Dong et al. relied on a mouse embryonic stem (ES) cell line where the cohesin subunit, RAD21, had been tagged with an auxin-inducible degron, thereby enabling them to induce depletion of cohesin in mouse ES cells. Next, they performed single-cell transcriptomics and single-cell ATAC-seq in control or cohesin-depleted mouse ES cells and used these data to define 776 ACDs across the mouse genome. In line with previous studies, the authors noted that cohesin loss had a minimal effect on chromatin accessibility or gene expression within individual ACDs. However, they also observed that depleting cohesin made it more likely for genes within distinct ACDs on the same chromosome to be coexpressed or exhibit concordant changes in chromatin accessibility. These results suggest that while cohesin does not affect gene expression at a population scale, it may regulate gene coactivation within individual mouse ES cells.

Next, the authors used intron sequential fluorescence in situ hybridization to visualize how cohesin loss affects active transcription in mouse ES cells. As a case study, they designed probes that targeted 208 genes across mouse chromosome 2 and looked for fluorescent puncta that represent bursts of transcriptional activity. While loss of cohesin did not affect the frequency of bursting events for individual genes, it did result in more co-bursting events, or coexpression of genes located within different ACDs. Cohesin loss also made it more likely for these coactivated genes to localize into shared transcriptional hubs marked by the Mediator complex proteins, MED1 and MED6. Finally, from single-molecule tracking, the authors noted that other key transcriptional proteins such as OCT4 or BRD4 exhibited broader ranges of activity across the genome after cohesin loss.

Dong 等人依赖于小鼠胚胎干细胞 （ES） 细胞系，其中黏连蛋白亚基 RAD21 已被生长素诱导型 degron 标记，从而使他们能够诱导小鼠 ES 细胞中黏连蛋白的耗竭。接下来，他们在对照或黏连蛋白耗尽的小鼠 ES 细胞中进行了单细胞转录组学和单细胞 ATAC-seq，并使用这些数据定义了小鼠基因组中的 776 个 ACD。与以前的研究一致，作者指出，黏连蛋白丢失对单个 ACD 中的染色质可及性或基因表达的影响很小。然而，他们还观察到，耗竭黏连蛋白使得同一染色体上不同 ACD 内的基因更有可能共表达或表现出染色质可及性的一致变化。这些结果表明，虽然黏连蛋白在群体规模上不会影响基因表达，但它可能会调节单个小鼠 ES 细胞内的基因共激活。

接下来，作者使用内含子顺序荧光原位杂交来可视化粘连蛋白丢失如何影响小鼠 ES 细胞中的活性转录。作为一个案例研究，他们设计了靶向小鼠 2 号染色体上 208 个基因的探针，并寻找代表转录活性爆发的荧光点。虽然黏连蛋白的缺失不会影响单个基因的爆发事件频率，但它确实导致了更多的共爆发事件，或位于不同 ACD 内的基因的共表达。黏连蛋白缺失也使这些共激活基因更有可能定位到以 Mediator 复合体蛋白 MED1 和 MED6 为标志的共享转录枢纽。最后，从单分子追踪中，作者注意到其他关键转录蛋白，如 OCT4 或 BRD4 在黏连蛋白丢失后在整个基因组中表现出更广泛的活性范围。