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

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.

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.

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.