Abstract
SummaryInhibition of glycogen synthase kinase-3 (Gsk3) supports mouse embryonic stem cells (ESCs) by modulating Tcf3, but the critical targets downstream of Tcf3 are unclear. We analyzed the intersection between genome localization and transcriptome data sets to identify genes repressed by Tcf3. Among these, manipulations of Esrrb gave distinctive phenotypes in functional assays. Knockdown and knockout eliminated response to Gsk3 inhibition, causing extinction of pluripotency markers and loss of colony forming capability. Conversely, forced expression phenocopied Gsk3 inhibition or Tcf3 deletion by suppressing differentiation and sustaining self-renewal. Thus the nuclear receptor Esrrb is necessary and sufficient to mediate self-renewal downstream of Gsk3 inhibition. Leukaemia inhibitory factor (LIF) regulates ESCs through Stat3, independently of Gsk3 inhibition. Consistent with parallel operation, ESCs in LIF accommodated Esrrb deletion and remained pluripotent. These findings highlight a key role for Esrrb in regulating the naive pluripotent state and illustrate compensation among the core pluripotency factors.
Highlights
Since the original derivation of mouse embryonic stem cells (ESCs) in 1981 (Evans and Kaufman, 1981; Martin, 1981), culture conditions for sustaining pluripotency ex vivo have been progressively refined
Following the demonstration that the cytokine leukemia inhibitory factor (LIF) could replace feeder cells (Smith et al, 1988; Williams et al, 1988) and that ESC differentiation is suppressed by inhibition of mitogen activated protein kinase (Erk) signaling (Burdon et al, 1999; Kunath et al, 2007), further addition of an inhibitor of glycogen synthase kinase-3 (Gsk3) has enabled robust ESC propagation in well-defined conditions (Ying et al, 2008)
Genome-Scale Identification of Candidate Genes Directly Regulated by Tcf3 Genome-wide mapping of transcription factor binding events is achievable by chromatin immunoprecipitation and deep sequencing (ChIP-seq)
Summary
Since the original derivation of mouse embryonic stem cells (ESCs) in 1981 (Evans and Kaufman, 1981; Martin, 1981), culture conditions for sustaining pluripotency ex vivo have been progressively refined. Following the demonstration that the cytokine leukemia inhibitory factor (LIF) could replace feeder cells (Smith et al, 1988; Williams et al, 1988) and that ESC differentiation is suppressed by inhibition of mitogen activated protein kinase (Erk) signaling (Burdon et al, 1999; Kunath et al, 2007), further addition of an inhibitor of glycogen synthase kinase-3 (Gsk3) has enabled robust ESC propagation in well-defined conditions (Ying et al, 2008). Understanding how intracellular signaling pathways engage with the core transcription factor circuitry to maintain or extinguish pluripotency remains fragmentary (Chen et al, 2008; Jaenisch and Young, 2008; Nichols and Smith, 2012; Niwa et al, 2009)
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