Long-range enhancer-controlled genes are hyper-sensitive to regulatory factor perturbations [CRISPRi screen].

Cell-type-specific gene activation is regulated by enhancers, sometimes located at large genomic distances from target gene promoters. Whether distal enhancers require specific factors to orchestrate gene regulation remains unclear. Here, we used enhancer distance-controlled reporter screens to find candidate factors. We depleted them and employed activity-by-contact predictions to genome-wide classify genes based on enhancer distance. Predicted distal enhancers typically control tissue-restricted genes and often are strong enhancers. We find cohesin, but also mediator, most specifically required for long-range activation, with cohesin repressing short-range gene activation and prioritizing distal over proximal HBB genes competing for shared enhancers. Long-range controlled genes are also most sensitive to perturbations of other regulatory proteins and to BET inhibitor JQ1, this being more a consequence of their distinct enhancer features than distance. Our work predicts that lengthening of intervening sequences can help limit the expression of target genes to specialized cells with optimal trans-factor environments. A CRISPRi screen in E0, E50, E100, EC100 K562 reporter cell lines with a custom-made guide RNA (sgRNA) library composed of ∼18 thousand sgRNAs targeting 3200 nuclear protein-encoding genes (5 sgRNAs per TSS, plus 624 non-targeting sgRNAs). After library transduction, we isolated the top and bottom 10% GFP expressing cells and calculated the enrichment of sgRNAs in the low versus high GFP populations, to analyze per cell line the impact of each factor on reporter gene expression.