Characterising a novel BRCA1-EGR1 interaction in breast cancer

• Main Author: Dickson, Naomi
• Published: Queen's University Belfast 2016
• Subjects: 616.99
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.705911

Described herein is the identification of a DNA damage responsive interaction between BRCA1 and the transcription factor EGR1. This study sought to elucidate whether these proteins were linked functionally in the regulation of common transcriptional pathways. NDRG1 was identified as a transcriptional target of BRCA1 by microarray analyses and this positive regulation was validated following manipulation of BRCA1 expression levels. The minimal NDRG1 promoter was both demonstrated to be BRCA1-regulated and exhibited significantly upregulated activity in response the anthracycline class of cytotoxics. Promoter analyses identified an EGR1 consensus motif within the BRCA1-responsive region of the NDRG1 promoter and subsequent promoter assays confirmed the requirement of this EGR1 motif for NDRG1 promoter activity. Indeed, BRCA1 and EGR1 were found to interact in a DNA damage-inducible manner. Recruitment of BRCA1 and EGR1 to the NDRG1 promoter was confirmed by ChIP assays. It was demonstrated that EGR1 specifically interacts with the C-terminus of BRCA1 which contains the tandem BRCT domains, and their DNA damage induced interaction was partially abrogated by treatment with a protein phosphatase. BRCA1 associated exclusively with the zinc finger domain of EGR1 which contains several residues predicted in silico to be targets of ATM and CHK1. Depletion of either protein in vitro (i) induced expression of markers of endogenous DNA damage signalling, (ii) increased intracellular ROS generation and (iii) caused coordinate changes in the expression of genes associated with the DNA damage response. To investigate transcriptional regulation downstream of this interaction the analysis of publically available ChIP-Seq data for both BRCA1 and EGR1 was performed and identified a list of gene promoters at which both proteins were localised. A subset of genes were selected for further investigation, of which one was consistently upregulated (GNS), and two consistently downregulated (GTSE1 and PKMYT1), in response to depletion of either BRCA1 or EGR1.