Synthetic genomic dissection of enhancer context sensitivity and synergy

Noncoding disease and trait-associated genetic variation is frequently interpreted in the context of genomic regulatory elements such as DNase I hypersensitive sites (DHSs). But while most DHSs lie within a few kilobases of another DHS, regulatory elements are typically analyzed individually without accounting for their neighbors. Based on our Big-IN technology for scarless genome engineering, we have developed a high-throughput multiplexed delivery pipeline to analyze >100 engineered payloads in hundreds of mouse embryonic stem cell (mESC) clones. We characterize multiple heterotypic DHS combinations from different critical mESC regulator loci, all delivered in a constant chromosomal context replacing the Sox2 Locus Control Region (LCR). We identify widespread examples of context-dependent enhancers which have no activity on their own but can double the activity of a neighboring DHS. We find pervasive context dependencies that depend on the specific DHS pairings. We further show that synergy between neighboring DHSs decays as a characteristic function of distance, with its influence extending up to several kilobases. We fine map this context dependency to the contribution of individual transcription factor recognition sequences. Our approach implicates the specific sequence and architectural features underpinning pervasive genomic context effects, and outlines key directions for modeling the functional impact of noncoding regulatory variation on common human traits and diseases.