A Quantitative Epistasis Framework For Interrogating the Network of Enhancers

The mammalian genome possesses a network of non-coding regulatory elements with key genes regulated by many enhancers. It remains unknown why these genes require multiple enhancers for regulation and what is the functional role of each enhancer contributing to a coordinated enhancer network. Here we develop a novel framework, named SEER (Systematic Enhancer Epistasis Regulatory network analysis), which leverages a suite of perturbative, mapping, and imaging approaches, combined with machine learning and Genome-Wide Association Study (GWAS) analysis to systematically and quantitatively anatomize the enhancer epistasis network. Applying the framework to the MYC locus, we revealed a hierarchical two-layer epistasis model and defined a class of synergistic regulatory elements (SREs) which can maintain both high expression and robustness upon perturbation. Via machine learning, we identified and validated that two features, spatial contacts and BRD4 coactivator condensation, are major factors in maintaining the synergistic interactions of SREs. We used SEER to predict the synergistic functions of non-coding variants in SREs for their clinical risks in cancer and autoimmune disorders. The SEER framework provides a novel approach and theory for delineating roles of the massive enhancer network in gene regulation and interpreting non-coding variants for clinical risks in complex diseases. To explore the ultralong-distance enhancer network, we examined the regulatory landscape of MYC, a key oncogene governing cellular proliferation and cancer pathogenesis. The MYC locus encompasses 7 enhancers (e1-e7) spanning a 1.8 Mb region. We conducted a multiplexed CRISPR interference (CRISPRi) screen using a pooled library consisting of 87,025 pairs of single guide RNAs (sgRNAs) targeting all single and pairwise combinations of 7 enhancers . We then transduced the pooled sgRNA library into K562 erythroleukemia cells stably expressing the nuclease-dead dCas9-KRAB fusion and cultured cells for 30 doublings (more than 1 month). Trac-looping experiment was used to exam the changes of enhancer-enhancer spatial contacts upon CRISPR individual and pairwise perturbation. H3K9me3 ChIP-seq experiment was used to exam the KRAB effect on chromatin status upon CRISPR individual and pairwise perturbation ATAC-seq experiment was used to exam the KRAB effect on chromatin status upon CRISPR individual and pairwise perturbation H3K27ac ChIP-seq experiment was used to exam the KRAB effect on chromatin status upon CRISPR individual and pairwise perturbation