Synergy between regulatory elements can render cohesin dispensable for distal enhancer function

Enhancers are critical genetic elements controlling transcription from promoters, yet how they convey regulatory information across large genomic distances remains unclear. Here, we engineer pluripotent stem cells in which cohesin loop extrusion can be inducibly disrupted without confounding cell cycle defects. Transcriptional dysregulation is cell type-specific, and not all loci with distal enhancers depend equally on cohesin extrusion. Using comparative genome editing, we demonstrate that enhancer-promoter communication over just 20 kilobases can require cohesin. However, promoter-proximal elements can support long-range, cohesin-independent enhancer action – even across strong CTCF insulators. Finally, transcriptional dynamics and the emergence of embryonic cell types remain largely robust despite disrupted extrusion. Beyond establishing novel strategies to study cohesin in enhancer biology, our work provides mechanistic insight into cell type- and genomic context-specificity. Overall design: Stranded mRNA-seq and CUT&Tag for H3K27ac and H3K27me3 in mouse ES cells, ES cells exiting pluripotency, neural progenitors, and neural progenitors differentiating into astrocytes in control (DMSO/untreated) and NIPBL-depleted (dTAG-13/IAA) cells. Bulk ATAC-seq from control (NIPBL) and NIPBL-depleted (dTAG-13) gastruloids. NIPBL depletion was initiated either 48, 72, or 96 hours after aggregation of mouse embryonic stem cells. ChIP-seq for CTCF in ES cells with a mutation of the CTCF-binding site in the Car2 promoter. 4C-seq in ES cells (WT SRR2 and homozygous SRR2 deletion) from control (DMSO/untreated) and NIPBL- or RAD21-depleted (dTAG or IAA) cells. Viewpoints include the Sox2 promoter, Sox2 SCR, Car2 promoter, Car2 enhancer 1, and Car2 enhancer 2. Cells harboring an dTAG-inducible (FKBP) or auxin-inducible degron (AID) on endogenous Nipbl or Rad21 alleles and were treated with either DMSO (control) or dTAG-13/IAA (NIPBL or RAD21 depletion). WT parental cells are E14Tg2a (129 X,Y background).

增强子是调控启动子转录的关键遗传元件，但其如何跨越庞大基因组距离传递调控信息的机制仍未阐明。本研究通过工程化改造多能干细胞，实现了黏连蛋白（cohesin）环挤出的可诱导性破坏，且不会引发混淆性的细胞周期缺陷。转录失调具有细胞类型特异性，并非所有携带远端增强子的基因座均同等依赖黏连蛋白环挤出过程。通过比较基因组编辑技术，本研究证实：即便仅跨越20千碱基对（kb）的增强子-启动子通讯，也可能依赖黏连蛋白。然而，启动子近端元件可支持长距离、不依赖黏连蛋白的增强子调控作用——即便该过程需跨越强效CCCTC结合因子（CTCF）绝缘子。最终，即便环挤出过程被破坏，转录动态变化与胚胎细胞类型的分化成熟仍基本保持稳定。本研究不仅建立了在增强子生物学领域研究黏连蛋白的全新策略，还为阐明细胞类型与基因组背景特异性的调控机制提供了新的认知。

整体实验设计：针对小鼠胚胎干细胞、退出多能性状态的胚胎干细胞、神经前体细胞以及分化为星形胶质细胞的神经前体细胞，分别设置对照组（二甲基亚砜/DMSO处理/未处理）与NIPBL耗竭组（dTAG-13/吲哚-3-乙酸/IAA处理），并对这些样本进行链特异性mRNA测序以及针对H3K27ac、H3K27me3的CUT&Tag实验。从对照组（NIPBL正常表达）与NIPBL耗竭组（dTAG-13处理）的类原肠胚中获取样本进行批量转座酶可及性测序（ATAC-seq），NIPBL耗竭的诱导时机设置为小鼠胚胎干细胞聚集后的48、72或96小时。针对Car2启动子区CCCTC结合因子（CTCF）结合位点发生突变的胚胎干细胞，开展CTCF的染色质免疫沉淀测序（ChIP-seq）。分别在对照组（DMSO处理/未处理）以及NIPBL或RAD21耗竭组（dTAG或IAA处理）的胚胎干细胞（野生型SRR2与纯合SRR2缺失型）中开展4C测序（4C-seq），其测序锚点包括Sox2启动子、Sox2 SCR、Car2启动子、Car2增强子1以及Car2增强子2。本研究使用的细胞系均在内源Nipbl或Rad21等位基因上整合了dTAG诱导型（FKBP）或生长素诱导型降解标签（AID），并分别用二甲基亚砜（DMSO，对照组）或dTAG-13/IAA（NIPBL或RAD21耗竭组）进行处理；野生型亲本细胞为E14Tg2a（129 X,Y背景）。