Abstract Cells need to overcome both intrinsic and extrinsic threats. Although pluripotency is associated with damage responses, how stem cells respond to DNA damage remains controversial. Here, we elucidate that DNA damage activates Chk2, leading to the phosphorylation of serine 164 on C-terminal binding protein 2 (Ctbp2). The phosphorylation of Ctbp2 induces the disruption of Ctbp2 tetramer, weakening interactions with zinc finger proteins, leading to the dissociation of phosphorylated Ctbp2 from chromatin. This transition to a monomeric state results in the separation of histone deacetylase 1 from Ctbp2, consequently slowing the rate of H3K27 deacetylation. In contrast to the nucleosome remodeling and deacetylase complex, phosphorylated Ctbp2 increased binding affinity to polycomb repressive complex (PRC)2, interacting through the N-terminal domain of Suz12. Through this domain, Ctbp2 competes with Jarid2, inhibiting the function of PRC2. Thus, the phosphorylation of Ctbp2 under stress conditions represents a precise mechanism aimed at preserving stemness traits by inhibiting permanent transcriptional shutdown.
Transcription factors and chromatin remodeling proteins control the transcriptional variability for ESC lineage commitment. During ESC differentiation, chromatin modifiers are recruited to the regulatory regions by transcription factors, thereby activating the lineage-specific genes or silencing the transcription of active ESC genes. However, the underlying mechanisms that link transcription factors to exit from pluripotency are yet to be identified. In this study, we show that the Ctbp2-interacting zinc finger proteins, Zfp217 and Zfp516, function as linkers for the chromatin regulators during ESC differentiation. CRISPR-Cas9-mediated knock-outs of both Zfp217 and Zfp516 in ESCs prevent the exit from pluripotency. Both zinc finger proteins regulate the Ctbp2-mediated recruitment of the NuRD complex and polycomb repressive complex 2 (PRC2) to active ESC genes, subsequently switching the H3K27ac to H3K27me3 during ESC differentiation for active gene silencing. We therefore suggest that some zinc finger proteins orchestrate to control the concise epigenetic states on active ESC genes during differentiation, resulting in natural lineage commitment.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. https://doi.org/10.7554/eLife.10877.001 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. https://doi.org/10.7554/eLife.10877.002 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 (Neganova et al., 2009; Van Hoof et al., 2009). Nanog controls S phase entry by targeting Cdc25c and Cdk6 (Zhang et al., 2009). 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 (Jang et al., 2012). 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 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 cycle-dependent manner. Figure 1 with 2 supplements see all Download asset Open asset Phosphorylated Oct4 at serine 229 is enriched in G2/M phase and dissociated from chromatin. (A) Immunostaining of E14 ESCs treated with or without nocodazole (NOC, 200 ng/ml) for 10 hr. Oct4 was stained with anti-Oct4 (green), p-Oct4(S229) was stained with anti-p-Oct4(S229) (red), and DNA was stained with DAPI (blue). White boxes represent cells at various stages. Shown are interphase (1), metaphase (2), and anaphase (3) cells. Scale bars were shown. (B) E14 ESCs were treated with nocodazole (200 ng/ml) for the indicated times and immunoblotted with the indicated antibodies. Phosphorylation levels of Oct4 at serine 229 were gradually induced during nocodazole treatment. (C) Histograms of the proportions of nocodazole-treated (200 ng/ml) E14 ESCs at various stages in the cell cycle. Cells were stained with PI and DNA contents were analyzed by FACS (1x104 cells/sample). (D and E) Fluorescence images of E14 ESCs expressing mKO2-Cdt1 and mAG-Geminin (FUCCI reporter). Shown are green (mAG-geminin) and red (mKO2-Cdt1) fluorescence. E14 ESCs expressing FUCCI reporter were left untreated or treated with nocodazole (NOC, 200 ng/ml) for 10 hr. p-Oct4(S229) was stained with anti-p-Oct4(S229) (red, Figure 1E; green, Figure 1F), and DNA was stained with DAPI (blue). Scale bars, 30 μm (F) ChIP-qPCR assay was performed with anti-IgG, anti-Oct4, and anti-p-Oct4(S229) in E14 ESCs with or without nocodazole (NOC, 200 ng/ml) for 10 hr. Values represent mean ± standard deviation (n≥3). (**p<0.01, ***p<0.001) https://doi.org/10.7554/eLife.10877.003 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 (Zhang et al., 2009). 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). To confirm the localization of p-Oct4(S229), we adapted the fluorescence ubiquitination cell cycle indicator (FUCCI) reporter system to ESCs (Sakaue-Sawano et al., 2008). We generated E14 ESCs that stably expressed GFP-mAG-geminin during S-G2-M phase and examined p-Oct4(S229) by confocal microscopy on nocodazole treatment (Figure 1D). As expected, p-Oct4(S229) overlapped with GFP-geminin in G2/M phase. In contrast, p-Oct4(S229) did not merge with Red-mKO2-Cdt1, which is highly expressed in G1 phase (Figure 1E). 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. Aurora kinase b binds Oct4 and phosphorylates Oct4 at serine 229 in a cell cycle dependent manner To identify the kinases that phosphorylate Oct4(S229), we selected 19 candidates using a group-based 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 (Figure 2—figure supplement 1B). Figure 2 with 1 supplement see all Download asset Open asset Aurkb binds and phosphorylates Oct4 at serine 229 during G2/M phase. (A) Radioactive in vitro kinase assay using recombinant Aurkb to phosphorylate GST-Oct4 WT and S229A mutant. Coomassie staining of purified proteins and autoradiogram showing incorporation of γ-32P ATP. (B) Cold in vitro kinase assay reactions using recombinant Aurkb with purified GST, GST-Oct4 WT, and S229A mutant as substrate followed by western blot. (C and D) Nocodazole-arrested E14 ESCs (200 ng/ml for 10 hr) were treated with the Aurora kinase inhibitors AT9283 (inhibits Aurka and Aurkb), hesperadin (inhibits Aurkb), and MLN8237 (inhibits AurkA). Gradual decreases in p-Oct4(S229) levels with increasing concentrations of Aurkb inhibitors in E14 ESCs were seen by western blot (C). FACS analysis was performed under the same condition (D) (1x104 cells/sample). (E) Coimmunoprecipitation of Oct4 with Aurka and Aurkb from E14 ESCs stably expressing Flag-tagged Aurora kinases. (F) Changes in Oct4 interaction with Aurkb during cell cycle progression. Whole-cell lysates from Flag-Oct4-expressing ZHBTc4 ESCs were pulled down with anti-Flag beads. Bound proteins were immunoblotted with the indicated antibodies. (G) DNA content analysis of Flag-Oct4 expressing ZHBTc4 ESCs by FACS. Flag-Oct4-expressing ZHBTc4 ESCs, treated with nocodazole (200 ng/ml) for 6 hr, were released for 2 and 4 hr and DNA contents were counted (1x104 cells/sample). https://doi.org/10.7554/eLife.10877.006 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 32P-ATP-labeled kinase assay and western blot with anti-p-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 (Figure 1—figure supplement 1E). 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. Protein phosphatase 1 binds Oct4 and dephosphorylates serine 229 in Oct4 in G1 phase When nocodazole treated ZHBTc4 ESCs were released into normal serum, the Aurkb-Oct4 interaction weakened and p-Oct4(S229) levels declined (Figure 2F), indicating that certain phosphatases catalyze the dephosphorylation of p-Oct4(S229) during the M/G1 transition. 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: PP1α, PP1β, and PP1γ. We found that Oct4 interacted more strongly with endogenous PP1β and PP1γ than with PP1α in ZHBTc4 ESCs (Figure 3C). Figure 3 with 1 supplement see all Download asset Open asset PP1 binds and dephosphorylates Oct4 at serine 229 during G1 phase. (A) Sequence alignment of Oct4. Oct4 contains a conserved PP1 docking motif (RVXF). (B) Three-dimensional structure of Oct4 and DNA complex (MMDB ID: 87311) was adapted from the Molecular Modeling Database (MMDB) of NCBI. Each yellow region indicates S229 and an RVWF PP1-binding domain. (C) Coimmunoprecipitation assay revealing the endogenous interaction between Oct4 and PP1 catalytic subunits. Proteins were immunoprecipitated from Flag-Oct4-expressing ZHBTc4 ESCs with Flag antibody, followed by western blot. (D) Changes in Oct4 interaction with PP1 catalytic subunits during cell cycle progression. Whole-cell lysates from Flag-Oct4-expressing ZHBTc4 ESCs were pulled down with anti-Flag beads. Immunoprecipitated proteins were immunoblotted with the indicated antibodies. (E) Purified GST-Oct4(WT) or GST-Oct4(F271A) mutant was incubated with purified (His)6-PP1β and PP1γ and then pulled down with GST beads. Immunoblot shows that PP1β and γ directly bind GST-Oct4(WT). PP1β and PP1γ show weaker interaction with GST-Oct4(F271A) than wild-type Oct4. (F) In vitro phosphatase assay using PP1β or PP1γ with phosphorylated Oct4 as substrate. Okadaic acid (OKA) treatment decreased PP1-mediated dephosphorylation of Oct4. https://doi.org/10.7554/eLife.10877.008 Next, we examined the interaction between Oct4 and PP1 isoforms during the M/G1 transition after nocodazole treatment and release into normal serum (Figure 3D). p-Oct4(S229) disappeared quickly during the M/G1 transition. In parallel, PP1β and PP1γ interacted strongly with Oct4 in G1 phase (2 hr after release). However, PP1α bound weakly to Oct4 regardless of cell cycle stage, suggesting that PP1α might be not a true p-Oct4(S229) phosphatase during the M/G1 transition. Thus, we focused on PP1β and PP1γ with regard to the dephosphorylation of Oct4 in subsequent experiments. We altered phenylalanine-271 to alanine in Oct4 [Oct4(F271A)] by site-directed mutagenesis and measured the in vitro interaction between PP1 and Oct4(WT) or Oct4(F271A) using bacterially purified recombinant (His)6-PP1β, (His)6-PP1γ, GST-Oct4(WT), and GST-Oct4(F271A). (His)6-PP1β and γ bound more robustly to GST-Oct4(WT) but weakly to GST-Oct4(F271A) (Figure 3E), indicating that Oct4 interacts directly with PP1 through a PP1-binding motif (RVWF). 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 PP1β and PP1γ, but not PP1α, dephosphorylated Aurkb-mediated phospho-Oct4(S229) (Figure 3—figure supplement 1B). Further, pretreatment with okadaic aid (OKA), a PP1 inhibitor, blocked the dephosphorylation of p-Oct4(S229) (Figure 3F), indicating that the interaction between Oct4 and PP1 is important for dephosphorylation of phospho-S229 in Oct4. Treatment of E14 ESCs with 50 nM OKA increased p-Oct4(S229) levels after 4 hr (Figure 3—figure supplement 1C). Rising concentrations of OKA (from 0–200 nM) gradually increased p-Oct4(S229) levels after 2 hr of treatment (Figure 3—figure supplement 1D), suggesting that PP1 activity regulates the phosphorylation state of S229 in Oct4. These findings demonstrate that PP1 isoforms have an opposite activity with Aurkb by binding to Oct4 and by dephosphorylating S229 of Oct4 during the M/G1 transition in ESCs. 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 PP1-mediated 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). Figure 4 with 1 supplement see all Download asset Open asset PP1-mediated dephosphorylation of Oct4(S229) correlates with the resetting of pluripotency genes in the next G1 phase. (A) p-Oct4(S229) levels after release of E14 ESCs from M-phase arrest. Shown are immunoblots for the indicated proteins. (B) Nocodazole-treated E14 ESCs were released and analyzed for DNA content by FACS (1x104 cells/sample). (C) OKA treatment retards dephosphorylation of p-Oct4(S229) during the M/G1 phase transition. The experimental strategy is shown (upper panel). The same strategy was applied to (D–F). Whole-cell lysates from E14 ESCs were collected and assessed by western blot. (D) Histogram shows cell cycle state of E14 ESCs without (upper panel) or with (lower panel) OKA treatment. (E) ChIP-qPCR analysis of E14 ESCs with anti-Oct4 in regions of pluripotency-associated Oct4 target genes 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 at pluripotency-associated Oct4 target genes in ESCs during the M/G1 transition with or without OKA. (*p<0.05, **p<0.01, ***p<0.001) (F) Nascent RNA of pluripotency-associated Oct4 target genes from E14 ESCs were collected and analyzed by real-time qPCR during the M/G1 phase transition with or without OKA. Levels of each nascent RNA were normalized by those in asynchronous E14 ESCs. https://doi.org/10.7554/eLife.10877.010 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 (Jang et al., 2012). 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. 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). Figure 5 Download asset Open asset Oct4 regulates cell cycle related genes by direct targeting and resetting during the M/G1 transition. (A) A Venn diagram shows overlapped genes between proximal genes of Oct4 binding sites (green, n=5824; (Whyte et al., 2013)) and downregulated genes (fold changes≤0.75) in ZHBTc4 ESCs after Oct4 depletion by doxycycline treatment for 2 days (red, n=3617; [Boo et al., 2015]). (B) Gene ontology (GO) functional categories for putative Oct4 target genes. Cell cycle related GO functional categories are enriched. (C) RNA-seq reads of Bub1 and Rif1 of E14 ESCs during ESC differentiation upon LIF withdrawal (upper panel; [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. https://doi.org/10.7554/eLife.10877.012 Figure 5—source data 1 Identification of putative Oct4 target genes. https://doi.org/10.7554/eLife.10877.013 Download elife-10877-fig5-data1-v3.xlsx Figure 5—source data 2 Cell-cycle related genes in putative Oct4 target genes. https://doi.org/10.7554/eLife.10877.014 Download elife-10877-fig5-data2-v3.xlsx 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 (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 in
Abstract For cells to exit from pluripotency and commit to a lineage, the circuitry of a core transcription factor (CTF) network must be extinguished in an orderly manner through epigenetic modifications. However, how this choreographed epigenetic remodeling at active embryonic stem cell (ESC) genes occurs during differentiation is poorly understood. In this study, we demonstrate that C-terminal binding protein 2 (Ctbp2) regulates nucleosome remodeling and deacetylation (NuRD)-mediated deacetylation of H3K27 and facilitates recruitment of polycomb repressive complex 2 (PRC2)-mediated H3K27me3 in active ESC genes for exit from pluripotency during differentiation. By genomewide analysis, we found that Ctbp2 resides in active ESC genes and co-occupies regions with ESC CTFs in undifferentiated ESCs. Furthermore, ablation of Ctbp2 effects inappropriate gene silencing in ESCs by sustaining high levels of H3K27ac and impeding H3K27me3 in active ESC genes, thereby sustaining ESC maintenance during differentiation. Thus, Ctbp2 preoccupies regions in active genes with the NuRD complex in undifferentiated ESCs that are directed toward H3K27me3 by PRC2 to induce stable silencing, which is pivotal for natural lineage commitment. Stem Cells 2015;33:2442–2455
Constitutive heterochromatin undergoes a dynamic clustering and spatial reorganization during myogenic differentiation. However the detailed mechanisms and its role in cell differentiation remain largely elusive. Here, we report the identification of a muscle-specific long non-coding RNA, ChRO1, involved in constitutive heterochromatin reorganization. ChRO1 is induced during terminal differentiation of myoblasts, and is specifically localized to the chromocenters in myotubes. ChRO1 is required for efficient cell differentiation, with global impacts on gene expression. It influences DNA methylation and chromatin compaction at peri/centromeric regions. Inhibition of ChRO1 leads to defects in the spatial fusion of chromocenters, and mislocalization of H4K20 trimethylation, Suv420H2, HP1, MeCP2 and cohesin. In particular, ChRO1 specifically associates with ATRX/DAXX/H3.3 complex at chromocenters to promote H3.3 incorporation and transcriptional induction of satellite repeats, which is essential for chromocenter clustering. Thus, our results unveil a mechanism involving a lncRNA that plays a role in large-scale heterochromatin reorganization and cell differentiation.
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.
Cellular skin substitutes such as epidermal constructs have been developed for various applications, including wound healing and skin regeneration. These cellular models are mostly derived from primary cells such as keratinocytes and fibroblasts in a two-dimensional (2D) state, and further development of three-dimensional (3D) cultured organoids is needed to provide insight into the in vivo epidermal phenotype and physiology. Here, we report the development of epidermal organoids (EpiOs) generated from induced pluripotent stem cells (iPSCs) as a novel epidermal construct and its application as a source of secreted biomolecules recovered by extracellular vesicles (EVs) that can be utilized for cell-free therapy of regenerative medicine. Differentiated iPSC-derived epidermal organoids (iEpiOs) are easily cultured and expanded through multiple organoid passages, while retaining molecular and functional features similar to in vivo epidermis. These mature iEpiOs contain epidermal stem cell the populations and retain the ability to further differentiate into other skin compartment lineages, such as hair follicle stem cells. By closely recapitulating the epidermal structure, iEpiOs are expected to provide a more relevant microenvironment to influence cellular processes and therapeutic response. Indeed, iEpiOs can generate high-performance EVs containing high levels of the angiogenic growth factor VEGF and miRNAs predicted to regulate cellular processes such as proliferation, migration, differentiation, and angiogenesis. These EVs contribute to target cell proliferation, migration, and angiogenesis, providing a promising therapeutic tool for in vivo wound healing. Overall, the newly developed iEpiOs strategy as an organoid-based approach provides a powerful model for studying basic and translational skin research and may also lead to future therapeutic applications using iEpiOs-secreted EVs.
Abstract Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly immediately after the transcription termination site. H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal. AKT1 was more effective than AKT2 in phosphorylating H3-T45. Blocking H3-T45 phosphorylation by inhibiting AKT or through amino acid substitution limited RNA decay downstream of mRNA cleavage sites and decreased RNA polymerase II release from chromatin. Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3′ end of DNA damage-activated genes to facilitate transcriptional termination.
2-oxogluatrate and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) was recently revealed to be a proline hydroxylase of RPS23 for translational termination. However, OGFOD1 is nuclear, whereas translational termination occurs in the cytoplasm, raising the possibility of another function of OGFOD1 in the nucleus. In this study, we demonstrate that OGFOD1 is involved in cell cycle regulation. OGFOD1 knockdown in MDA-MB-231 breast cancer cells significantly impeded cell proliferation and resulted in the accumulation of G1 and G2/M cells by decreasing the mRNA levels of G1/S transition- and G2/M-related transcription factors and their target genes. We also confirmed that OGFOD1 is highly expressed in breast cancer tissues by bioinformatic analysis and immunohistochemistry. Thus, we propose that OGFOD1 is required for breast cancer cell proliferation and is associated with poor prognosis in breast cancer.
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