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Originally published In Press as doi:10.1074/jbc.M412224200 on January 19, 2005

J. Biol. Chem., Vol. 280, Issue 13, 12976-12987, April 1, 2005
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Cell Cycle and Developmental Regulations of Replication Factors in Mouse Embryonic Stem Cells*{boxs}

Hiroko Fujii-Yamamoto{ddagger}§, Jung Min Kim{ddagger}, Ken-ichi Arai¶, and Hisao Masai{ddagger}||

From the Departments of {ddagger}Cell Biology and Integrative Life Science, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613 and the §Department of Biochemistry and Molecular Biology, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received for publication, October 28, 2004 , and in revised form, December 28, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Embryonic stem (ES) cells can grow rapidly and permanently while maintaining their differentiation capacity. To gain insight into how the cell cycle progression of undifferentiated murine ES cells is regulated, we have examined the expression patterns of various replication and cell cycle regulators. Most factors including cyclins, Cdc6, and geminin are rather constitutively expressed during the cell cycle of ES cells. Furthermore, the transcript levels of almost all the cell cycle regulators we investigated except for p21 and p27 are higher in undifferentiated ES cells than in murine embryonic fibroblasts (MEFs), and the increased stability of mRNA in ES cells may be partially responsible for this at least with some of the factors. More strikingly, the transcriptional levels of these factors are strongly correlated with the acetylated state of histone H3 at their promoter regions. However, the methylation state of histone or CpG methylation of the promoter region is not generally correlated significantly with the expression pattern of these factors in both cell types. On the protein level, Cdc6, ASK, cyclin A2, and cyclin B1 are extremely abundant in ES cells compared with MEFs. Furthermore, they are rapidly down-regulated upon induction of differentiation of ES cells. The significance of these findings is discussed in relation to the unusual proliferative properties of ES cells in an undifferentiated state.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Murine embryonic stem (ES)1 cells are pluripotent cell lines established from the inner cell mass of the blastocysts, which are thought to be the in vitro counterparts of epiblast. It has been well documented that ES cells show unusual proliferative features (1). Although they retain a normal diploid karyotype, its growth property resembles that of transformed cells; the growth of ES cells are insensitive to serum starvation, and their proliferation is not restricted by contact inhibition or anchorage dependence. Furthermore, their cell cycle properties are also unique in that ES cells devote more than a half of their entire cycle to S phase and has short gap phases, which explains their rapid proliferation rate (2, 3). These unusual properties of the ES cell cycle may be related to their ability to maintain the undifferentiated state.

In mammalian somatic cells, cell cycle progression is tightly regulated by a series of phosphorylation events, which involves Cdks and Dbf4/ASK-dependent Cdc7 kinase (4–7). At G1 to S phase transition, Cdk4/6-cyclin D and Cdk2-cyclin E phosphorylate Rb and liberate E2F (8, 9). The resulting activation of E2F promotes transcription of various cell cycle regulators, including cyclin E1 (10–12), cyclin A2 (13), Cdc6 (14–16), and ASK (17), an activation subunit of mammalian Cdc7 kinase. Preceding this process, preRC (prereplicative complex), composed of ORCs, Cdc6, and MCMs, is assembled onto chromatin during late M to early G1 phase (18, 19). Phosphorylation of MCM components by Cdk and Cdc7-ASK (20, 21) may activate preRC for initiation of replication by facilitating the conformational change of the MCM complex to promote its helicase activity and/or interaction with other replication factors.

However, in ES cells, the situation seems significantly different. Recent studies on ES cell cycle regulation revealed that activities of Cdks are robust throughout the cell cycle, and cell cycle oscillation of the kinase activity was detected only with cyclin B/Cdc2 (3). Rb is constitutively in the phosphorylated state (2), and E2F target genes are actively transcribed throughout the cell cycle (3). On the other hand, conditional knock-out of Cdc7 in ES cells resulted in growth arrest at S phase DNA content (6), strongly suggesting that Cdc7-ASK plays an essential role in the progression of ES cell cycle.

In this report, we have characterized the expression and chromatin binding patterns of various factors involved in regulation of DNA replication, especially the components and activators of preRC complex, during the cell cycle of undifferentiated ES cells. To further explore the molecular mechanisms underpinning the proliferative feature of undifferentiated ES cells, we also compared the expression of these replication regulators between ES and differentiated somatic cells at both protein and mRNA levels. We found that all the factors we have investigated except for Cdk inhibitors are highly transcribed in ES cells, and that elevated transcriptional activity is strongly correlated with the histone acetylation status of their promoter region. However, at the protein level, only a subset of the factors are highly overexpressed in ES cells, which include Cdc6, ASK, cyclin A2, and cyclin B1. The expression levels of these factors sharply decreased upon induction of differentiation. These results will be discussed in relation to the specific mode of cell cycle regulation of ES cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Synchronization—ES cell lines, CCE28 and D3, were cultured on gelatin-coated culture dishes without feeder layer. The culture medium consisted of Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 0.1 mM {beta}-mercaptoethanol, and mouse leukemia inhibitory factor (LIF, ESGRO; 104 units/ml for cell maintenance and 103 units/ml for synchronization or drug treatment). Primary mouse embryonic fibroblasts (passage 2) and mouse fibroblast cell line L929 were maintained in DMEM supplemented with 10% fetal bovine serum and 2 mM L-glutamine.

For synchronous release from M phase arrest, ES cells were first treated with 2.5 mM thymidine (Sigma) for 12 h, then washed twice with prewarmed PBS and incubated in the presence of 150 ng/ml of TN-16 (Biomol) for 5 h. These cells were washed with PBS and then released into prewarmed medium for the indicated time. For release from G1/S phase, cells were first treated with 2.5 mM thymidine (Sigma) for 12 h, then washed twice with prewarmed PBS and released into prewarmed medium for 6 h. These cells were treated with 0.5 mM mimosine for 6 h, and then, after being washed with PBS, the G1/S-arrested cells were released into prewarmed medium for the indicated time. For synchronization of L929 cells from M phase arrest, they were first treated with 2.5 mM thymidine for 15 h then released in the medium containing 150 ng/ml of TN-16 for 8 h. These cells were synchronously released in the prewarmed medium as described above. Other synchronization protocols were described in the legend. For in vitro differentiation of ES cell line CCE28, cells were cultured in the medium without LIF, which was supplemented with 1 µM retinoic acid (Sigma) for indicated periods.

Inhibitors—MG132 (proteasome/calpain inhibitor, Calbiochem) and ALLN (calpain inhibitor, Sigma) were used at 10 µM. cycloheximide (50 µg/ml, Calbiochem) and actynomicin D (5 µg/ml, Calbiochem) were used to inhibit mRNA or protein synthesis, respectively.

Antibodies—The rabbit polyclonal antibody against murine ASK was developed against His-tagged full-length ASK protein generated in Escherichia coli and affinity-purified with GST-ASK fusion protein. The rabbit antisera against murine MCM4 was developed against GST-MCM4 fusion protein (based on the plasmid originally constructed by Hiroshi Kimura). Antibodies against cyclin E1 (sc-481), cyclin A2 (sc-596), cyclin B1 (sc-245), Cdc6 (sc-9964), Cdk2 (sc-6248), Cdc2 (sc-954), PCNA (sc-56), MCM2 (sc-9839), geminin (sc-13015), Lamin B (sc-6217) were from Santa Cruz Biotechnology, anti-MCM5 and anti-MCM7 antibodies from Katsuyuki Tamai (MBL), and anti-tubulin antibody (B-5-1-2) from Sigma. For chromatin immunoprecipitation, anti-acetylated histone H3 antibody (06-599), and anti-dimethylated histone H3K9 antibody (ab7312) were purchased from Upstate and Abcam, respectively.

Immunoblot Analyses—Cells were collected with trypsinization and washed in ice-cold PBS. To prepare whole cell lysates, cell pellets were resuspended in IP lysis buffer (50 mM HEPES-KOH, pH 7.6, 150 mM NaCl, 2.5 mM EGTA, 1 mM EDTA, 0.5% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM Na3VO4, 50 mM NaF, 1 tablet of complete, EDTA-free protease inhibitor mixture (PI tablets, Roche Applied Science)/50 ml) and were sonicated for 10 pulses at output 3 and 50% duty cycle of Branson Sonifier 250. Lysates were centrifuged at 15,000 rpm for 10 min at 4 °C and protein concentrations were determined by Bradford assay using bovine serum albumin as a standard (Bio-Rad). To fractionate cells into Triton X-100-soluble and insoluble fractions, CSK buffer extraction was performed as described previously (22) with minor modifications; cells were lysed on ice for 5 min in CSK buffer containing 0.1% Triton X-100, 100 mM NaCl, 1 mM Na3VO4, 50 mM NaF, and PI tablets. Soluble fractions (30 µg) and insoluble fractions of equivalent numbers of cells were run on SDS-PAGE and transferred onto polyvinylidene difluoride membranes for immunoblot analysis.

Northern Blot Analysis—Total RNA from ES cells and MEFs were extracted using TRIzol reagent (Invitrogen, Life Technologies, Inc.). RNA (15 µg) was separated on 1% agarose-formaldehyde gel and was transferred onto Hybond-N+ membranes (Amersham Biosciences). Probes were generated by RT-PCR using the primer sets shown in Table SI (see Supplemental Materials). These primer pairs were designed to amplify the regions including intron to avoid contamination of the DNA amplified from genomic DNA. After the prehybridization of membranes in prehybridization solution (6x SSPE, 5x Denhardt's solution, 0.5% SDS, 50% formaldehyde, and 100 µg/ml denatured DNA (Nacalai)) for at least 3 h, 32P-labeled probes (prepared using the Megaprime DNA labeling system from Amersham Biosciences) were added and hybridization was conducted at 42 °C for overnight. Membranes were washed in 1x SSC, 0.1% SDS at room temperature for 20 min, and then washed three times in 0.2x SSC, 0.1% SDS at 68 °C for 20 min each.

Semi-quantitative RT-PCR—RNA (5 µg) from ES cells and MEFs were converted to cDNA with expand reverse transcriptase (Roche Applied Science) following the manufacturer's instructions. 1:40 volume of reverse transcription mixtures were used to amplify each cDNA fragment with primer pairs shown in Table SI (Supplemental Materials). All the cDNA fragments except for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using TaqDNA polymerase (Sigma) with 15 cycles of 94 °C for 30 s, 66 °C for 30 s, and 72 °C for 15 s. The GAPDH cDNA fragment was amplified with 10 cycles of the same PCR condition. Under this condition, the target cDNA fragments were amplified linearly at least up to 20 cycles (data not shown). Amplified DNA fragments separated on an agarose gel were detected by Southern blotting using the same cDNA fragments as probes which were 32P-labeled as above.

Chromatin Immunoprecipitation (ChIP)—ChIP analyses were performed as described previously (23) with minor modifications. ES cells and MEFs were trypsinized and cross-linked in cell culture medium containing 1% formaldehyde at room temperature for 10 min. After the reaction was stopped by addition of glycine to a final concentration of 0.125 M and was incubated for 5 min, 1 x 107 cells were washed in PBS and resuspended in 400 µl of SDS buffer (100 mM NaCl, 50 mM Tris-Cl, pH 8.1, 5 mM EDTA, and 0.5% SDS), supplemented with PI tablet, and stored at -80 °C. For sonication, 200 µl of Triton dilution buffer (100 mM Tris-Cl, pH 8.6, 100 mM NaCl, 5 mM EDTA, and PI tablet) was added, and samples were processed with the Branson sonifier 250 for 50 s at output 5 and 100% duty cycle to yield the average DNA length of 800 bp. The chromatin solution was adjusted to 5 x 106 cells/500 µl in IP buffer (2:1 mixture of SDS buffer and Triton dilution buffer) for each sample, and were precleared by centrifugation at 15,000 rpm for 20 min with protein A-Sepharose beads. Then, the supernatant was subjected to immunoprecipitation with 1 µg of antibody. Immunoprecipitates were washed and eluted as described (9). Eluted DNA, after ethanol precipitation, was resuspended in 200 µl of water and 10 µl was used for PCR (26–33 cycles). Linear range for detection with each primer set was predetermined using diluted input DNA. All the experiments were conducted at least twice with independently immunoprecipitated samples. The primer sets used are shown in Table SII (Supplemental Materials). The primer sets for the promoters of p21, p27, and Oct-3/4 were the same as previously described (24, 25).

Methylation Assays—Genomic DNA (2 µg) from undifferentiated ES cells, retinoic acid-treated ES cells, MEFs, and tail were digested with HpaII or MspI, and 5 ng of the resulting digest were used for PCR. The primer sets used in this study were the same as those used in the ChIP assay (Table SII) or otherwise shown in Table SIII (Supplemental Materials).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Cycle-dependent Expression of Replication Regulators in Murine Embryonic Stem Cells—To analyze the cell cycle regulation of factors regulating DNA replication of ES cells, we arrested the CCE28 ES cell line at the G2/M with TN-16 treatment following release from double thymidine block and then synchronously released into cell cycle (Fig. 1A). On the basis of the FACS analyses of DNA content, it was estimated that these cells spend ~1 h in G2/M, another 1 h in G1, and 7 h in S phase. This cell cycle profile is consistent with previous reports on different murine ES cell lines. We have fractionated the cells into Triton-soluble and -insoluble fractions (22); the former contains both cytoplasmic and nuclear soluble proteins and the latter contains nuclear insoluble proteins including chromatin-bound ones (Fig. 1B).



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  		FIG. 1.
Expression of replication and cell cycle regulators during synchronized cell cycle of ES cells. A, C, and E, ES cells CCE28, synchronously released from TN-16-mediated M phase arrest (A), ES cells, CCE28, and D3, synchronously released from mimosine-induced G1/arrest (C), or L929 cells synchronously released from TN-16-mediated M phase arrest (E), were harvested at the times indicated after release. DNA contents of the PI-stained cells were analyzed by FACS. B, D, and F, extracts, fractionated with CSK buffer from equivalent numbers of cell cycle-synchronized cells shown in A, C, and E, respectively, were analyzed by Western blotting using the antibodies against the proteins indicated. Stages of the cell cycle are indicated above the panels. S and P represent Triton X-100-soluble and -insoluble fractions, respectively.

 
Cdc6 protein was detected mostly in the Triton-insoluble fraction throughout the cell cycle, and its level was rather constant. The mobility-shifted form observed at time 0 (immediately after release from TN-16 arrest), was likely to be caused by the phosphorylation, because it disappeared upon phosphatase treatment (data not shown). ASK, the activation subunit of Cdc7 kinase, was more enriched in the Triton-soluble fraction throughout the cell cycle. It was not detected in the Triton-insoluble fractions at the time of release, and reappeared in the chromatin-enriched fraction within 1 h from the release. ASK protein appeared as a series of multiple bands generated by phosphorylation, since {lambda} phosphatase treatment resulted in disappearance of all the shifted bands and convergence into a single band of ~75 kDa (see Fig. 6A). It was previously reported that transcription of ASK in somatic cells was induced when they were released from serum starvation and progressed into S phase (26). Our results indicate that ASK protein in ES cells dissociates from chromatin during G2/M phase and reassociates at G1/S phase, although the expression level of the total protein is rather constant during the cell cycle. MCM2 and MCM4 proteins were also not detected in the chromatin-enriched fractions at the time of release, but were loaded onto chromatin within 1 h after release. The levels of the chromatin-bound MCM proteins gradually decreased as the S phase proceeded. MCM2 appeared as a doublet in Triton-soluble fractions, the lower of which represents phosphorylated forms. The level of this mobility-shifted form decreased as cells entered S phase, and increased again at late S through G2 phase. A portion of MCM4 in the Triton-soluble fractions also became mobility-shifted because of phosphorylation during G2/M-early G1 phase. Similar phosphorylation-induced mobility shift of MCM4 protein during G2/M phase was previously observed in human somatic cells (27). The level of chromatin-bound PCNA protein increased during the S phase, consistent with its association with the sites of DNA replication (28, 29).



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  		FIG. 6.
Cdc6 is destabilized in differentiated cells. A, ES cells and MEFs were treated with 50 µg/ml of CHX for increasing times, and 15 µg of the whole cell lysates were subjected to Western blot analysis with antibodies indicated. +PPase indicate that the lysates were pretreated with {lambda}-phosphatase (New England BioLabs, 30 units for 30 min). For MEFs, the blot with five times more lysate is also shown (indicated as MEF (x5)). B, intensities of the bands in A are quantified, and the values relative to that at time 0 are presented for each gene. Data shown are the averages of two independent experiments. C, ES cells and MEFs were treated with 10 µM MG132 (M) or ALLN (A) for 6 h. Whole cell lysates (5 µg) were subjected to Western blot analysis with antibodies indicated. For MEFs, the blot with ten times more lysate are also shown (indicated as MEF (x10)). D, intensities of the bands in C were quantified and the ratio of drug-treated/untreated samples are presented for Cdc6 with each inhibitor. Data shown are the averages of two independent experiments.

 
It was previously reported that cyclin B1 is the only cyclin the level of which markedly fluctuates during the cell cycle of ES cells (3). We observed rather moderate fluctuation of cyclin B1 protein, which was abundant at the time of TN-16 arrest, slightly decreased during G1/S phase and reincreased at G2/M phase. Cyclin A2 also weakly fluctuated in a manner similar to cyclin B1. On the other hand, we did not detect significant change of cyclin E1 protein level, consistent with the previous report (3). The conflict with previous reports could be caused by the difference of the synchronization protocols. Therefore, we synchronized ES cells by release from mimosine-arrest at G1/S phase. In this experiment, we used two different ES cell lines, CCE28 and D3, and pattern of cell cycle oscillation of various factors were examined (Fig. 1, C and D). Basically, we obtained almost identical results with the two cell lines. Consistent with above results, the levels of chromatin-bound ASK protein increased during early S phase, and decreased as cells completed S phase and entered G2 phase. We also noticed that the extent of the mobility shift of ASK decreased during S phase, and reincreased at G2 phase, which may be caused by phosphorylation by Cdc2. Cdc6 protein level did not change much during the cell cycle and was detected mostly in the chromatin bound fraction. Cyclin A2 and cyclin B1 protein levels did not significantly change during this synchronization procedure. Total MCM2 protein level was constant, but that in the Triton-insoluble fraction decreased and the mobility-shifted phosphorylated form increased in the Triton-soluble fraction, as S phase progressed. These results are consistent with those of cells synchronized from G2/M block.

We also examined the cell cycle profile of expression of these factors in mouse fibroblast cell line L929, which were synchronously released from M-phase arrest (Fig. 1E). Cells completed M phase within 2 h after release, and entered S phase at about 4 h after release. S phase was completed by 10 h after release and entered next G1 phase by 12 h. In this cell line, cyclin B1, detected mostly in Triton-soluble fraction, significantly fluctuated during the cell cycle and peaked in G2/M phase. Cyclin A2 weakly fluctuated, increasing in late S to G2/M phase. Geminin also disappeared in G1 phase and reappeared during S to G2/M phase, consistent with previous report. Thus, the levels of these factors oscillated to a larger extent than in ES cells. However, the protein levels of ASK and Cdc6 appeared to be almost constant during the cell cycle. In this cell line, the cyclin E1 protein level only slightly increased during G1 and the extent of oscillation was much smaller. The level of phosphorylation of some of these factors (MCM2, ASK, and Cdc6) was enhanced during G2/M as indicated by the extent of mobility shift. The majority of MCM2 and ASK was in the Triton-soluble fractions at the time of TN-16 arrest, but a significant fraction was detected in Triton-insoluble fractions within 2 h after release. These changes were similar to what were observed in ES cells. Thus, the nearly constitutive expression of cell cycle and replication factors during cell cycle appears to be a unique characteristic of undifferentiated ES cells.

ASK, Cdc6, Cyclin A2, and Cyclin B1 Proteins Are Extremely Overexpressed in Undifferentiated ES Cells—To investigate the factors responsible for cell cycle regulation specific to undifferentiated ES cells, we compared the expression patterns of replication factors between ES and MEFs. The protein levels of various factors involved in DNA replication were determined in asynchronously growing ES and MEFs by Western blotting using specific antibodies. Among the proteins examined, ASK, Cdc6, cyclin A2, and cyclin B1 proteins were expressed at a high level in ES cells, whereas they were expressed at a much lower level in MEFs. On the other hand, MCM2, MCM3, MCM4, MCM7, cyclin E1, Cdk2, and Cdc2 were expressed at a similar level in both cells. PCNA, MCM5, and geminin were expressed in ES cells at a level slightly higher than that in MEFs (data not shown). In agreement with the recent report, cyclin D3 was expressed at a similar level in undifferentiated ES cells and MEFs (30), whereas cyclin D1 and D2, expressed at a high level in MEF, were expressed only at a very low level in ES cells, consistent with low or very little transcription of these genes in ES cells (30, 31). The Cdk inhibitors p21 and p27 were not expressed to a detectable level in ES cells, but at a significant level in MEFs (3).

Because cell cycle profile of asynchronously growing ES and MEFs were different, the observed differential expression of cell cycle factors could be caused by difference of cell cycle distribution. To examine this possibility, we synchronized ES cells at G1 phase by mimosine treatment or release from TN-16 arrest, which resulted in cell populations with DNA content similar to that of asynchronously growing MEFs (Fig. 2B). Under these conditions as well, the levels of the four proteins (Cdc6, ASK, cyclin A2, and cyclin B1) are much higher in ES cells of any cell cycle population than in MEFs, indicating that they are intrinsically overexpressed in ES cells (Fig. 2C).



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  		FIG. 2.
ASK, Cdc6, cyclin A2, and cyclin B1 proteins are overexpressed in ES cells. A, ES cells and MEFs were fractionated as in Fig. 1. Soluble (S) and insoluble (P) fractions of cells were separated on SDS-PAGE and immunoblotted with antibodies indicated. B, cell cycle profiles of asynchronously growing CCE28 ES cells (ES/Asyn), ES cells treated with 0.5 mM mimosine for 12 h (ES/Mim), ES cells released for 1 h after the TN-16 arrest as described in Fig. 1 (ES/TN->R), and asynchronously growing MEFs (MEF/Asyn) were analyzed by FACS. The percentage of the G1/S, S, and G2/M phase populations were estimated by CELL Quest software (BD Biosciences). C, fractionated extracts from the cells shown in B were immunoblotted as described in A.

 
Decreased Expression of Replication Factors in Response to Differentiation—Mouse ES cells can be induced into differentiation upon removal of leukemia inhibitory factor (32, 33). We have examined the expression of the critical replication factors after induction of differentiation by removing LIF from and adding retinoic acid to the medium. Upon induction of differentiation, population of the cells in G1/S phase significantly increased, resulting in cell cycle profiles similar to MEFs (Fig. 3A). Oct-3/4, a transcription factor known to be specifically expressed in undifferentiated ES cells (34), is down-regulated within 2 days after addition of retinoic acid (Fig. 3B). Similarly, the protein levels of ASK and Cdc6 were below detection at 6 days after induction of differentiation. The cyclin A2 and B1 proteins were also significantly down-regulated under the same conditions. On the other hand, MCM2 protein stayed at a relatively constant level, although the amount of the shifted, phosphorylated band detected mainly in the soluble fractions decreased. This may reflect decreased Cdk and ASK-Cdc7 activities upon differentiation or may simply be because the G1 phase population decreased. On the other hand, p21 and p27 were significantly up-regulated during the same treatment. These results indicate that expression of Cdc6, ASK, cyclin A2, and cyclin B1 proteins is under the regulation of the differentiation state of the cells, and their overexpression is correlated with the undifferentiated state of ES cells.



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  		FIG. 3.
ASK, Cdc6, cyclin A2, and cyclin B1 proteins are down-regulated upon in vitro differentiation of ES cells. A, ES cells CCE28, cultured in the presence of 104 units/ml of LIF (D0), were induced into differentiation by removing the LIF and adding 1 µM retinoic acid for 2 days (D2) and 6 days (D6), then DNA contents of these cells as well as MEFs (MEF) were analyzed by FACS. B, Triton-soluble (S) and -insoluble (P) fractions prepared from the equivalent numbers of the cells in A were analyzed by Western blotting with the antibodies specific to the proteins indicated.

 
Transcription Levels of Many Cell Cycle Regulators Are Elevated in ES Cells—In order to examine whether the elevated levels of these replication factors in ES cells are caused by regulation at the transcriptional level, we examined the transcription levels of various cell cycle regulators in asynchronously growing ES cells and MEFs (Fig. 4). Unexpectedly, all the mRNAs we have tested except for p21 and p27 were more abundantly expressed in ES than in MEFs regardless of its expression profile at the protein level. Some of these genes such as MCMs (35), ASK (17), Cdc6 (14–16), cyclin E1 (10, 11), cyclin A2 (13), Cdc2 (36), PCNA (37), and Cdc45 (38) are known to be targets of the E2F transcription factor, and others (cyclin B1 and Cdk2) are not. In contrast, transcription of p21, which is reported as an E2F target and whose promoter activity is enhanced by E2F1 and E2F3 (39, 40), was repressed in ES cells. Thus, although E2F may be constitutively active because of the absence of the hypophosphorylated form of Rb in ES cells, transcriptional regulation of replication factors in undifferentiated ES cells may involve additional factors.



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  		FIG. 4.
Northern blot analyses of replication/cell cycle regulators in ES cells and MEFs. Total RNA (15 µg each) from ES cells and MEFs were resolved by agarose gel electrophoresis and subjected to Northern blot analyses with indicated probes. Ethidium bromide staining of the gel, showing rRNA, is presented to indicate that equal amounts of RNA are loaded.

 
Stability of mRNAs May Play a Role in Enhanced Expression of Replication Proteins and Cell Cycle Regulators in ES Cells— The abundance of mRNA may be determined by the rate of transcription as well as by its stability (41, 42). Expression of some replication factors including cyclin A2 and B1 are known to be regulated at the post-transcriptional level by the RNA-binding protein Elav (embryonic lethal abnormal visual)/HuR (43, 44). This protein binds to AU-rich elements in the 3'-untranslated regions of target mRNAs and protects them from degradation (45). The HuR protein level and the half-lives of its target mRNAs are lower in replicative senescent cells, and overexpression of HuR in fibroblasts resulted in attenuation of its senescent phenotype (46), strongly suggesting that stabilization of certain mRNAs could affect the proliferation of the cells. As in the case of cyclin A2 and B1, which were overexpressed in ES cells, ASK also possesses AU-rich sequences in the 3'-untranslated region of its mRNA. Furthermore, microarray experiments revealed that mRNA encoding HuR is upregulated in ES cells compared with MEFs.2 Therefore, stabilization of their mRNAs by HuR could also explain abundant expression of some of the transcripts. To test this possibility, we treated ES cells and MEFs with actinomycin D, a specific inhibitor of transcription, and measured the stability of these mRNAs (Fig. 5, A, left panels and B). As expected, mRNAs encoding cyclin A2 and cyclin B1 were more stable in ES cells than in MEFs. Consistent with the presence of the HuR target sequence, mRNA for ASK was also stabilized in ES cells compared with MEFs. Unexpectedly, the stability of mRNA encoding Cdc6, which does not possess apparent AU-rich elements, also slightly increased in ES cells. In control, the stability of glyceraldehyde-3-phosphate dehydrogenase mRNA was similar between the two cells. We also conducted similar experiments using {alpha}-amanitin, a specific inhibitor for RNA polymerase II, and obtained results indicating that mRNAs for all these factors are more stable in ES cells than in MEFs (Fig. 5, A, right panels and B). Thus, the results suggest that the mRNAs for multiple cell cycle and replication regulators may be stabilized in ES cells in a manner dependent on an AU-rich element and/or under a different mechanism.



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  		FIG. 5.
mRNA encoding replication/cell cycle regulators are stabilized in ES cells. A, ES cells and MEFs were treated with 5 µg/ml of actinomycin D or 20 µg/ml of {alpha}-amanitin (right panels) for increasing times (0.5, 1, 3, 6, and 10 h). Each mRNA was detected by semiquantitative RT-PCR followed by hybridization with cDNA probes indicated. S and L represent the short and long exposures, respectively, of the same gels. B, intensity of each band was quantified, and the values relative to that at time 0 are presented for each gene. The data represent the averages of three independent experiments for actinomycin D-treated cells (left) and those of two for {alpha}-amanitin-treated cells (right).

 
Stability of Replication Proteins in ES and MEFs—Although there was significant difference in the abundance of mRNA encoding replication factors between ES and MEFs, it was not able to fully explain the difference in the protein level. To further investigate the molecular mechanisms underlying the overexpression of certain replication proteins in the ES cells, we next tested the possibility of selective degradation and/or stabilization of the polypeptides. To evaluate protein stability, asynchronously growing ES and MEFs were treated with 50 µg/ml of cycloheximide, an inhibitor of protein synthesis, for increasing time periods and then were subjected to immunoblot analysis (Fig. 6A). The results revealed no significant difference in the stability of Mcm2, ASK, cyclin A2, and cyclin B1 proteins between ES cells and MEFs (Fig. 6B). However, Cdc6, which was reported to be a rather unstable protein in somatic cells (47, 48), was significantly more stable in ES cells than in MEFs. Although the estimated half-life of Cdc6 in MEFs was 2 h, roughly consistent with previous reports (48), that in ES cells was ~8 h.

Next, the roles of degradation pathways on Cdc6 was examined by using a proteasome/calpain inhibitor MG132 and a calpain inhibitor ALLN (Fig. 6, C and D). Cdc6 was stabilized by MG132 in MEFs to a level greater than in ES cells, indicating the possibility that Cdc6 is selectively degraded by the proteasome pathway in differentiated cells, which may be at least partially responsible for differential expression of Cdc6 protein in ES and MEFs.

Transcription of Replication Proteins Is Regulated by Epigenetic Modification—Epigenetic regulation of gene expression is critical for the control of cell proliferation and differentiation (49–51). Among them, acetylation of lysines 9 and 14 of histone H3 are known to be associated with transcriptional activation, while deacetylation of these residues or methylation of lysine 9 correlates with transcriptional repression (50, 52). To investigate epigenetic control of chromatin structures of replication/cell cycle factors, we have conducted chromatin immunoprecipitation assays using antibodies specific to acetylated H3K9/K14 and dimethylated H3K9 (Fig. 7, A and B). The promoter region of Oct-3/4, the transcription factor specifically expressed in undifferentiated ES cells was used as a control because it is known to be regulated by these modifications (25, 53).



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  		FIG. 7.
Epigenetic modifications of the promoter regions of the replication/cell cycle regulators. A and B, chromatins prepared from ES cells and MEFs were immunoprecipitated with antibodies specific to the acetylated histone H3K9/K14 (A) or with that specific to dimethylated histone H3K9 (B). The resulting immunoprecipitates were used for amplification with primer pairs designed to detect the promoter region of each gene. Input indicates PCR reactions containing 0.125% of the total amount of chromatin used in immunoprecipitation. Control reactions lacking antibody (-Ab) or chromatin (Mock) are also shown. Each experiment was performed at least two times with the same conclusion. Only representative data are shown. C, genomic DNA (2 µg) from undifferentiated ES cells, retinoic acid-treated ES cells, MEFs, and tail were digested with indicated restriction enzymes, and 5 ng were used for PCR with the primer sets designed for each gene indicated. ND indicates PCR reactions on the undigested DNA. The promoter region of {alpha}-tubulin does not possess HpaII/MspI sites. The data for ASK and cyclin A2 are missing in C, because these promoter segments were not cleaved with MspI, possibly because of secondary structure of DNA in these regions.

 
The results indicate that histone H3K9/K14 is strongly acetylated in the promoter segment of Oct-3/4 in ES cells, whereas its acetylation level is significantly decreased in MEFs (Fig. 7A). Similarly, the promoter regions of Mcm2, Mcm5, ASK, Cdc6, cyclin A2, and cyclin B1 genes, which were actively transcribed in undifferentiated ES cells, were associated with acetylated H3K9/K14 in ES cells but not in MEFs (Fig. 7A). In contrast, on the promoter regions of p21 and p27, actively transcribed in MEFs but not in ES cells, acetylated H3K9/K14 was accumulated in MEFs but not in ES cells. Thus, active transcription appears to be always associated with acetylation of H3K9/K14.

We next investigated the methylation of histone H9 on these genes (Fig. 7B). Consistent with a previous report, the DNA segment for the promoter region of Oct-3/4 was precipitated by anti-dimethylated H3K9 antibody in MEFs but not in ES cells. However, the promoter segments of any replication/cell cycle factors examined, except for ASK, were not significantly precipitated by the antibody in either cells. This result indicates that histone methylation may not play a major role in repression of most of the replication/cell cycle factors in MEFs.

Because histone deacetylases are known to form transcription repressor complex with methyl-CpG-binding proteins and/or DNA methyltransferases (54–58), we next investigated the methylation status of cytosines in the CpG islands present in these promoter regions. Most of the promoter sequences examined are associated with GC-rich regions, which often overlap with the E2F and SP-1 sites. We performed a PCR-based methylation assay in which genomic DNA is cleaved by HpaI or MspI (both recognizing CCGG), and the segment spanning the target sequences is amplified. MspI cleaves both methylated and unmethylated CCGG, resulting in loss of amplification. HpaI, on the other hand, does not cleave the methylated segments, and therefore, the fragments are amplified if the sequences on them are methylated. Consistent with previous reports, the promoter region of Oct-3/4 was not methylated in undifferentiated ES cells, while the level of methylation increases as the cells undergo differentiation and a high level of methylation was indicated in MEFs or tail cells (25, 53). In contrast, the promoter regions of replication/cell cycle genes appear to be unmethylated in undifferentiated ES cells, retinoic acid-induced differentiated cells, or MEFs. However, they were methylated at a high level in genomic DNA derived from the mouse tail (Fig. 7C). These results indicate that cytosine methylation of the promoter regions of cell cycle regulators may not occur during differentiation of ES cells or in early developmental stage, but it occurs extensively in terminally differentiated cells such as in the tail.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Embryonic stem cells are capable of self-renewal as well as of differentiation into any cell types given an appropriate signal. Since proliferation and differentiation could be on the opposite sides of a coin, tautipotency of ES cells may be intimately linked to their growth properties. The most unique feature of cell cycle progression of undifferentiated mouse ES cells is very short gap phases (G1 and G2) (2, 3). Furthermore, their proliferation is not restricted by serum starvation or contact inhibition (1). In this report, we have conducted a detailed characterization of various replication/cell cycle factors during the cell cycle of mouse ES cells and compared their properties with those of differentiated cells.

Cell Cycle Regulation of Replication Factors in Mouse ES Cells—Our analyses of replication and cell cycle regulators during synchronized cell cycle of mouse ES cells revealed the presence of a more or less constant amount of most of the factors throughout the cell cycle. Cyclin E1, cyclin A2, and cyclin B1 proteins were present at all the cell cycle stages and did not show striking fluctuation, whereas the level of these cyclins oscillated in L929 cells, as expected. We also did not detect significant cell cycle oscillation of kinase activities dependent on these three cyclins in ES cells (data not shown).

Cell cycle-specific protein degradation also plays a major role in the regulation of the cell cycle. In ES cells, however, proteasome-dependent degradation of Cdc6, which normally occurs in the early G1 phase of somatic cells (48, 59), did not seem to take place. However, cell cycle-dependent phosphorylation and chromatin association/dissociation of replication factors indeed occurred in ES cells in a manner similar to that in differentiated cells (as were observed with Cdc6, MCM, and ASKs). Therefore, cell cycle-regulation of phosphorylation events in ES cells is similar to that in differentiated cells. We noticed that the phosphorylated forms of MCM2 and MCM4 (detected by phosphopeptide-specific antibodies recognizing Cdk or Cdc7 recognition sites) were much more abundant in ES cells than in MEFs, while the total protein levels were similar in both cells (data not shown). This may reflect the elevated activities of these kinases in ES cells because of abundant expression of cyclin and ASK molecules.

Transcriptional Activities of Replication/Cell Cycle Factors in ES Cells Are Regulated by Histone and DNA Modifications— Northern blot analyses indicate that the transcriptional levels of most replication/cell cycle factors were enhanced in ES cells compared with MEFs. It has already been reported that hypophosphorylated Rb is absent during the cell cycle of ES cells (2), and most E2F exists as free forms (3). We showed that not only E2F targets but also non-targets such as cyclin B1 and Cdk2 were also transcriptionally up-regulated in ES cells. Moreover, p21, a known target of E2F, is not actively transcribed in ES cells. Therefore, transcriptional regulation of replication/cell cycle factors in ES cells may be influenced by more general mechanisms.

Our data indicate that the chromatin structures regulated by histone acetylation play important roles in this process, since the acetylation/deacetylation patterns of histone H3K9/K14 at the promoter regions of various factors examined strongly correlated with their activated/repressed status, respectively, of transcription. It is interesting to note that p300/CBP, a general transcriptional coactivators, was identified as one of the targets of the Wnt signaling pathway in human embryonic carcinoma (60), which is involved in maintaining the undifferentiated state of the stem cells (61). The inability of Rb to interact with E2F because of its hyperphosphorylation in ES cells may facilitate the maintenance of the hyperacetylated state of the histones located proximal to the E2F-regulated promoters, since Rb is known to recruit histone deacetylases to the E2F target promoters (62, 63).

On the other hand, methylation of histone H3K9 is known to be involved in transcription repression of Oct-3/4 during ES cell differentiation (25). Moreover, this modification is also reported to play some roles in transcriptional silencing of the cell cycle regulators such as cyclin E1 and cyclin A2 during the course of the cellular senescence of fibroblasts (64) or terminal differentiation of myoblasts (65). However, the methylation status did not appear to change between ES and MEFs in all the regulators except for ASK. The ASK promoter region is extremely rich in GC, and appears to be regulated also by methylation of H3K9 during early differentiation. The dual regulation of ASK expression by acetylation and methylation may suggest its distinct roles in maintenance and regulation of undifferentiated state of ES cells.

Cytosine methylation plays a role in establishing silent chromatin and is known to be involved in differentiation-induced silencing of Oct-3/4 (25, 53, 66). Furthermore, in some cancer cells, transcription of Cdk inhibitors such as p21 and p27 are silenced by cytosine methylation (67–69). However, we did not detect methylation in the promoter regions of replication and cell cycle regulators during the course of ES cell differentiation or in MEFs. On the other hand, the promoter regions for these factors are fully methylated in terminally differentiated cells. Therefore, cytosine methylation of the promoter of replication/cell cycle factor genes occurs only in the later stage of differentiation.

Our results indicate that histone acetylation plays a major role in activating the transcription of replication/cell cycle genes in undifferentiated ES cells, and is also a major initial target for transcriptional repression upon induction of differentiation. It appears that chromatin structures in undifferentiated ES cells are regulated so that a set of positive factors are coordinately transcribed and a set of cell cycle inhibitors are repressed. Upon differentiation, the chromatin structures undergo a major transition, now repressing the positive factors and activating the negative regulators. The mechanisms underlying the coordinated transition of chromatin structures associated with differentiation are not known. Oct-3/4 is a marker for undifferentiated ES cells, and its transcript and protein levels respond rapidly to differentiation signals. In keeping with this, the transcription of Oct-3/4 is regulated by epigenetic modifications involving cytosine methylation and histone methylation/acetylation (25, 53, 66). Thus, elaborate and multilayer mechanisms ensure the rapid and efficient down-regulation of expression of key transcription factors required for self-renewal and maintenance of undifferentiated state. In contrast, chromatin regulation of replication/cell cycle factors during differentiation appears to be simpler. Whereas expression of Oct-3/4 was repressed within 2 days after induction of differentiation, it took 6 days before that of Cdc6, ASK, cyclin A2 and cyclin B1 was repressed. It would be an interesting possibility that transcription factors including Oct-3/4 may facilitate histone acetylation of cell cycle regulators by recruiting histone acetylase.

Four Cell Cycle Regulators Overexpressed in Undifferentiated Embryonic Stem Cells—Another unexpected discovery in this communication was the identification of four factors that were overexpressed specifically in undifferentiated mouse ES cells, and which underwent rapid down-regulation upon induction of differentiation. They were Cdc6, ASK, cyclin A2, and cyclin B1 proteins. Although the extent of overexpression of these factors on the transcription level was similar to that of other factors, they were expressed at a much higher level on the protein level. Except for Cdc6, which was significantly stabilized in the presence of proteasome/calpain inhibitors in MEFs, stability of these proteins was similar between ES and MEFs. Therefore, accumulation in undifferentiated ES cells and differentiation-induced decrease of these factors must be regulated at steps after transcription of their mRNAs. Differential translation efficiency in ES cells and differentiated cells may account for this. This could be caused by more abundant translation machinery in undifferentiated ES cells. Alternatively, it could be mediated by non-coding RNA or antisense RNA, which are recently discovered in many genes and have been suggested to regulate the expression of target genes (70, 71)

The four critical factors identified in this study are known to play critical roles in progression of cell cycle. Cdc6 serves as a "loading factor" at late M to early G1 phase for preRC, prerequisite for initiation of DNA replication. ASK is an activation subunit of Cdc7 kinase, which triggers firing of replication origins during S phase. Cyclin A2 activates Cdk2 or Cdc2 and is required for late S to G2 phases. Cyclin B1, in complex with Cdc2, plays an essential role in mitosis. Thus, these factors are the critical regulators for driving the cell cycle, and their overexpression may drive the embryonic cell cycle, which is dominated by alternating S and M phases. It will be of interest to examine whether combined overexpression of these factors could induce in differentiated cells a mode of cell cycle progression similar to the embryonic state, and whether it may affect their differentiation capabilities.


   FOOTNOTES
 
* This work was supported by grants-in-aid for scientific research on priority areas from the Japanese Ministry of Education, Culture, Sports, Science and Technology (to H. M.). It was also supported in part by a grant from the Tokyo Metropolitan Small Business Promotion Agency and by Ginkgo Biomedical Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Supplemental Materials. Back

|| To whom correspondence should be addressed. Tel.: 81-3-5685-2264; Fax: 81-3-5685-2932; E-mail: hmasai{at}rinshoken.or.jp.

1 The abbreviations used are: ES, embryonic stem cell; Rb, retino-blastoma; PBS, phosphate-buffered saline; FACS, fluorescent-activated cell sorter; MEF, murine embryonic fibroblast; preRC, prereplicative complex; LIF, leukemia inhibitory factor; Cdk, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation assay; PI, propidium iodide; RT-PCR, reverse transcriptase-PCR; MCM, minichromosome maintenance. Back

2 H. Fujii-Yamamoto, Y. Ishikawa, and H. Masai, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Mitsuharu Sato and Nobuaki Yoshida for the gift of ES cell line, D3 and Oct-3/4 antibody, Katsuyuki Tamai for MCM antibodies, and Hiroshi Kimura for MCM4 antibody. We thank Masako Oda for helpful suggestions on promoter analyses of replication genes and Miyuki Kawashima for help in preparation of probes for Northern blot analyses. We thank Hiroto Okayama for helpful suggestions and encouragement and Naoko Arai for useful discussion.



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