Geminin escapes degradation in G1 of mouse pluripotent cells and mediates the expression of Oct4, Sox2, and Nanog

Abstract

Geminin is an essential cell-cycle protein that is only present from S phase to early mitosis in metazoan somatic cells. Genetic ablation of geminin in the mouse results in preimplantation embryonic lethality because pluripotent cells fail to form and all cells differentiate to trophoblast. Here we show that geminin is present in G1 phase of mouse pluripotent cells in contrast to somatic cells, where anaphase-promoting complex/cyclosome (APC/C)-mediated proteasomal destruction removes geminin in G1. Silencing geminin directly or by depleting the APC/C inhibitor Emi1 causes loss of stem cell identity and trophoblast differentiation of mouse embryonal carcinoma and embryonic stem cells. Depletion of cyclins A2 or B1 does not induce this effect, even though both of these APC/C substrates are also present during G1 of pluripotent cells. Crucially, geminin antagonizes the chromatin-remodeling protein Brg1 to maintain expression of Oct4, Sox2, and Nanog. Our results define a pluripotency pathway by which suppressed APC/C activity protects geminin from degradation in G1, allowing sustained expression of core pluripotency factors. Collectively, these findings link the cell cycle to the pluripotent state but also raise an unexplained paradox: How is cell-cycle progression possible in pluripotent cells when oscillations of key regulatory proteins are lost?

Figure 1

Silencing Geminin or Emi1 Mimics Depletion of the Pluripotency Factor Oct4 in P19 Mouse Embryonal Carcinoma Cells (A) Geminin, Oct4, and Emi1 were depleted by siRNA in P19 embryonal carcinoma (EC) cells, and whole-cell lysates were harvested for analysis by western blotting. β-actin was used as a loading control. In mouse cells, a cross-reacting band lies below the Emi1 band marked with an asterisk. (B) Geminin depletion by siRNA in P19 EC cells results in upregulation of trophectoderm markers Troma-1 and P-cadherin (both in green) and nuclear enlargement at 2 days and 6 days posttransfection. Total DNA is shown in red. Scale bars represent 20 μm. (C) Trophectoderm markers Troma-1, P-cadherin, and Cdx2 (in green) are induced in P19 EC cells that lack geminin (purple). The mean proportion of cells lacking geminin was 67.8% of cells at 24 hr versus 43% at 48 hr as a result of outgrowth of untransfected cells (data not shown). (D) Bar chart showing lineage analysis of control, geminin-, Oct4-, and Emi1-depleted P19 EC cells. The proportion of cells labeled by immunofluorescence with markers of extraembryonic (Troma-1, P-cadherin, Cdx2) and embryonic (β-III tubulin, Sox1, Gata6, and Brachyury) differentiation is shown. Eomes is a marker of both mesoderm and trophoblast differentiation. At least 1000 cells were scored, and all cells in a given field were scored. Error bars represent 5% standard error. (E) Upregulation of trophectoderm markers is observed in P19 EC cells depleted of Oct4 at 2 days and 6 days posttransfection. (F) Emi1 inhibits APC/C^Cdh1 activity to drive progression through the mitotic cell cycle by inhibiting the degradation of geminin and cyclins A2 and B1. Emi1 inactivation (or depletion) results in unopposed APC/C^Cdh1 activity and ubiquitin-mediated proteasomal destruction of its substrates geminin and cyclins. (G) Emi1 depletion phenocopies depletion of geminin in P19 EC cells with upregulation of trophectoderm markers and gross nuclear enlargement observed at 2 days and 6 days. Total DNA is shown in red. Scale bars represent 20 μm. For additional related data, see Figure S1.

Figure 2

Emi1 Depletion Induces Trophoblast Differentiation in Mouse EC Cells by Downregulation of Geminin, but Not by Downregulation of Cyclins A2 or B1 (A) Emi1 depletion in P19 EC cells resulted in downregulation of geminin and cyclins A2 and B1 by immunoblotting, with stabilization of each of these APC/C substrates following treatment with the proteasome inhibitor MG132. (B) P19 EC cells were transfected with cyclin A2 or cyclin B1 siRNA, and whole-cell lysates were harvested for analysis by western blotting. β-actin was used as a loading control. Giant cell formation and upregulation of Troma-1 and P-cadherin are not evident following depletion of cyclin A2 or cyclin B1. Total DNA is shown in red. Scale bars represent 20 μm. (C) Scatter dot plots show relative DNA content in geminin-, cyclin A2-, cyclin B1-, Emi1-, and Oct4-depleted P19 EC cells relative to control siRNA-treated cells. (D) Codepletion of Emi1 and Cdc6 abolishes nuclear enlargement, but not upregulation of trophectoderm markers Troma-1 and P-cadherin (both green), in P19 EC cells at 2 days posttransfection. Total DNA is shown in red. Scale bars represent 20 μm. Bar chart shows the proportion of control, Emi1-depleted, and Emi1- and Cdc6-codepleted P19 EC cells labeled with Troma-1 and P-cadherin by immunofluorescence. At least 1000 cells were scored for each time point. Error bars represent 5% standard error. For additional related data, see Figure S2.

Figure 3

Mouse Embryonic Stem Cells Lose Expression of the Pluripotency Markers Oct4, Sox2, and Nanog and Express Trophoblast Markers on Depletion of Geminin and Emi1 (A) Giant cell formation and induction of trophectoderm markers Troma-1 and P-cadherin (both in green) is evident in B6/Blu-1 mouse embryonic stem (ES) cells depleted of Emi1, geminin, and Oct4 at 2 days. Total DNA is shown in red. Scale bars represent 20 μm. (B) Emi1 and geminin depletion in mouse B6/Blu-1 ES cells result in downregulation of the pluripotency transcription factors Oct4, Sox2, and Nanog (in white). Total DNA is shown in red. Scale bars represent 20 μm. (C) In B6/Blu-1 ES cells, geminin is coexpressed with Oct4 (geminin and Oct4 both in white). Total DNA is shown in red. Scale bars represent 20 μm. The bar chart shows that geminin is present in a higher proportion of asynchronous ES and EC cells than in 3T3 fibroblasts, and notably in a far great proportion of cells that are not actively replicating DNA. Error bars represent 5% standard error. (D) Table summarizing cell-cycle analyses by immunofluorescence and flow cytometry. In asynchronous populations, the majority of 3T3 fibroblasts are in G1, in contrast to ES and EC cells in which G1 comprises a small fraction of the cell cycle and the majority of cells are in S phase. (E) Geminin is present during G1 in ES cells harvested at different time points following mitotic shake-off. Approximately 50% of ES cells enter S phase at 2 hr, suggesting that the duration of G1 phase is likely to be between 2 and 4 hr. For additional related data, see Figure S3.

Figure 4

Geminin Antagonizes Brg1 to Inhibit Repression of Oct4, Sox2, and Nanog in B6/Blu-1 ES Cells (A) Codepletion of geminin and Brg1 abolishes the upregulation of trophectoderm markers (in green) observed in geminin-depleted ES cells, but not nuclear enlargement (total DNA, red). Geminin and Brg1 codepletion also blocks the loss of Oct4, Sox2, and Nanog (in white) seen in geminin-depleted ES cells. Scale bars represent 20 μm. (B) Bar chart shows that codepletion of geminin and Brg1 rescues the loss of Oct4, Sox2, and Nanog induced by depletion of geminin alone, particularly in enlarged nuclei. (C) Codepletion of geminin and Brg1 rescues loss of Sox2 and Nanog in geminin-depleted ES cells. Error bars indicate standard error of the mean using four housekeeping genes for normalization. (D) Model of how geminin maintains genomic stability and the pluripotent state in ES cells. Suppressed APC/C^Cdh1 activity, possibly mediated by Emi1, stabilizes geminin in ES cells to prevent overreplication of DNA and inhibit repression of the core pluripotency factors Oct4, Sox2, and Nanog by antagonizing the chromatin-remodeling protein Brg1. For additional related data, see Figure S4.