Transcriptional responses to Wnt pathway stimulation in mouse embryonic stem cells cultured in serum+LIF condition

The objective of this study was to identify genes regulated by canonical Wnt signaling in mouse embryonic stem cells (ESCs).Canonical Wnt signaling supports the pluripotency of mouse ESCs but also promotes differentiation of early mammalian cell lineages. To explain these paradoxical observations, we explored the gene regulatory networks at play. Canonical Wnt signaling is intertwined with the pluripotency network comprising Nanog, Oct4, and Sox2 in mouse ESCs. In defined media supporting the derivation and propagation of mouse ESCs, Tcf3 and β-catenin interact with Oct4; Tcf3 binds to Sox motif within Oct-Sox composite motifs that are also bound by Oct4-Sox2 complexes. Further, canonical Wnt signaling up-regulates the activity of the Pou5f1 distal enhancer via the Sox motif in mouse ESCs. When viewed in the context of published studies on Tcf3 and β-catenin mutants, our findings suggest that Tcf3 counters pluripotency by competition with Sox2 at these sites, and Tcf3 inhibition is blocked by β-catenin entry into this complex. Wnt pathway stimulation also triggers β-catenin association at regulatory elements with classic Lef/Tcf motifs associated with differentiation programs. The failure to activate these targets in the presence of a MEK/ERK inhibitor essential for mouse ESC culture suggests that MEK/ERK signaling and canonical Wnt signaling combine to promote mouse ESC differentiation. Triplicates of mouse embryonic stem cells cultured with GSK3 inhibitor CHIR99021 or with Wnt pathway inhibitor XAV939.

本研究旨在鉴定小鼠胚胎干细胞（mouse embryonic stem cells, ESCs）中受经典Wnt信号通路（canonical Wnt signaling）调控的基因。经典Wnt信号通路既可维持小鼠胚胎干细胞的多能性，又可促进早期哺乳动物细胞谱系的分化。针对这一矛盾现象，本研究对其背后的基因调控网络进行了深入探究。小鼠胚胎干细胞内的经典Wnt信号通路与由Nanog、Oct4及Sox2构成的多能性网络相互交织。在支持小鼠胚胎干细胞分离与扩增的化学成分限定培养基中，转录因子3（Tcf3）与β-连环蛋白（β-catenin）可与Oct4发生相互作用；Tcf3可结合于Oct-Sox复合基序内的Sox基序，而该基序亦可被Oct4-Sox2复合物所结合。此外，在小鼠胚胎干细胞中，经典Wnt信号通路可通过Sox基序上调Pou5f1远端增强子的活性。结合已发表的Tcf3与β-连环蛋白突变体相关研究，本研究结果表明：Tcf3可通过在这些位点与Sox2竞争，从而拮抗多能性；而β-连环蛋白进入该复合物可阻断Tcf3的抑制作用。Wnt通路激活还可促使β-连环蛋白结合于带有经典淋巴增强因子/T细胞因子（Lef/Tcf）基序的调控元件，此类基序与细胞分化程序相关。在小鼠胚胎干细胞培养必需的MEK/ERK信号通路（mitogen-activated protein kinase kinase/extracellular regulated protein kinase signaling）抑制剂存在时，上述靶基因无法被激活，这表明MEK/ERK信号通路与经典Wnt信号通路协同作用，共同促进小鼠胚胎干细胞的分化。本研究针对两种培养条件——分别采用糖原合成激酶3（glycogen synthase kinase 3, GSK3）抑制剂CHIR99021或Wnt通路抑制剂XAV939培养小鼠胚胎干细胞——各设置三次生物学重复。