Geminin Escapes Degradation in G1 of Mouse Pluripotent Cells and Mediates the Expression of Oct4, Sox2, and Nanog

Summary Geminin is an essential cell-cycle protein that is only present from S phase to early mitosis in metazoan somatic cells [1, 2]. Genetic ablation of geminin in the mouse results in preimplantation embryonic lethality because pluripotent cells fail to form and all cells differentiate to trophoblast [3, 4]. 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 [1, 2, 5]. 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?

, Related to Figure 1

. Lineage Analysis of P19 EC Cells Depleted of Geminin, Emi1, and Oct4
Depletion of geminin, Emi1, and Oct4 at 2 days induces upregulation of trophoblast markers Troma-1, P-cadherin, Cdx2, and Eomes but not markers indicative of differentiation towards embryonic lineages. Total DNA is shown in red. Scale bar represents 20 μm.

Figure S2, Related to Figure 2. Emi1 Depletion Results in Nuclear Enlargement, Upregulation of Trophectoderm Markers, and Unopposed APC/C Cdh1 Activity in Mouse EC Cells
(A) Emi1, Cdh1 and both were depleted by siRNA in P19 EC cells. Codepleting Emi1 and Cdh1 antagonizes the effect of depleting Emi1 alone in P19 EC cells, inhibiting both nuclear enlargement (total DNA is shown in red) and induction of trophectoderm markers Troma-1 and P-cadherin (both in green) at two days posttransfection. Scale bar represents 20 μm. Whole cell lysates were harvested for analysis by western blotting with Emi1 and Cdh1 antibodies. β-actin was used as a loading control. Bar chart shows the proportion of control, Emi1-depleted, and Emi1 and Cdh1 codepleted P19 EC cells labelled with Troma-1 and P-cadherin by immunofluorescence. At least 1000 cells were scored for each time point. Error bars represent 5% standard error. (B) Emi1 depletion results in massive nuclear enlargement (total DNA is shown in red) and upregulation of trophectoderm markers, Troma-1 and P-cadherin (in green) specifically in cells in which the APC/C Cdh1 substrates geminin or cyclin A2 (in purple) are downregulated. Scale bar represents 20 μm. (C) Geminin, cyclin A2, and cyclin B1 (in green) are downregulated following depletion of Emi1 in P19 EC cells and dramatically stabilized following MG132 treatment. Total DNA is shown in red. Scale bar represents 20 μm. (D) Cyclin E (green), which is not a target of the APC/C, is upregulated by immunofluorescence in enlarged nuclei (total DNA is shown in red) following depletion of Emi1 in pluripotent cells. Scale bar represents 20 μm. (E) Emi1, Cdc6 and both were depleted by siRNA in P19 EC cells and whole cell lysates were harvested for analysis by western blotting. β-actin was used as a loading control. Scatter dot plots show relative DNA content analysis of control, Emi1-depleted, and Emi1 and Cdc6 codepleted P19 EC cells. Z stacks of DAPI-stained nuclei were collected and compiled into a single 3Dreconstructed image for analysis. Red bars represent the mean value. (F) Cdx2 (in white) is induced following depletion of Emi1 at 24 hours posttransfection and remains upregulated in Emi1-depleted cells treated with aphidicolin for 12 hours at 12 hours posttransfection. Total DNA is shown in red. Scale bar represents 20 μm.

Figure S3, Related to Figure 3. Mouse Pluripotent Cells Lose Expression of Pluripotency Markers and Express Trophoblast Markers on Depletion of Geminin and Emi1
(A) Lineage analysis of B6/Blu-1 ES cells depleted of geminin, Emi1, and Oct4. Total DNA is shown in red. Scale bar represents 20 μm. (B) Trophectoderm markers Troma-1 and P-cadherin (in green) are present at 6 days in B6/Blu-1 ES cells depleted of geminin, Emi1, and Oct4. Total DNA is shown in red. Scale bar represents 20 μm. (C) Loss of Oct4 and Sox2 is observed following depletion of geminin or Emi1 in P19 EC cells. Nanog was not found to be present in control P19 EC cells.  (C) Validation of rabbit polyclonal anti-geminin antibody by immunoblotting. Western blots of whole cell lysates from mouse 3T3 cells transfected with control or geminin siRNA were incubated with immunoaffinity-purified R1 anti-mouse geminin antibody. β-actin was used as a loading control. CCCGGAAGAGAAAGCGAACTA, AACGAGAAGAGTATGAGGCTA and AACCTTCAGGAGATATGCAAA. All other siRNAs were obtained from the Qiagen HP genome-wide siRNA databank (http://www.qiagen.com).

Quantitative Reverse Transcriptase (RT)-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen), RNA concentrations and quality determined using the Nanodrop UV Spectrophotometer (Nanodrop Technologies) and cDNA synthesized using the Quantitect Reverse Transcription kit (Qiagen). Quantitative RT-PCR was performed using SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma-Aldrich) and reactions were run on an Opticon™ continuous fluorescence detector PCR machine (MJ Research).