Abstract
Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1–Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.
Highlights
Mouse embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst and can be maintained indefinitely in a self-renewing state in culture [1,2]
Previous reports indicated that overexpression of Klf2, Klf4, or Klf5 achieves LIFindependent self-renewal of mouse ES cells [10,11,28,29], it is still unknown whether other Krüppel-like transcription factors (Klfs) family members might have similar activity
Our analysis identified that only Klf2, Klf4, and Klf5 have the abilities to both maintain the undifferentiated state of mouse ES cells and to reprogram Epiblast stem cells (EpiSCs) into Induced pluripotent stem cells (iPSCs) when overexpressed
Summary
Mouse embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst and can be maintained indefinitely in a self-renewing state in culture [1,2]. The utilization of ES cells for therapeutic purposes will require a better understanding of the molecular mechanisms underlying the regulation of pluripotency [4,5]. A reduction in extracellular-signal-regulated kinase (ERK) activity strongly promotes pluripotency. Activation of the fibroblast growth factor (Fgf)4–Fgf receptor(R)–ERK pathway destabilizes the pluripotent state and promotes a primed state [16,17,18]. The simultaneous inhibition of ERK and glycogen synthase kinase-3 (Gsk3) beta (with the inhibitor 2i) dramatically stabilizes the selfrenewal process of mouse ES cells [19]. The precise molecular mechanisms of selfrenewal remain elusive
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