Esrrb Is a Pivotal Target of the Gsk3/Tcf3 Axis Regulating Embryonic Stem Cell Self-Renewal

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.

Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation

“…Dynamic restoration of heterogeneity in transcription factor expression was also assessed upon release of wild‐type ESCs from LIF/2i. An initial reduction in ESRRB expression upon removal of GSK inhibition (Martello et al , 2012) coincided with the previously observed expansion of the dynamic range of NANOG expression. After this, the levels of both TFs decreased with NANOG‐negative cells appearing at day 2 and ESRRB‐negative cells later at day 3 (Fig 2B and C).…”

“…This suggests that when unsupported by the transcriptional activity of NANOG, the naïve pluripotency gene regulatory network is weakened. While alternative positive inputs into Esrrb expression have been documented (Martello et al , 2012), our results indicate that such inputs ultimately fail in the absence of NANOG. ESCs may then initiate downregulation of other naïve pluripotency TFs including Klf4 and Esrrb .…”

Negative feedback via RSK modulates Erk‐dependent progression from naïve pluripotency

“…ES cells in 2iLIF were compared to cells cultured without MEK inhibition in medium containing GSK3 inhibitor, Chir99021 (CH), plus LIF (CHLIF). This allows analysis of the ERK‐responsive phosphoproteome independent of a change in identity because ES cells withstand ERK signalling and remain undifferentiated in CHLIF 7, 19, 20.…”

“…Notably, it can phosphorylate GSK3 leading to a decrease in activity 80. Inactivation of RSK could therefore lead to decreased intracellular β‐catenin and increased repression of Esrrb and other naïve factors by Tcf3 19. However, the observations that RSK mutant ES cells differentiate even in the presence of GSK3 inhibitor and that Esrrb downregulation is not markedly accelerated indicate that the major effect is through pErk.…”

“…The individual 2iLIF components each reduce and delay differentiation but a pairwise combination is required for long‐term self‐renewal and all three are optimal 7, 10. The most important effect of partial inhibition of GSK3 is to abrogate the capacity of the transcriptional repressor Tcf3 (gene name Tcf7l1 ) to downregulate key components of the ES cell gene regulatory network 19, 20, 21. Conversely, LIF acts by boosting expression of members of the network 22, 23, 24.…”