Investigation of BRCA1 target gene and role of Dicer mutation in breast cancer

Abstract

BRCA1 is a multifunctional tumor suppressor involved in DNA damage repair, cell cycle regulation, metabolism and other essential cellular processes. Many of these functions are driven by or related with its transcriptional/epigenetic regulator activity. Despite of this, there has been no comprehensive study to reveal the transcriptional/epigenetic targets of the BRCA1. Here, report a comprehensive analysis of genomics / transcriptomics data to identify genome-wide, BRCA1 target genes. We first analyzed ENCODE data where BRCA1 ChIP-seq results from four different cells lines are available. To identify a set of genes whose promoter is occupied by BRCA1, we collected 3,085 loci with BRCA1 ChIP signal found in all of the 4 cell lines and calculated distance from the loci to the nearest gene transcription start site (TSS). The result revealed 66.5% of the BRCA1 bound loci fall into 2kb around TSS, suggesting the role of BRCA1 in transcriptional regulation. Among these loci, we selected 20 genes based on the expression correlation data from two GEO datasets and TCGA containing human breast cancer expression profiles. The selected genes have significant correlation coefficient ranging from 0.25 to 0.54 (spearman correlation) to the BRCA1 Expression. According to the known function of selected genes, we further studied three genes and verified FADD as a novel direct target of BRCA1 by ChIP, real-time PCR and luciferase reporter assay. Altogether, our data demonstrate a genome-wide transcriptional regulation by BRCA1 and suggest target genes as biomarker candidates for BRCA1 associated breast cancer. Dicer is an indispensable part of the microRNA biogenesis machinery. Mutations in this protein have been confirmed in recent literatures about many cases of cancers such as pleuropulmonary blastoma, breast, ovarian tumors, and the others. The level of expression and activity of the mature miRNAs, which are products of the dicing process, are often found to be dysregulated in cancer. In this research, we aimed to investigate the effect of mutations on the C-terminal domain of Dicer found from clinical data, in both in-vitro and in-vivo studies. Recently, a Dicer domain was expressed in E.coli, then purified and functionally tested by in-vitro interaction and kinetic assays. The three selected mutants R1851C, A1870S and A1914V showed their impact on miRNA synthesis in breast cancer cell via luciferase reporter assay. Future study may help us to understand the role of Dicer mutations in miRNA synthesis pathway, which might lead to the discovery of regulatory factors amenable to pharmacological intervention in cancer.