Enhancer regulatory networks globally connect non-coding breast cancer loci to cancer genes

Background: Genetic studies have associated thousands of enhancers with breast cancer (BC). However, the vast majority have not been functionally characterized. Thus, it remains unclear how BC-associated enhancers contribute to cancer. Results: Here, we perform single-cell CRISPRi screens of 3513 regulatory elements associated with breast cancer to measure the impact of these regions on transcriptional phenotypes. Analysis of > 500,000 single-cell transcriptomes in two breast cancer cell lines shows that perturbation of BC-associated enhancers disrupts breast cancer gene programs. We observe BC-associated enhancers that directly or indirectly regulate the expression of cancer genes. We also find one-to-multiple and multiple-to-one network motifs where enhancers indirectly regulate cancer genes. Notably, multiple BC-associated enhancers indirectly regulate TP53. Comparative studies illustrate subtype specific functions between enhancers in ER + and ER − cells. Finally, we develop the pySpade package to facilitate analysis of single-cell enhancer screens. Conclusions: Overall, we demonstrate that enhancers form regulatory networks that link cancer genes in the genome, providing a more comprehensive understanding of the contribution of enhancers to breast cancer development.