(222c) Profiling Cell-Type-Specific Epigenomic Changes Associated with BRCA1 Mutation in Breast Tissues Using a Low-Input Microfluidic Technology

Previous studies have shown that genes can be switched on or off by age, environmental factors, diseases, and lifestyles. The open or compact structures of chromatin is a crucial factor that affects gene expression. Epigenetics refers to hereditary mechanisms that change gene expression and regulations without changing DNA sequences. Epigenetics plays critical roles in cell differentiation and disease processes. Breast cancer is the second mortal cancer among American women, following lung cancer. BRCA1 and BRCA2 are extremely influential in repairing DNA double-strand break through homologous recombination and suppressing DNA replication stress. Gene mutations in BRCA1 and BRCA2 increase the risk of breast cancer. Human female mammary epithelium is a two-layer tissue, including the inner (luminal) layer and outer (basal) layer. The luminal progenitors (LP) and mature luminal cells (ML) are from the inner layer and basal cells (B) are from the outer layer. The fourth cell type is stromal cells (S) from mesodermal tissue. Recent advances in epigenetics enable us to use next-generation sequencing to elucidate epigenomic dynamics during disease and development. Conventional chromatin immunoprecipitation followed by sequencing (ChIP-seq) necessitates the use of up to 1 - 10 million cells. Using microfluidic-oscillatory-washing-based ChIP-Seq (MOWChIP-Seq) developed in our lab, we profile epigenomes using as few as 100 cells as starting material. We used MOWChIP technology to examine four cell types from breast tissues of cancer-free patients, including normal (non-carrier) and BRCA1 mutant (carrier).

Cancer-free BRCA1 mutant (carrier) or normal (non-carrier) breast tissues were obtained from adult female patients who underwent cosmetic reduction of mammoplasty, diagnostic biopsies, or mastectomy. Four different cell types from the same patient were sorted by FACS. The coated beads and sheared chromatin (from 50k cells) were loaded to MOWChIP device for ChIP process. We examined genome-wide H3K4me3 and H3K27ac. After ChIP, DNA was purified, used for library preparation and sequenced by Illumina HiSeq 4000. The digital data were analyzed with Bowtie, MACS, and DiffBind.

Using the MOWChIP device enables us to perform ChIP on rare samples in less time and with improved quality of sequencing data. Stromal cells displayed the most differences in H3K27ac peak signal intensity between carrier and non-carrier groups, with the vast majority of peak regions having higher H3K27ac levels in non-carriers. Similarly, non-carrier mature luminal cells also had increased H3K27ac peak signal intensity, though very few peaks were significantly different between the two groups. In contrast, non-carriers had lower H3K27ac peak signal intensity across thousands of regions in both basal and luminal progenitor cells. These differences support widespread and cell-specific epigenomic changes associated with the BRCA1 mutation. Our work suggests strong genetic and epigenetic interactions due to mutations.