High-Throughput Mapping of Diverse Functional Genomic Elements Governing BRCA2 Expression

Abstract

The goal of this work is to develop a high-throughput approach to quantify the functional impact of the regulatory genome on target gene expression, and apply this system to catalogue functional cis-regulatory elements (CREs) that drive expression of BRCA2, a mutational driver of breast and ovarian cancer progression. The interactions between transcription factors and regulatory DNA modules underlie transcriptional outputs, but current techniques of cis-regulatory characterization utilize correlative features of enhancers, such as chromatin state, to assume CRE activity and do not measure the contributory effects of each CRE to a given target gene. Here we develop a CRISPR/Cas-based high-throughput screen to comprehensively and directly identify cis-regulatory sites that are necessary for BRCA2 expression by intercalating mutations across 185 kilobases of genomic space and using a fluorescence reporter to obtain measurements of diminished BRCA2 expression levels. We spatially map the distribution of required cis-regulatory sequences, and find evidence that proximal and distal elements exert controlling influences on BRCA2 expression. Multiple statistical evaluations of individual site and regional significance enable clarification of a diversified and spatially dispersed functional regulatory architecture governing BRCA2 transcription.