Single-cell ATAC-seq analysis of mouse embryonic stem cells before and after Cohesin depletion

The contrast between the disruption of genome topology upon cohesin loss and the lack of downstream gene expression changes instigates intense debates regarding the structure-function relationship between genome and gene regulation. Here, by analyzing transcriptome at the single-cell level, we discover that, instead of dictating population-wide gene expression levels, cohesin supplies a general function to neutralize stochastic co-expression tendency of cis-linked genes in single cells. Notably, through single-cell ATAC-seq analysis of mouse embryonic stem cells, we found that cohesin loss induces chromatin co-opening tens of million bases apart in cis. Our results support that cohesin arranges nuclear topology to control gene co-expression in single cells. Overall design: JM8.N4 mouse embryonic stem cells (mESCs) from the C57BL/6N strain were edited by using CRISPR/Cas9 to fuse cohesin subunit RAD21 to the auxin-induced degron (AID) system. The derived cells were used in Smart-SCRB (Smart-Single Cell RNA Barcoding, a highly sensitive single-cell transcriptome assay) analysis and single-cell ATAC-seq analysis under control and acute cohesin loss (6hrs after the auxin treatment) conditions.

黏连蛋白（cohesin）缺失引发的基因组拓扑结构破坏，与下游基因表达无显著变化之间的矛盾，引发了学界关于基因组结构与基因调控间结构-功能关系的激烈争论。本研究通过单细胞水平转录组分析发现，黏连蛋白并非决定全群体的基因表达水平，而是通过普遍性功能抵消单细胞内顺式连锁基因的随机共表达趋势。值得注意的是，通过对小鼠胚胎干细胞开展单细胞转座酶可及性测序（single-cell ATAC-seq）分析，我们发现黏连蛋白缺失会诱导顺式作用区域内数千万碱基对间距的染色质发生共开放现象。本研究结果证实，黏连蛋白通过排布细胞核拓扑结构，实现对单细胞内基因共表达的调控。实验设计概述：选取C57BL/6N品系的JM8.N4小鼠胚胎干细胞（mESCs），利用CRISPR/Cas9技术将黏连蛋白亚基RAD21与生长素诱导降解子（AID）系统进行融合编辑。将获得的衍生细胞分别在正常对照及急性黏连蛋白缺失（生长素处理6小时后）条件下，开展Smart-SCRB（Smart-Single Cell RNA Barcoding，一种高灵敏度单细胞转录组检测方法）分析与单细胞转座酶可及性测序分析。