MK2 promotes Tfcp2l1 degradation via ß-TrCP ubiquitin ligase to regulate mouse embryonic stem cell self-renewal.

Tfcp2l1 can maintain mouse embryonic stem cell (mESC) self-renewal. However, it remains unknown how Tfcp2l1 protein stability is regulated. Here, we demonstrate that ß-transducin repeat-containing protein (ß-TrCP) targets Tfcp2l1 for ubiquitination and degradation in a mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2)-dependent manner. Specifically, ß-TrCP1 and ß-TrCP2 recognize and ubiquitylate Tfcp2l1 through the canonical ß-TrCP-binding motif DSGDNS, in which the serine residues have been phosphorylated by MK2. Point mutation of serine-to-alanine residues reduces ß-TrCP-mediated ubiquitylation and enhances the ability of Tfcp2l1 to promote mESC self-renewal while repressing the speciation of the endoderm, mesoderm, and trophectoderm. Similarly, inhibition of MK2 reduces the association of Tfcp2l1 with ß-TrCP1 and increases the self-renewal-promoting effects of Tfcp2l1, whereas overexpression of MK2 or ß-TrCP genes decreases Tfcp2l1 protein levels and induces mESC differentiation. Collectively, our study reveals a posttranslational modification of Tfcp2l1 that will expand our understanding of the regulatory network of stem cell pluripotency.

ß-catenin stimulates Tcf7l1 degradation through recruitment of casein kinase 2 in mouse embryonic stem cells.

Activation of the Wnt/ß-catenin signaling pathway by the inhibition of glycogen synthase kinase-3 (GSK-3) will induce Tcf7l1 protein degradation to effectively promote embryonic stem cell (ESC) self-renewal. However, the exact mechanism remains unclear. Here, we found that inhibition of casein kinase 2 (Csnk2) by TBB or DMAT was sufficient to block the reduction of the Tcf7l1 protein induced by CHIR99021, a specific inhibitor of GSK-3. Similarly, downregulation of Csnk2 increased the Tcf7l1 level. In contrast, overexpression of Csnk2 significantly decreased Tcf7l1 protein stability in mouse ESCs. Notably, Csnk2α1 controls Tcf7l1 turnover to a greater degree than the other two isoforms of Csnk2, Csnk2α2 and Csnk2ß, as Csnk2α1-overexpressing mouse ESCs exhibited the lowest level of Tcf7l1. Csnk2α1 interacted with and phosphorylated Tcf7l1. In addition, the association of Csnk2α1 and Tcf7l1 was enhanced by CHIR99021. Our study demonstrated, for the first time, that Csnk2 is involved in Tcf7l1 turnover mediated by the Wnt/ß-catenin signaling pathway. These results expand our understanding of the function and circuit of Wnt/ß-catenin signaling pathway in ESCs.

The transcription factor TFCP2L1 induces expression of distinct target genes and promotes self-renewal of mouse and human embryonic stem cells.

TFCP2L1 (transcription factor CP2-like 1) is a transcriptional regulator critical for maintaining mouse and human embryonic stem cell (ESC) pluripotency. However, the direct TFCP2L1 target genes are uncharacterized. Here, using gene overexpression, immunoblotting, quantitative real-time PCR, ChIP, and reporter gene assays, we show that TFCP2L1 primarily induces estrogen-related receptor ß (Esrrb) expression that supports mouse ESC identity and also selectively enhances Kruppel-like factor 4 (Klf4) expression and thereby promotes human ESC self-renewal. Specifically, we found that in mouse ESCs, TFCP2L1 binds directly to the Esrrb gene promoter and regulates its transcription. Esrrb knockdown impaired Tfcp2l1's ability to induce interleukin 6 family cytokine (leukemia inhibitory factor)-independent ESC self-renewal and to reprogram epiblast stem cells to naïve pluripotency. Conversely, Esrrb overexpression blocked differentiation induced by Tfcp2l1 down-regulation. Moreover, we identified Klf4 as a direct TFCP2L1 target in human ESCs, bypassing the requirement for activin A and basic fibroblast growth factor in short-term human ESC self-renewal. Enforced Klf4 expression recapitulated the self-renewal-promoting effect of Tfcp2l1, whereas Klf4 knockdown eliminated these effects and caused loss of colony-forming capability. These findings indicate that TFCP2L1 functions differently in naïve and primed pluripotency, insights that may help elucidate the different states of pluripotency.

Modulation of STAT3 phosphorylation by PTPN2 inhibits naïve pluripotency of embryonic stem cells.

STAT3 phosphorylation at tyrosine 705 (STAT3pY705 ), triggered by the addition of the leukemia inhibitory factor (LIF), can maintain mouse embryonic stem cell (mESC) self-renewal and reprogram mouse epiblast stem cells (EpiSCs) to enter a naïve pluripotent state. The activation of STAT3pY705 occurs mainly through Janus kinases. However, it remains unclear how STAT3pY705 levels are decreased in mESCs. Our study shows that upregulation of the protein tyrosine phosphatase (PTPN2) inhibits STAT3 activity by reducing its phosphorylation level and promotes mESC differentiation, whereas PTPN2 knockout by CRISPR/CAS9 delays mESC differentiation. Consistently, PTPN2 knockdown facilitates the generation of mESC-like colonies in STAT3-overexpressing EpiSCs. PTPN2-mediated STAT3 activity, thus, contributes to the exit of ESCs from the pluripotent ground state. These findings expand the current understanding of the regulatory network of naïve pluripotency.