Long-range enhancer-controlled genes are hyper-sensitive to regulatory factor perturbations [High-throughput bulk RNA-seq].

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. High-throughput RNA-seq of EC50 K562 reporter cells after depletion of various factors using CRISPRi.

细胞类型特异性基因激活由增强子（enhancer）调控，而增强子有时会位于距靶基因启动子（promoter）甚远的基因组区域，目前远端增强子是否需要特定因子来协调基因调控仍不明确。本研究通过增强子距离可控的报告基因筛选实验寻找候选调控因子，随后对这些因子进行敲低处理，并采用活性-接触（activity-by-contact）预测模型在全基因组范围内依据增强子与靶基因的距离对基因进行分类。预测得到的远端增强子通常调控组织限制性基因，且多为强增强子。本研究发现，黏连蛋白（cohesin）与中介体复合物（mediator）是长距离基因激活最为特异性所需的调控因子，其中黏连蛋白会抑制短距离基因激活，并在竞争共享增强子的过程中优先选择远端而非近端的HBB基因；受长距离调控的基因对其他调控蛋白的扰动以及BET抑制剂JQ1也更为敏感，这一现象更多源于其独特的增强子特征而非基因与增强子间的距离本身。本研究预测，增加调控区间的间隔序列长度可将靶基因的表达限制在具备最优反式作用因子环境的特化细胞中，本数据集包含经CRISPRi（成簇规律间隔短回文重复序列干扰）敲除多种因子后的EC50 K562报告细胞的高通量RNA测序（RNA-seq）数据。