Abstract 2049: Characterization of a novel BRCA1-EGR1 interaction

Abstract

Abstract The tumor suppressor protein BRCA1 orchestrates DNA repair and cell cycle arrest following DNA damage, with transcriptional regulation integral to these processes. We have identified a novel, DNA-damage responsive, interaction between BRCA1 and the zinc-finger transcription factor EGR1. EGR1 is rapidly and transiently expressed in response to a wide variety of stress stimuli and growth factors, and like BRCA1, decreased expression of EGR1 correlates with tumor formation in mammary cell lines and tissues. The stress response gene, NDRG1, was identified as a transcriptional target of BRCA1 by microarray analysis and subsequent RQ-PCR validation. NDRG1 is a known suppressor of breast cancer metastasis and we have observed a significant reduction of NDRG1 expression in breast cancer cell lines (relative to normal cell lines). Promoter analyses identified EGR1 binding motifs within the NDRG1 proximal promoter region, and EGR1 was confirmed as the transcription factor of interest by promoter assays. Downregulation of either BRCA1 or EGR1 (using a siRNA approach) resulted in a reduction in both basal and DNA-damage induced NDRG1 promoter activity, suggesting a co-regulatory mechanism. Indeed, recruitment of BRCA1 and EGR1 to the NDRG1 promoter was confirmed by ChIP assays. GST-fused BRCA1 deletion constructs showed that EGR1 specifically interacts with a C-terminal region of BRCA1 (which contains both BRCT domains). However, following DNA damage, we observed EGR1 interaction along the entire length of the BRCA1 protein, suggesting that both BRCA1 and EGR1 reside within a larger transcriptional complex upon DNA damage. In agreement with this, siRNA knockdown of either BRCA1 or EGR1 in breast cell lines (i) induced endogenous DNA damage signalling, (ii) increased intracellular ROS generation and (iii) upregulated numerous stress-response genes. To investigate transcriptional regulation downstream of this interaction we performed analyses of publically available ChIP-Seq data for both BRCA1 and EGR1, and identified a list of genomic locations (corresponding to gene promoters) at which both proteins localise. We will use these findings, in combination with our own ChIP-Seq experiments performed in the non-tumorigenic mammary cell line, 184A1, to investigate the roles of this novel complex in the maintenance of genomic stability and within normal breast biology. Citation Format: Naomi A. Dickson, Nyree T. Crawford, Paul B. Mullan. Characterization of a novel BRCA1-EGR1 interaction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2049. doi:10.1158/1538-7445.AM2015-2049