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Abstract
Pluripotency of embryonic stem cells (ESC) is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb). Esrrb contributes to the relaxation of the G1 to S-phase (G1/S) checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs) displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation.
Highlights
Developmental transitions during very early embryogenesis are characterized by major rearrangements of the cell cycle [1,2,3]
Dub3 is abundant in mESCs, a further increase in expression occurs after UV damage, suggesting a role of Dub3 during S-phase, possibly in checkpoint recovery by facilitating G2 to M–phase transition by dephosphorylation of CDK1 [8]
Expression of Dub3 is observed in cells in which p53 expression has been abrogated, suggesting an alternative DNA damage induced transcriptional control that may implicate the nuclear family of estrogen receptors (ERRs) and their co-activators
Summary
Developmental transitions during very early embryogenesis are characterized by major rearrangements of the cell cycle [1,2,3]. Very recent data indicate that cell fate decisions are intimately linked to the cell cycle and in particular to the length of the G1-phase [5,6]. ESCs have a relaxed checkpoint at the G1/S transition, due to persistent abundance of Cdc25A, a phosphatase that by controlling the activity of CDKs (Cycle Dependent Kinase) regulates cell cycle transitions. Persistence of Cdc25A in G1 leads to constitutive CDK2 dephosphorylation so that the length of the G1 phase remains unaffected, even after DNA damage, thereby ensuring that mESCs remain pluripotent [6]. Cdc25A protein levels are tightly regulated through the cell cycle of somatic cells, and its turnover is the result of the opposite activities of the Dub deubiquitylase [7] and of the two ubiquitin ligase complexes, APC/CCdh and SCFbTrCP [8]. It was found that the pluripotency factor estrogen-related receptor b (ERRb, Esrrb) contributes to the transcriptional regulation of Dub in ESCs [6], regulation of Dub expression during an unchallenged pluripotent cell cycle of ESCs still remains unexplored
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