Aurkb/PP1-mediated resetting of Oct4 during the cell cycle determines the identity of embryonic stem cells

Pluripotency transcription programs by core transcription factors (CTFs) might be reset during M/G1 transition to maintain the pluripotency of embryonic stem cells (ESCs). However, little is known about how CTFs are governed during cell cycle progression. Here, we demonstrate that the regulation of Oct4 by Aurora kinase b (Aurkb)/protein phosphatase 1 (PP1) during the cell cycle is important for resetting Oct4 to pluripotency and cell cycle genes in determining the identity of ESCs. Aurkb phosphorylates Oct4(S229) during G2/M phase, leading to the dissociation of Oct4 from chromatin, whereas PP1 binds Oct4 and dephosphorylates Oct4(S229) during M/G1 transition, which resets Oct4-driven transcription for pluripotency and the cell cycle. Aurkb phosphor-mimetic and PP1 binding-deficient mutations in Oct4 alter the cell cycle, effect the loss of pluripotency in ESCs, and decrease the efficiency of somatic cell reprogramming. Our findings provide evidence that the cell cycle is linked directly to pluripotency programs in ESCs. DOI: http://dx.doi.org/10.7554/eLife.10877.001


Introduction
Embryonic stem cells (ESCs) have unique transcriptional programs for self-renewal and pluripotency which differentiates into all types of cells. Core transcription factors-Oct4, Sox2, Nanog (OSN)govern such pluripotency transcriptional programs (Jaenisch and Young, 2008;Young, 2011). ESCs grow rapidly and undergo an unusual cell cycle, characterized by a very short G1 phase and a long S phase in mouse and human (Kapinas et al., 2013;Savatier et al., 1994;White and Dalton, 2005). The duration of G1 in mouse ESCs and human ESCs determines their fate with regard to differentiation and pluripotency (Coronado et al., 2013;Mummery et al., 1987;Pauklin and Vallier, 2013). A recent study revealed that the S and G2 phases tend to maintain the pluripotent state at early time of differentiation (Gonzales et al., 2015). Thus, cell cycle regulation in ESCs should be linked to pluripotency in maintaining ESC identity.
Our understanding of the molecular associations between the cell cycle and pluripotency in ESCs is limited. Robust Cdk2 activity shortens G1 phase by inducing rapid G1-S transition and promotes pluripotency and self-renewal in ESCs Van Hoof et al., 2009). Nanog controls S phase entry by targeting Cdc25c and Cdk6 . Despite the growing evidence on the direct connection between the cell cycle and pluripotency, it remains unknown how ESCs preserve their pluripotency through cell cycle progression and reset pluripotency transcriptional programs during the transition from mitosis to G1 phase.
Oct4 is considered a master regulator of ESC pluripotency through its cooperation with other core transcription factors (Jerabek et al., 2014). Post-translational modifications to Oct4 affect its transcriptional activity and lead to ESC pluripotency. For example, we previously reported that O-GlcNAcylatoin of murine Oct4(T228) is important for ESC pluripotency and somatic cell reprogramming . Also, Oct4 is controlled by phosphorylation (Brumbaugh et al., 2012;Saxe et al., 2009;Spelat et al., 2012), but there is no evidence that phosphorylation-mediated regulation of Oct4 during the cell cycle affects Oct4-mediated pluripotency programs in ESCs.
The Aurora kinase b (Aurkb)-protein phosphatase 1 (PP1) axis is critical for kinetochore assembly/ disassembly during the cell cycle, regulating the balance between phosphorylation and dephosphorylation of kinetochore substrates (Emanuele et al., 2008;Kim et al., 2010). Specifically, PP1 mediates the M/G1 transition and ensures proper resetting of the subsequent G1 phase by dephosphorylating cell cycle machinery (Ceulemans and Bollen, 2004). When the cell cycle resets, transcriptional programs for ESC pluripotency should be reset, because transcriptional programs are generally switched off at the onset of mitosis and subsequently reestablished during entry into the next G1 phase (Delcuve et al., 2008;Egli et al., 2008;Martinez-Balbas et al., 1995). Thus, during the cell cycle, the Aurkb-PP1 axis might be linked directly to the post-translational modification of pluripotency factors with regard to the resetting of pluripotency in ESCs.
In this study, we demonstrate that the Aurkb-PP1 axis regulates Oct4 during the cell cycle over time and by location. We found that Oct4 contains a well-conserved Aurkb phosphorylation residue (S229) and PP1 binding motif (RVXF) in its homeodomain. Aurkb phosphorylates Oct4(S229) during eLife digest Embryonic stem cells can give rise to any type of cell in the body -an ability known as pluripotency. These cells rapidly divide and self-renew until they are exposed to signals that cause them to mature into a particular specialized cell type.
As cells prepare to divide, they transition through a series of phases known as the cell cycle. In embryonic stem cells, these phases are often shorter than in other cell types. This altered timing is thought to be important for maintaining the pluripotency of the stem cells.
Proteins called core transcription factors also help stem cells to remain pluripotent. Evidence suggests that the activity of some of these proteins affects the timing of the different cell cycle phases. However, it is not clear exactly how they do so or how the activity of the transcription factors is controlled.
A core transcription factor called Oct4 is thought to be a "master regulator" of pluripotency that controls the activity of many of the other core transcription factors. Shin, Kim, Kim, Kim et al. have now studied the activity of Oct4 around the point of cell division. This revealed that a protein called aurora kinase B modifies Oct4 by adding a phosphate group to it just before a cell divides. This modification causes Oct4 to detach from chromatin, the protein structure in which DNA is packaged inside cells.
Following cell division, another protein called PP1 removes the phosphate group from Oct4. This "resets" the pluripotency of the stem cell, allowing it to continue to self-renew. Cells that contain only mutant forms of Oct4 that cannot bind to aurora kinase B or PP1 lose their pluripotency. The mutant Oct4 proteins also alter the cell cycle of the stem cells.
Overall, Shin et al.'s findings suggest that Oct4 regulates the cell cycle of embryonic stem cells as well as their pluripotency. How Oct4 activity affects the specialization of the stem cells into mature cell types remains to be investigated in future studies. G2/M phase and dissociates p-Oct4(S229) from chromatin, and PP1 dephosphorylates p-Oct4(S229) during the M/G1 transition, which prompts Oct4 to reset pluripotency transcription on re-entry into the following G1 phase. We found that mutating the Aurkb-phosphorylation residue S229 and the PP1-binding residue F271 of Oct4 in ESCs led to a significant loss of pluripotency and altered the cell cycle. Transduction of these mutants into MEFs significantly decreased the reprogramming efficiency.
Based on these findings, we propose that the spatiotemporal regulation of Oct4 by the Aurkb-PP1 axis during the cell cycle is critical for resetting pluripotency and cell cycle genes in determining the identity of ESCs.

Results
Phosphorylated Oct4 at serine 229 is highly enriched in G2/M phase and dissociated from chromatin To understand the function of the phosphorylation of Oct4, we examined its phosphorylation sites by transient transfection of Flag-Oct4 into E14 ESCs, analyzed the phosphorylation state of purified Oct4 by mass spectrometry, and identified 4 phosphorylation sites (Figure 1-figure supplement 1A and B). We then generated phosphor-mimetic mutants and measured their transcriptional activities by transfecting them into NIH-3T3 cells that stably harbored Oct4-driven luciferase reporter genes ( Figure 1-figure supplement 1C). Only the S229D mutant significantly reduced Oct4 transcriptional activity. Notably, serine 229 lies in the N-terminal region of the homeodomain of Oct4 and is well conserved throughout many species (Figure 1-figure supplement 1D and E).
Next, we generated a rabbit polyclonal antibody against phosphorylated Oct4(S229) [thereafter p-Oct4(S229)] and confirmed its specificity by dot blot and western blot (Figure 1-figure supplement 2A and B).
We then examined p-Oct4(S229) expression by confocal microscopy in undifferentiated E14 ESCs ( Figure 1A upper panel). p-Oct4(S229) in E14 ESCs was detected locally around mitotic cells. From this result above, we wondered whether Oct4 phosphorylation at serine 229 occurs in a cell cycledependent manner.
To this end, we treated cells with various agents that are related to the cell cycle and DNA damage. Notably, treatment of ESCs with nocodazole significantly enhanced Oct4 phosphorylation at S229, but aphidicolin and adriamycin decreased p-Oct4(S229) levels (Figure 1-figure supplement 2C-E and Figure 1B). In previously published data, 18 hr incubation with 200ng/ml of nocodazole was enough to synchronize hESCs at G2/M phase without inducing differentiation . In the case of E14 ESCs, nocodazole treatment during 10 hrs completely arrested cells at G2/ M phase. On treatment with nocodazole, p-Oct4(S229) began to rise in late S phase or early G2/M phase, peaking at G2/M phase ( Figure 1A-C).
We then confirmed that Oct4 dissociates from the binding region of Oct4 and Nanog at G2/M phase by ChIP-qPCR ( Figure 1F). Under the same conditions, p-Oct4(S229) rarely bound to the same locus, despite p-Oct4(S229) was successfully pulled down with the antibody (Figure 1-figure  supplement 2F). This result is consistent with a previous report that a human phosphor-mimetic form of Oct4(S325E) [homolog of mouse Oct4(S229)] binds to DNA more weakly than Oct4(WT) by in vitro EMSA (Brumbaugh et al., 2012). The loss of DNA binding affinity of Oct4 by phosphorylation might be induced by steric and electrostatic clashes (Saxe et al., 2009). Based on these findings, Oct4 is specifically phosphorylated at serine 229, and p-Oct4(S229) dissociates from chromatin in G2/M phase. To identify the kinases that phosphorylate Oct4(S229), we selected 19 candidates using a groupbased prediction system (Xue et al., 2008) and among Oct4-interacting kinases (Ding et al., 2012) ( Figure 2-figure supplement 1A). We examined the phosphorylation of S229 by these 19 recombinant kinases by in vitro kinase assay and western blot with anti-pOct4(S229)-6 kinases could phosphorylate S229 ( To identify the kinases that mediate Oct4(S229) phosphorylation during G2/M phase, we screened the kinases by administering nocodazole to ESCs that were knocked down with cognate lentivirally expressed shRNAs of kinases (Figure 2-figure supplement 1C and D). p-Oct4(S229) levels declined significantly on knockdown of Aurkb. Further, we confirmed that recombinant Aurkb phosphorylated GST-Oct4(S229) by in vitro 32 P-ATP-labeled kinase assay and western blot with antip-Oct4(229) (Figure 2A and B).
To verify the Aurkb-mediated phosphorylation of Oct4(S229), we treated nocodazole-pretreated E14 ESCs (10 hr) with various aurora kinase inhibitors for 15 min. An Aurkb-specific inhibitor, hesperadin, completely blocked the phosphorylation, but an Aurka-specific inhibitor, MLN8237, did not. AT9283, an inhibitor of both Aurka and Aurkb, prevented phosphorylation ( Figure 2C). Under this condition, Aurkb inhibition did not alter cell cycle profile ( Figure 2D). Aurkb preferentially phosphorylates serine when arginine lies 2 residue upstream of a phosphoserine (-2 position) (Sugiyama et al., 2002). In Oct4, we found arginine-227, residing 2 residues upstream of S229 ( We then observed that Flag-Aurkb interacts with endogenous Oct4 in E14 ESCs by immunoprecipitation ( Figure 2E). To determine the cell cycle phases during which Oct4 preferentially interacts with Aurkb, Flag-Oct4-expressing ZHBTc4 ESCs were pretreated with nocodazole for 6 hr, maintaining them in G2/M phase, and released on removal of nocodazole for the cell cycle progression. Notably, Flag-Oct4 interacted strongly with endogenous Aurkb in G2/M phase in Flag-Oct4-expressing ZHBTc4 ESCs ( Figure 2F and G), consistent with our result that Oct4(S229) is heavily phosphorylated in G2/M phase ( Figure 1). These findings demonstrate that Aurkb is the kinase that phosphorylates Oct4(S229) in G2/M phase.
In examining the amino acid sequence of Oct4, we found that it contains a protein phosphatase 1 (PP1)-binding sequence (268-RVWF-271) in its homeodomain, near the S229 Aurkb phosphorylation site in the 3-dimensional structure ( Figure 3A and B). This motif is well conserved among many species ( Figure 3-figure supplement 1A). Thus, we studied the interaction of Oct4 with 3 isoforms of PP1: PP1a, PP1b, and PP1g. We found that Oct4 interacted more strongly with endogenous PP1b and PP1g than with PP1a in ZHBTc4 ESCs ( Figure 3C).
We then determined whether PP1 dephosphorylates the Aurkb-catalyzed p-Oct4(S229) by preincubating recombinant GST-Oct4 with recombinant Aurkb, adding purified PP1, and measuring the phosphorylation state of p-Oct4(S229) by western blot. Recombinant PP1b and PP1g, but not PP1a, dephosphorylated Aurkb-mediated phospho-Oct4(S229) ( PP1-mediated dephosphorylation of Oct4(S229) correlates with the resetting of pluripotency in the next G1 phase Based on the findings that p-Oct4(S229) dissociates from condensed chromatin ( Figure 1) and PP1 dephosphorylates p-Oct4(S229) during the M/G1 transition (Figure 3), We hypothesized that PP1mediated dephosphorylation of Oct4(S229) is required for the resetting of Oct4 for pluripotency gene expression during the M/G1 transition. To examine this possibility, we arrested E14 ESCs in G2/M phase with nocodazole and released them into normal serum ( Figure 4A and B). The high amounts of p-Oct4(S229) that accumulated in G2/M phase vanished quickly after G1 phase (after 1 hr release), reappeared at late S phase (after 6 hr release) and enriched at the next M phase (10 hr after release). In addition, to confirm weather Oct4 binding to target genes are regulated by phosphorylation dependent manner throughout the S-G2/M phase, we chased p-Oct4 level and binding of Oct4 to target genes ( Figure 4-figure supplement 1A and B). Binding of Oct4 to target chromatin declined through S-G2/M phase (7-9 hr after release), and increased at the M/G1 transition (10 hr after release) in parallel with the level of p-Oct4(S229) accumulated. This result is consistent with recent report that Aurkb is active during S phase in ESCs (Mallm and Rippe, 2015).
Interestingly, the overall levels of Oct4 protein did not change significantly throughout the cell cycle. However, unlike Oct4, the levels of Nanog, which has a short half-life (Ramakrishna et al., 2011), rose in G1 phase, declined through S-G2/M phase, and reappeared at the start of the next G1 phase. This finding indicates that the synchronization of the phosphorylation state of Oct4(S229) with the cell cycle is linked to the resetting of Oct4 to its target genes.
To test that PP1 is required for resetting the transcription of Oct4 target genes, we administered 50 nM OKA to nocodazole-pretreated E14 ESCs for 4 hr and released them into normal serum. As expected, OKA retarded the dephosphorylation of p-Oct4(S229) and re-entry into the next G1 phase ( Figure 4C and D).
To examine the binding of Oct4 to its targeting pluripotency genes on chromatin during the M/ G1 transition, we performed the ChIP-qPCR assay. As a result, Oct4 bound weakly to target genes in G2/M phase, strengthening its association during entry into G1 phase. On treatment with OKA, Oct4 binding to target genes declined significantly during entry into G1 phase ( Figure 4E). In addition, to elucidate the cell cycle effect induced by OKA treatment to Oct4 binding, we performed ChIP-qPCR assay in Zhbtc4 ESCs stably expressing wild-type Oct4 (WT) and phosphor-defect mutant (S229A). When cells were released into G1 phase, OKA treatment significantly prevented the binding of Oct4(WT) to target genes. On the other hand, binding of Oct4(S229A) was relatively less affected even in treatment of OKA (Figure 4-figure supplement 1C). However, Oct4(S229A) mutant affects the O-GlcNAcylation of Oct4, which is critical for Oct4 activity, thereby Oct4(S229A) mutant fail to self-renew .
To analyze Oct4-depenent transcriptional resetting during the M/G1 transition, we measured nascent RNA levels ( Figure 4F). When E14 ESCs were arrested in G2/M phase, nascent RNA levels of a subset of Oct4-targeting pluripotency genes declined significantly versus asynchronous ESCs.  [Xiao et al., 2012]) and ChIP-seq binding profiles of Oct4 at the Bub1 and Rif1 locus in undifferentiated E14 ESCs (lower panel; [Whyte et al., 2013]). (D) ChIP-qPCR analysis of E14 ESCs with anti-Oct4 in regions of Bub1 and Rif1 during the M/G1 phase transition with or without OKA treatment. IgG was used as a control. Values represent mean ± standard deviation (n!3). t-test was used to calculate the statistical significance of differences in enrichment levels of Oct4 in ESCs during the M/G1 transition with or without OKA treatment. (*p<0.05, **p<0.01, ***p<0.001) (E) Nascent RNA levels of Bub1 and Rif1 in E14 ESCs were nalyzed by real-time qPCR during the M/G1 phase transition with or without OKA treatment. Levels of nascent RNA were divided by those in asynchronous state of E14 ESCs. DOI: 10.7554/eLife.10877.012 The following source data is available for figure 5: Source data 1. Identification of putative Oct4 target genes. DOI: 10.7554/eLife.10877.013 Source data 2. Cell-cycle related genes in putative Oct4 target genes. DOI: 10.7554/eLife.10877.014 Nascent RNA levels of certain Oct4 target genes were upregulated when cells entered G1 phase (until 2 hr after release). Complementing the ChIP data, the nascent RNA levels of target genes were retarded after OKA treatment. Thus, we conclude that dephosphorylation by PP1 is critical for the transcriptional resetting of Oct4 to pluripotency genes during the M/G1 transition.

Oct4 targets and resets cell cycle related genes in the next G1 phase
We next wondered whether Oct4 resets a subset of cell cycle related genes during the M/G1 transition. To address this, we first narrowed down the putative 1258 Oct4 target genes by crossover between 5824 genes co-occupied by OSN and 4617 genes decreased by Oct4 depletion in ZHBTc4 ESCs ( Figure 5A and Figure 5-source data 1) using publically-available ChIP-seq and RNA-seq data (Boo et al., 2015;Whyte et al., 2013). By gene ontology analysis of the putative Oct4 target genes using DAVID (http://david.abcc.ncifcrf.gov), we identified some Oct4 target genes associated with various cell cycle related functional categories ( Figure 5B and Figure 5-source data 2).
Intriguingly, we found that Oct4 governs the cell cycle genes related to S/G2/M phase. Thus, among these genes related to S/G2/M phase, we focused on Bub1 and Rif1 because loss of Bub1 and Rif1 are known to induce differentiation in the ESC-based knockdown experiment (Dan et al., 2014;Lee et al., 2012) and expression levels of both genes decrease upon ESC differentiation  Values represent mean ± standard deviation (n!3). t-test was used to calculate the statistical significance of differences in G1 phase of ZHBTc4-Oct4(WT) versus -Oct4(S229D) and -Oct4(F271A). (*p<0.05, ***p<0.001) (C) Anti-Oct4 ChIP-qPCR of ZHBTc4 ESCs that express exogenous Oct4 at 2 days after doxycycline treatment. F271A and S229D mutants showed decreased binding to target genes compared with Oct4 WT. Oct4-depleted ZHBTc4 Figure 7 continued on next page ( Figure 5C upper) in previously published RNA-seq study (Xiao et al., 2012). Furthermore, both Oct4 enrichment score and expression level of Bub1 and Rif1 were one of the top10 genes among putative Oct4 targeting cell cycle related genes (lower, Figure 5C and Figure 5-source data 2).
To investigate whether Bub1 and Rif1 are reset by PP1-mediated dephosphorylation of Oct4 during the M/G1 transition, we first investigated the resetting of Oct4 to target genes during the M/G1 transition by ChIP-qPCR ( Figure 5D). Oct4 binding to both genes on chromatin was weakly sustained in G2/M phase and rapidly increased during the M/G1 transition (0.5 hr after release into normal serum), and thereafter Oct4 binding is either saturated or declined, while induced Oct4 binding to both genes during the M/G1 transition were relatively retarded when cells were released into normal serum with treatment of OKA ( Figure 5D). Likewise, nascent RNA levels of Bub1 and Rif1 were downregulated when cells were arrested in G2/M phase and upregulated during the M/G1 transition, but rapid increase of nascent RNA levels of both genes during entry into G1 phase was retarded by OKA treatment ( Figure 5E). Considered together, we concluded that Oct4 directly controls cell cycle related genes by its resetting to cell cycle related genes during the M/G1 transition.

Oct4 ChIP-seq reveals that resetting regions enriched in pluripotency and cell cycle related genes
To identify which regions are reset by Oct4, we mapped the genomewide occupancy of Oct4 at G2/ M and G1 phase in E14 ESCs by ChIP-seq. We found high-confidence peaks (with p value<10 À5 ) at G2/M (9204) and G1 (24548) phase ( Figure 6A and Figure 6-source data 1). Expectedly, Oct4 bound much more regions of genome at G1 phase rather than G2/M phase ( Figure 6A). Furthermore, mean peak density of Oct4 bound regions was higher in G1 phase than G2/M phase ( Figure 6B) implicating that Oct4 genome-widely resets the target genes at G1 phase. Next to identify reset region by Oct4, we discovered 9092 of genomic regions enriched by Oct4 more than twofold increase in G1 phase compared to G2/M phase ( Figure 6-source data 2). We categorized these as the Oct4 resetting peaks, which are significantly enriched in not only pluripotency but also cell cycle categories ( Figure 6C). For example, we showed that Oct4 more strongly binds to the regions of both Nanog and Sox2 at G1 phase rather than G2/M phase, supporting that Oct4 resets its target genes at G1 phase ( Figure 6D).
Under the same conditions (dox for 2 days), we analyzed the cell cycle patterns in these ESCs ( Figure 7B). Oct4-depleted ZHBTc4 ESCs (Mock) harbored significantly more cells in G1 phase and Oct4(S229D)-backup cells contained larger G1-phase populations than Oct4(WT)-backup cells but fewer than Oct4(F271A)-backup cells, indicating that the PP1-binding motif in Oct4 is essential for maintaining the pluripotency and cell cycle progression of ESCs. The binding of Oct4 mutants to target genes-including pluripotency-related and cell cycle genes-decreased significantly versus Oct4 (WT) after dox treatment for 2 days ( Figure 7C).
To determine the reset patterns of Oct4 mutants during the M/G1 transition, we analyzed nascent RNA levels of a subset of Oct4 target genes in ZHBTc4 ESCs harboring Oct4(WT) and Oct4 mutants that were pretreated with dox for 2 days, given nocodazole (6 hr), and released into normal serum ( Figure 7D).Nascent RNA transcripts of certain Oct4 target genes in Oct4(WT)-backup cells were upregulated during the M/G1 transition, whereas those in Oct4-mutated cells did not increase versus ZHBTc4 Oct4(WT), indicating that Oct4 mutations (S229D, F271A) impede the prompt resetting of Oct4 during the entry into the next G1 phase.
To determine the long-term effects of Oct4 mutations on ESC pluripotency and cell cycle progression, we cultured ZHBTc4 ESCs containing Mock, Oct4(WT), Oct4(S229D), and Oct4(F271A) for 7 days with dox. Ectopic Oct4(WT) and Oct4 mutants (S229D, F271A) were continuously expressed until dox treatment for 7 days ( Figure 7A). Total mRNA expression of a subset of Oct4 target genes in Oct4(WT) cells was continuous, whereas that in Oct4-depleted and Oct4 mutant-backup cells was significantly downregulated ( Figure 7E). Consistent with this finding, alkaline phosphatase (AP) positive colonies in Oct4-depleted and Oct4 mutant-backup cells was much lower than in Oct4(WT)backup cells ( Figure 7F).
To determine the effects of mutation on Oct4 transcriptional activity, we infected retroviral wildtype and mutant Oct4 into NIH-3T3 cells in which a 10X Oct4 response element (RE)-driven luciferase reporter was stably incorporated and measured luciferase activity. Oct4(F271A) and Oct4 (S229D) showed little luciferase activity (Figure 7-figure supplement 1A). Oct4(S229A) also had weaker activity than Oct4(WT). Consistent with these data, during Oct4 depletion in ZHBTc4 ESCs, retroviral infection with Oct4(F271A) and Oct4(S229D) failed to rescue the maintenance of ZHBTc4 ESC self-renewal compared with Oct4(WT) infected cells (Figure 7-figure supplement 1B and C). In addition, these Oct4 mutants impeded somatic cell reprogramming ( Figure 7G).
Based on our findings, we propose that the recycling of Oct4 by Aurkb/PP1 over time and by location is pivotal for the transcriptional resetting of Oct4 during entry in to the subsequent G1 phase, ultimately maintaining ESC pluripotency and cell cycle progression (Figure 8).

Discussion
In this study, we have demonstrated that Oct4, a master pluripotency transcription factor, is spatiotemporally regulated by the Aurkb-PP1 axis during the cell cycle. In G2/M phase, Aurkb phosphorylates Oct4 extensively at serine 229, leading to its dissociation from chromatin (Figure 1 and Figure 2). The detachment of Oct4 from chromatin is consistent with findings from previous reports. Most transcriptional machinery proteins, such as RNA pol II (Gottesfeld and Forbes, 1997;Parsons and Spencer, 1997), and many transcription factors dissociate from condensed chromatin at the onset of mitosis, and many studies have implied that mitotic dissociation of transcription factors occurs through phosphorylation (Delcuve et al., 2008;Dephoure et al., 2008).
Aurka regulates ESC pluripotency through phosphorylation-mediated inhibition of p53 (Lee et al., 2012) but is not involved in the phosphorylation-mediated recycling of Oct4 ( Figure 2 and Figure 2-figure supplement 1). Thus, both Aurk isoforms-Aurka and Aurkb-appear to be related to ESC pluripotency, but in a different molecular mechanism.
We also found that PP1 mediates the resetting of Oct4 during the M/G1 transition. PP1 governs the resetting of cell cycle machinery (Ceulemans and Bollen, 2004), binding specific sequencesthe RVxF motif-and dephosphorylating interactors (Hendrickx et al., 2009). We identified an RVWF motif in the C-terminal POU-h domain ( Figure 3A) and an Aurkb phosphorylation site (S229) in the N-terminal POU-h domain of Oct4, which come into close proximity in the 3-dimensional structure ( Figure 3B). This arrangement convinces us that PP1 binds Oct4 through the RVxF motif and dephosphorylates Oct4 in vitro ( Figure 3E and F).
In addition, we revealed that PP1 inhibition by OKA treatment delayed the Oct4 dephosphorylation at S229 residue and consequently resetting to the target genes in E14 ESCs during M/G1 transition ( Figure 4C and E). We further found that OKA treatment relatively less affected the binding of phosphor-defect Oct4(S229A) to target genes, significantly impeding the resetting of Oct4(WT) at M/G1 transition (Figure 4-figure supplement 1C), implying that dephosphorylation of S229 residue by PP1 is paramount for the resetting of Oct4 to target genes. Nonetheless, we cannot perfectly rule out the possibility that OKA treatment may affect the function of broad spectra of cell cycle regulators during M/G1 transition.
We confirmed that Aurkb-phosphor-mimetic and PP1-binding-defective mutations lead to a loss of pluripotency, even in the presence of LIF, and that introduction of these mutants into somatic cells lowers the reprogramming efficiency ( Figure 7G), supporting that Aurkb/PP1 is a critical pair of regulators in resetting Oct4 on chromatin during the cell cycle.
Mitotic phosphorylation of a transcription factor affects its transcriptional activity and other properties. For example, Oct1, a member of the POU domain transcription factors, is phosphorylated during mitosis and localizes to the spindle matrix, forming a complex with lamin B1 at the midbody (Kang et al., 2011). Mitotic phosphorylation of Sp1 protects itself from ubiquitin-dependent degradation (Chuang et al., 2008). Notably, we found that Oct4 protein levels were unchanged, even though nascent Oct4 RNA levels fluctuated during the cell cycle ( Figure 4A and F), implying that phosphorylated Oct4 is probably protected from degradation during the cell cycle. We are interested in determining the mechanism of how phosphorylated Oct4 escapes protein degradation.
The molecular association between pluripotency and the cell cycle in ESCs has garnered attention with regard to identifying the mechanism by which the cell fate of ESCs is determined. In particular, the protraction of G1 in naive ESCs by knockdown of cyclin E1 causes spontaneous differentiation; in contrast, promoting G1/S transition through overexpression of cyclin E1 enhances self-renewal (Coronado et al., 2013). G1 phase regulators, cyclin D proteins control the differentiation of hESCs into various lineages via the TGF-b-Smad2/3 pathway (Pauklin and Vallier, 2013). Sox2 was identified as a cell cycle regulator that suppresses p21 and p27 and induces cyclin D3 expression (Herreros-Villanueva et al., 2013). Oct4 downregulation lengthens G1 phase and upregulates p21 in ESCs (Lee et al., 2012). A nontranscriptional function of Oct4 in mitotic entry has recently been reported (Zhao et al., 2014). A recent study performed functional screening of human embryonic stem cells under various differentiation conditions and identified that genes involved in S and G2 phases are gatekeepers of differentiation (Gonzales et al., 2015). Furthermore, S and G2 phases of cell cycle possess an intrinsic propensity for maintaining pluripotency.
In this study, in addition to observing that Aurkb/PP1 control the dynamics of Oct4 during the cell cycle, we found that Oct4 governs the cell cycle of ESCs by directly targeting genes that are related to cell cycle regulation and pluripotency. In particular, 2 cell cycle genes-Bub1 and Rif1are reset by Oct4 during the cell cycle ( Figure 5E and Figure 7D). Considering that Bub1 and Rif1 are important cell cycle regulators that control the checkpoints for mitosis and replication (Bolanos-Garcia and Blundell, 2011;Yamazaki et al., 2013) and are crucial for maintaining pluripotency (Dan et al., 2014;Lee et al., 2012), the regulation of S-G2-M phase by Oct4 might also be critical for ESC pluripotency.
Our study might provide insights into why ESCs reset Oct4 during the cell cycle. Like, this mechanism might bifurcate the fate of ESCs: tight resetting of Oct4 on chromatin during the cell cycle strengthens the pluripotency of ESCs at the ground state, but on differentiation, loose resetting of Oct4 at the next M/G1 transition leads to lineage differentiation.
During reprogramming by nuclear transfer, mitotic chromosomal condensation is required to reset the origins of replication of differentiated donor cells in embryonic DNA replication (Lemaitre et al., 2005), transfer of a mitotic genome into a zygote in mitosis-enhanced reprogramming (Egli et al., 2007), and mitotic chromatin induces core pluripotency factors more rapidly than interphase nuclei (Halley-Stott et al., 2014), suggesting that a genome can be exchanged during mitosis which is an open window that allows transcription factors to occupy target genes on mitotic exit and thus enabling postmitotic cell fate changes to be induced.
We have provided evidence that cell cycle machinery cooperates with pluripotency transcriptional programs. The resetting of Oct4 occurs rapidly during the exit from mitosis and that delayed resetting alters the cell cycle and effects the loss of pluripotency-ie, prompt resetting of Oct4 prevents postmitotic cell fate changes in ESCs. Based on our results, we suggest that the potential of ESCs to differentiate might be derived from the small window of the M/G1 transition by which the resetting of Oct4 is the central mechanism to determine the maintenance of ESC pluripotency or lineage commitments.

Mitotic arrest-release and cell cycle analysis
For G2/M phase synchronization, E14 cells were treated with 200 ng/ml nocodazole (Calbiochem, Germany ) for 10 hr and ZHBTc4 cells were treated with same concentrations of nocodazole for 6 hr. In order to release, synchronized cells were washed three times with PBS, and incubated for the indicated time in fresh culture media.
For cell cycle analysis, the collected cells at the indicated time were immediately fixed in 70% ethanol and stained with propidium iodide (PI; Sigma, P4170) for 1 hr at room temperature in the dark. Cell cycles were analyzed using FACSCalibur flow cytometer and LSRII (SORP) (Becton Dickinson, Franklin Lakes, New Jersey). Analysis of cell cycle data was performed with FlowJo (Tree Star Inc., Ashland, Oregon).
Confocal micro-images were obtained by a confocal laser scanning microscope (Carl Zeiss, Germany, LSM 510 META).

DNA constructs
The plasmid pGAE-mKO2:Cdt1 and pGAE-mAG:Geminin were generously provided by Savatier,P. (INSERM U846,France). For stable expression in ESCs, fragments containing mKO2:Cdt1 and hmAG1:Geminin coding sequences, respectively, were generated by PCR amplification and were sub-cloned between the SalI and AgeI sites into pCAG-IP vector to generate pCAG-mKO2:Cdt1-IP and pCAG-mAG:Geminin-IP. PP1a, b and g were amplified by PCR from cDNA of E14 ESCs. The PCR products were digested with XhoI and AgeI then subcloned into pCAG-Flag-IP vector. All Oct4 mutants were generated by site-directed mutagenesis (Intron, Korea).

Generation of stably expressing Flag-Oct4 ZHBTc4 ESCs
For long-term transgene expression in ZHBTc4 ESCs, Flag-tagged Oct4 was cloned into pCAG-Flag-IP, which was generated by inserting a Flag tag into pCAG-IP, kindly provided by Hitoshi Niwa (RIKEN, Japan). To generate ESCs stably expressing Flag-Oct4, pCAG-Flag-Oct4-IP was transfected into ESCs using Lipofectamine (Invitrogen). After 48 hr of transfection, selection with 2mg/ml of puromycin was performed to determine stable integration. Puromycin resistant cells were expanded and analyzed for Oct4 by Western blot.

Reporter gene assay
The reporter gene assay was done as described . Briefly, Ten copies of Oct4responsive element(10X Oct4 RE)-driven luciferase reporter gene was incorporated into the genome of NIH 3T3 cells by retroviral infection. To stably incorporate reporter gene into genomic DNA, cells were selected with puromycin for at least 2 weeks. These stable cells were infected with retroviruses expressing Oct4. Luciferase activity was measured 4 days after infection of Oct4.
In vitro kinase assay 0.2 mg of purified GST-Oct4 proteins were used in cold in vitro kinase assays with purified recombinant kinases. All kinases were purchased from Proqinase (Germany). Kinase reactions were performed in kinase buffer (60mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 mM Naorthovanadate, 1.2 mM DTT, 0.5mM ATP) for 30 min at 30˚C. Then reactions were stopped by the addition of 5X SDS-PAGE loading buffer and assessed by Westernblot.
For radioactive in vitro kinase assay GST-Oct4 was incubated with Aurkb in kinase buffer (60 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3mM MnCl2, 3 mM Na-orthovanadate, 1.2 mM DTT, 0.25 mM ATP) with 0.1 mM g-32 P-ATP (NEG002A250UC, purchased from PerkinElmer, Waltham, Massachusetts) for 30 min at 30˚C. Reactions were then stopped by the addition of 5X SDS-PAGE loading buffer and loaded for separation on 8% SDS-PAGE gel. After staining with Coomassie Blue, the gels were dried and exposed to films.

In vitro phosphatase assay
In vitro phosphatase assay was performed as described (Kassardjian et al., 2012). Briefly, phosphorylated GST-Oct4 was pulled down with Glutathione Sepharose 4B (Sigma) for 4 hr at 4˚C and suspended in phosphatase buffer. GST-Oct4 beads phosphorylated by Aurkb were incubated with 1 mg of purified His-tagged PP1 for 1 hr at 30˚C, with mild shaking.