Abstract 4460: BRCA1-associated R-loop accumulation at noncoding putative ERα enhancer area regulates expression of adjacent genes

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, I 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, I've deleted an ERα enhancer area with BRCA1-associated R-loops accumulation in MCF7 cells. The deletion of this non-coding putative ESR1 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 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 affect gene transcription at these loci. To investigate whether BRCA1-assocaited R-loop accumulation directly contributes to luminal differentiation blockage, I overexpressed RNASEH1 in breast epithelial cells freshly isolated from BRCA1 mutation carriers, flow cytometry comparing different breast lineage reveal a luminal population shift toward more mature luminal. All in all, the results infer 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, Chi Zhang, Xiayan Zhao, Jingwei Li, Yanfen Hu, Rong Li. BRCA1-associated R-loop accumulation at noncoding putative ERα enhancer area regulates expression of adjacent genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4460.