24 h differentiation in N2B27 of wild-type and Med12-mutant mESCs [bulkRNA-seq]

Cell differentiation results from coordinated changes in gene transcription in response to combinations of signals. FGF, Wnt, and mTOR signals regulate the differentiation of pluripotent mammalian cells towards embryonic and extraembryonic lineages, but how these signals cooperate with general transcriptional regulators is not fully resolved. Here, we report a genome-wide CRISPR screen that reveals both signaling components and general transcriptional regulators for differentiation-associated gene expression in mESCs. Focusing on the Mediator subunit Med12 as one of the strongest hits in the screen, we show that it regulates gene expression in parallel to FGF and mTOR signals. Loss of Med12 is compatible with differentiation along both the embryonic epiblast and the extraembryonic primitive endoderm lineage, but pluripotency transitions are slowed down, and the transcriptional separation between epiblast and primitive endoderm identities is enhanced in Med12-mutant cells. These cellular phenotypes correlate with reduced biological noise upon loss of Med12. These findings suggest that Med12 regulates cellular plasticity through the priming of transcriptional changes during differentiation, thereby modulating the effects of a broad range of signals. Gene expression in wild-type and Med12 mutant mESCs was compared in pluripotency and after 24h of differentiation in N2B27. Triplicates were obtained by seeding different passages on different days

细胞分化源于基因转录的协同变化，以响应多种信号的组合刺激。成纤维细胞生长因子（Fibroblast Growth Factor, FGF）、Wnt信号通路（Wnt）以及哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）信号可调控多能哺乳动物细胞向胚胎谱系与胚外谱系分化，但上述信号如何与通用转录调控因子协同发挥作用，目前尚未完全阐明。本研究通过全基因组CRISPR筛选（genome-wide CRISPR screen），鉴定出了小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）中参与分化相关基因表达的信号通路组分与通用转录调控因子。本研究以筛选中得分最高的靶点之一——中介体复合物（Mediator complex）亚基Med12为研究对象，证实其可与FGF及mTOR信号协同调控基因表达。敲除Med12不会阻碍细胞向胚胎上胚层与胚外原始内胚层谱系分化，但会减缓多能性转变进程，且Med12突变细胞中，上胚层与原始内胚层细胞身份间的转录分离程度显著增强。上述细胞表型与Med12缺失后细胞内生物学噪声（biological noise）的降低密切相关。本研究结果表明，Med12可通过在分化过程中预启动转录变化来调控细胞可塑性（cellular plasticity），进而调节多种信号通路的作用效果。本研究在多能状态下，以及使用N2B27培养基诱导分化24小时后，分别对野生型与Med12突变的小鼠胚胎干细胞的基因表达进行了比较。生物学重复样本通过在不同日期接种不同传代批次的细胞获得。