Single-cell RNA-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 RNA-seq profiling of genes in mouse embryonic stem cells, we found that cohesin loss induces widespread gene co-activation in cis. Our results support that cohesin arranges nuclear topology to control gene co-expression in single cells. 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）缺失引发基因组拓扑结构破坏，但下游基因表达却未出现显著变化，这一矛盾现象引发了学界关于基因组结构与基因调控间构效关系的激烈争论。本研究通过单细胞水平转录组分析发现，黏连蛋白并非主导群体水平的全局基因表达，而是通过一种通用功能抵消单细胞中顺式连锁基因的随机共表达倾向。值得注意的是，通过对小鼠胚胎干细胞中的基因进行单细胞RNA测序（single-cell RNA-seq）分析，我们发现黏连蛋白缺失会诱导顺式区域内广泛的基因共激活现象。本研究结果证实，黏连蛋白通过塑造细胞核拓扑结构来调控单细胞内的基因共表达。本研究使用CRISPR/Cas9技术对C57BL/6N品系的JM8.N4小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）进行编辑，将黏连蛋白亚基RAD21与生长素诱导降解子（auxin-induced degron, AID）系统进行融合。将所得的编辑细胞分别在正常培养条件以及急性黏连蛋白缺失（生长素处理6小时后）条件下，进行Smart-SCRB（Smart-Single Cell RNA Barcoding，一种高灵敏度单细胞转录组检测技术）分析与单细胞ATAC-seq（single-cell ATAC-seq）分析。