription. To investigate whether BRCA1-assocaited R-loop accumulation directly contributes to luminal differentiation blockage, we overexpressed RNASEH1 in breast epithelial cells freshly isolated from BRCA1 mutation carriers; flow cytometry comparing different breast lineage revealed a luminal population shift toward more mature luminal. All in all, the results imply that BRCA1 promotes luminal epithelial differentiation by alleviating R-loop accumulation at enhancer of ERα. Aberration in luminal transcription could contribute to lineage-specific BRCA1-associated tumorigenesis. Citation Format: Huai-Chin Chiang, Xiaowen Zhang, Yanfen Hu, Rong Li. BRCA1-associated R-loop accumulation at noncoding putative ERα enhancer area blocks luminal epithelial differentiation [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A61.