Previous reports have shown that inhibition of GSK by

Previous reports have shown that inhibition of GSK3 by CHIR promotes mESC self-renewal through activation of Wnt/β-catenin signaling pathway (Martello et al., 2012). In the absence of Wnt ligand or CHIR, T cell factor 3 (TCF3) occupies the promoter regions of many pluripotency genes (e.g., Nanog, Esrrb, etc.) and functions as a transcriptional repressor (Martello et al., 2012). Upon CHIR treatment, cytosolic β-catenin is stabilized and then translocates into the nucleus, where it binds to TCF3 and relieves its transcriptional repressing effect, leading to the upregulation of Wnt/β-catenin downstream target genes Nanog and Esrrb as well as other pluripotency genes suppressed by TCF3 such as Oct4 and Sox2 (Martello et al., 2012). In mESCs, CHIR alone can only maintain short term (<1 week) self-renewal, after which mESCs will gradually undergo non-neural differentiation. LIF or PD, when supplemented with CHIR, is able to block such differentiation induced by CHIR and therefore maintains long-term self-renewal of mESCs (Ying et al., 2008). How CHIR collaborates with LIF or PD to maintain ESC self-renewal, however, remains poorly understood. We speculated that activation of Wnt/β-catenin signaling by CHIR can upregulate the oxyntomodulin of both self-renewal-promoting genes and genes that induce differentiation, and addition of LIF or PD can repress the expression of differentiation genes induced by CHIR. In this study, we sought to dissect the effect of CHIR on promoting self-renewal from its effect on inducing differentiation. We identified Klf2 and Tfcp2l1 as the two key self-renewal-promoting targets of CHIR. When simultaneously overexpressed, these two genes can recapitulate the effect of 2i or LIF/CHIR on promoting mESC self-renewal.
Results
Discussion In this study, we have shown that Klf2 and Tfcp2l1 are the two key downstream targets of Wnt/β-catenin responsible for mediating mESC self-renewal. Overexpression of Klf2 and Tfcp2l1 can recapitulate the self-renewal-promoting effect of 2i in mESCs, whereas downregulation of Klf2 and Tfcp2l1 impairs ESC self-renewal. Klf2 and Tfcp2l1 can also facilitate the reprogramming of EpiSCs back to the naive pluripotent state. Our study therefore establishes KLF2 and TFCP2L1 as two key mediators of mESC self-renewal promoted by 2i. Inhibition of GSK3 has dual effects on mESCs: promoting self-renewal and inducing non-neural differentiation (Ying et al., 2008). Previous reports have shown that CHIR exerts its function on promoting self-renewal through activation of canonical Wnt/β-catenin signaling pathway (Martello et al., 2012). Upon Wnt ligand or CHIR treatment, β-catenin, the central effector of Wnt/β-catenin signaling pathway, induces the expression of several pluripotency genes, such as Oct4, Sox2, and Nanog, via suppression of TCF3 (Martello et al., 2012). In recent reports, Esrrb has been shown as a Wnt/β-catenin target in 129-derived mESCs (Martello et al., 2012). However, we did not observe such increase of Esrrb expression in our microarray data from C57BL/6-derived mESCs after CHIR treatment (GEO: GSE50393). Moreover, overexpression of Esrrb is not sufficient to support ESC self-renewal under serum-free condition (data not shown). On the contrary, Klf2 and Tfcp2l1, two downstream targets of Wnt/β-catenin signaling pathway (Martello et al., 2012), were upregulated upon CHIR treatment in our microarray data (Figure 1B) and play a pivotal role in mediating ESC self-renewal promoted by Wnt/β-catenin signaling (Figures 2D, 2E, and 3C). Both KLF2 and TFCP2L1 can upregulate Nanog expression to promote ESC self-renewal (Jiang et al., 2008; Ye et al., 2013) and Esrrb is a direct NANOG target gene that can substitute for NANOG function in mESCs (Festuccia et al., 2012). Therefore, it would be of great interest in future studies to determine the connection between Klf2/Tfcp2l1 and Esrrb in mediating ESC self-renewal.