Abstract
Although the differentiation of pluripotent cells in embryonic stem cells (ESCs) is often associated with protein kinase-mediated signaling pathways and Tousled-like kinase 1 (Tlk1) is required for development in several species, the role of Tlk1 in ESC function remains unclear. Here, we used mouse ESCs to study the function of Tlk1 in pluripotent cells. The knockdown (KD)-based Tlk1-deficient cells showed that Tlk1 is not essential for ESC self-renewal in an undifferentiated state. However, Tlk1-KD cells formed irregularly shaped embryoid bodies and induced resistance to differentiation cues, indicating their failure to differentiate into an embryoid body. Consistent with their failure to differentiate, Tlk1-KD cells failed to downregulate the expression of undifferentiated cell markers including Oct4, Nanog, and Sox2 during differentiation, suggesting a negative role of Tlk1. Interestingly, Tlk1 overexpression sufficiently downregulated the expression of core pluripotency factors possibly irrespective of its kinase activity, thereby leading to a partial loss of self-renewal ability even in an undifferentiated state. Moreover, Tlk1 overexpression caused severe growth defects and G2/M phase arrest as well as apoptosis. Collectively, our data suggest that Tlk1 negatively regulates the expression of pluripotency factors, thereby contributing to the scheduled differentiation of mouse ESCs.
Highlights
Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocysts and possess self-renewal and pluripotency capabilities[1,2]
The expression of other differentiation-associated genes (GATA4 and GATA6 for the endoderm) was moderately increased (Fig. 1D). Consistent with this mRNA expression profile, the Western blotting analysis revealed that the Oct[4], Nanog, and Sox[2] levels in Tousled-like kinases (Tlk)[1] KD cells were not significantly changed relative to the control KD cells (Fig. 1E and F). These results suggest that, it might not be necessary for mouse ESCs (mESCs) pluripotency and self-renewal, Tlk[1] might regulate the expression of endoderm-associated genes
Our results indicated that the overexpression of Flag-tagged Tlk[1] or Flag-Tousled-like kinase 1 (Tlk1)-D607A abolished the ability of mESCs to proliferate, suggesting that the precise control of Tlk[1] expression is critical for mESC survival irrespective of the kinase activity of Tlk[1] (Fig. 6A)
Summary
Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocysts and possess self-renewal and pluripotency capabilities[1,2]. ESC pluripotency is controlled by the modulation of pluripotency transcription factors (TFs), including the core pluripotency TFs Oct[4], Sox[2], and Nanog, TF associated proteins, and chromatin modifications[2,5,6,7,8,9]. Nanog is a homeodomain-containing TF that maintains the self-renewal of ESCs independently of leukemia inhibitory factor (LIF) and contributes to the determination of the fate between the epiblast and primitive endoderm in blastocysts[12,13,14]. Nanog cooperatively regulate their target genes required for maintaining pluripotency and self-renewal and occupy the promoters of developmental genes associated with lineage specification whose expression is silenced in undifferentiated ESCs2,16,17. Tlk1-depleted ESCs exhibited delayed silencing of pluripotency-related genes and maintained an undifferentiated state with high alkaline phosphatase (AP) activity even after the induction of differentiation. Our data suggest that Tlk[1] acts as a negative regulator of core pluripotency factors in mESCs
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