In silico tandem affinity purification refines an Oct4 interaction list
© Cheong et al.; licensee BioMed Central Ltd. 2011
Received: 31 January 2011
Accepted: 13 May 2011
Published: 13 May 2011
Octamer-binding transcription factor 4 (Oct4) is a master regulator of early mammalian development. Its expression begins from the oocyte stage, becomes restricted to the inner cell mass of the blastocyst and eventually remains only in primordial germ cells. Unearthing the interactions of Oct4 would provide insight into how this transcription factor is central to cell fate and stem cell pluripotency.
In the present study, affinity-tagged endogenous Oct4 cell lines were established via homologous recombination gene targeting in embryonic stem (ES) cells to express tagged Oct4. This allows tagged Oct4 to be expressed without altering the total Oct4 levels from their physiological levels.
Modified ES cells remained pluripotent. However, when modified ES cells were tested for their functionality, cells with a large tag failed to produce viable homozygous mice. Use of a smaller tag resulted in mice with normal development, viability and fertility. This indicated that the choice of tags can affect the performance of Oct4. Also, different tags produce a different repertoire of Oct4 interactors.
Using a total of four different tags, we found 33 potential Oct4 interactors, of which 30 are novel. In addition to transcriptional regulation, the molecular function associated with these Oct4-associated proteins includes various other catalytic activities, suggesting that, aside from chromosome remodeling and transcriptional regulation, Oct4 function extends more widely to other essential cellular mechanisms. Our findings show that multiple purification approaches are needed to uncover a comprehensive Oct4 protein interaction network.
Octamer-binding transcription factor 4 (Oct4) , also termed Oct3 or Pou5f1 , is an early developmental stage transcription factor. Oct4 expression begins in the oocyte from maternal sources and is continued by zygotic expression after the four-cell stage. Thereafter it becomes restricted to the inner cell mass, the epiblast and eventually the germ cells . During this time, Oct4 expression serves to regulate pluripotency and cell fate development . Oct4-null mouse embryos become restricted to a trophectoderm lineage at the blastocyst stage, leading to peri-implantation lethality . Such cell fate restriction is also observable in mouse embryonic stem (ES) cells when Oct4 levels decrease to less than 50% of the normal diploid expression. On the other hand, an increase in Oct4 levels by 50% converts ES cells to a primitive endodermal and mesodermal fate [6, 7]. Hence, the maintenance of pluripotency requires Oct4 to be present within a very narrow concentration range, and a change in Oct4 levels directs cells to different developmental fates. Oct4 with combinations of the following factors (Klf4, c-Myc, Sox2 and Esrrb) were also shown to be sufficient to induce pluripotency in various differentiated cell types [8–10]. Therefore, Oct4 is one of the key transcription factors involved in both the maintenance of ES cell pluripotency [11, 12] and somatic cell reprogramming [10, 13–17]. Oct4 performs its role via switching target genes on or off. Chromatin immunoprecipitation experiments and in silico analyses of Oct4 have identified at least 420 target genes with putative Oct4-binding motifs [18–21]. These target genes span multiple biological processes and developmental stages. Regulation of these different genes (including Pou5f1 itself) has been shown to be mediated via Oct4 interaction with other transcription factors [22–24]. To better understand how Oct4 regulates a large number of genes, several studies on its protein interaction network have been attempted [25–28], and they have shown that Oct4 associates with other transcription factors and epigenetic regulators [25–28]. Here we aim to further elucidate the Oct4 interaction network using a different approach. Unlike earlier studies, our study targets the endogenous Oct4 allele. This approach eliminates the altering of Oct4 from its physiological levels. Although previous studies strove to keep changes in Oct4 levels within perceived limits for ES cell maintenance, it is unknown whether this minor increase in dosage would affect embryonic development. This is a very real concern, since modulating Oct4 levels is an intrinsic mechanism used by the embryo to control cell fate .
The two most recent studies on the Oct4 interactome [26, 27] showed an overlap of about 40% of the smaller set. Is the real Oct4 interactome therefore a union or intersection of these data , and are these data sets sufficiently saturated to describe the Oct4 interactome? Since identical tags were used, differences between the data sets were attributed to the different preparations of ES cells. Pardo et al. extracted total ES cell lysate in a gentle buffer using mechanical disruption, and van den Berg et al. extracted only the nuclear extract using a high salt extraction method. Differences in data processing were another factor. These suggest that the type of interactors discovered is highly dependent on all of the experimental conditions and parameters. Therefore, the need for future studies to boost the confidence of proteins in these data sets remained . Our study indicates that the Oct4 interactome can be expanded by varying purification conditions through the use of different tags to the same endogenous Oct4 protein. In all, 33 Oct4-associated proteins were identified in our study, and they associate with proteins beyond transcriptional regulatory modules. This indicates that Oct4 may utilize self-modification as a means of transcriptional regulation or may even be involved in other types of cellular processes alongside transcriptional regulation.
Materials and methods
Gene targeting of ES cell lines via homologous recombination
V6.4 (C57BL/6 × 129/Sv) ES cells  were used for gene targeting as previously described . The targeting vector was constructed by inserting a floxed neo selection cassette (loxP-PGK-Gb2-neo-loxP) in the 5' untranslated region of the Pou5f1 (Oct3/4) gene in a mouse bacterial artificial chromosome (BAC) and insertion of a unique Fse I site immediately downstream of the translation start site of the Pou5f1 gene. Dual tags were inserted in-frame with Pou5f1 via the Fse I site. Four different constructs bearing the dual tags protein A calmodulin-binding peptide (CBP), biotin acceptor peptide (BAP)-6xHIS, S-CBP and 2xFLAG-6xHIS were made. These final constructs were used to generate the four separately tagged Oct4 ES cell lines expressing N-terminal tandem affinity purification (NTAP)-Oct4, N-terminal BAP-HIS (NBH)-Oct4, N-terminal S peptide CBP (NSC)-Oct4 and N-terminal FLAG-HIS (NFH)-Oct4. The neo selection cassette was removed by transient expression of Cre recombinase. For the NBH-Oct4 cell line, a second targeting vector (pROSA26-hBirA-lacZ-loxP-neo, courtesy of M. Lee) was introduced. This vector bears a "humanized" BirA ligase (hBirA) gene, as well as neo and lacZ markers, at the Rosa26 locus.
Generation of genetically modified mice
NTAP-Oct4 and NSC-Oct4 ES cells were injected into blastocysts and used to generate tagged Oct4 chimeras that were then used to derive heterozygous and homozygous animals. The Institutional Animal Care and Use Committee at our institution approved all animal protocols used in this study.
ES cell lines for protein purification were grown without Mouse Embryo Fibroblasts (MEF). Nuclear proteins were extracted using NE-PER Reagents (Pierce Biotechnology/Thermo Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions. Nuclear extracts were buffer-exchanged using Zeba Spin Desalting Columns (Pierce Biotechnology/Thermo Scientific) prior to purification. For S-tag purification, nuclear extract was incubated with S-protein agarose beads (Novagen, Darmstadt, Germany) in binding buffer (20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 0.2% Triton X-100 and 5% glycerol), washed with four column volumes (CVs) of binding buffer and eluted with 1.4 mg/mL S peptide, KETAAAKFERQHMDS (customized peptide synthesis by Sigma, St. Louis, Missouri), in binding buffer. For FLAG-tag purification, nuclear extract was incubated with ANTI-FLAG M2 Affinity Gel (Sigma) in binding buffer (50 mM Tris·HCl, pH 7.4, and 150 mM NaCl), washed with five CVs of binding buffer and eluted with 0.1 mg/mL of 3XFLAG peptide, MDYKDHDGDYKDHDIDYKDDDDK (Sigma), in binding buffer. For HIS-tag purification, nuclear extract was incubated with Ni-NTA Superflow (Qiagen, Venlo, Netherlands) in binding buffer (20 mM Tris·HCl, pH 7.9, 350 mM NaCl, 0.2% Triton X-100 and 10 mM imidazole), washed in one CV of wash buffer, followed by two CVs of binding buffer with increased imidazole (20 mM) before elution with 300 mM imidazole in pH 7.0 binding buffer. For CBP-tag purification, nuclear extract was incubated with calmodulin-agarose (Millipore, Billerica, Massachusetts, USA) in binding buffer (10 mM Tris·HCl, pH 8.0, 150 mM NaCl, 1 mM MgOAc, 1 mM imidazole, 2 mM CaCl2, 0.1% Nonidet P (NP)-40 and 10 mM β-mercaptoethanol), washed in three CVs of wash buffer (50 mM Tris·HCl, pH 7.0, 350 mM NaCl, 0.2% NP-40 and 5 mM imidazole) before elution via release of the HIS-tagged calmodulin with elution buffer (50 mM Tris·HCl, pH 7.0, 350 mM NaCl, 0.1% NP-40 and 350 mM imidazole). All buffers used in protein extraction and affinity purification were supplemented with Complete Protease Inhibitor EDTA-free Cocktail (Roche, Indianapolis, IN, USA). All resins were equilibrated in binding buffer before use. The incubation time of nuclear extract with resin ranged from 1.5 hours to overnight at 4°C.
Western blot analysis
Analyses were performed by resolving ES nuclear extraction or unbound, washed and eluted fractions from the purifications on gradient (4% to 15%) or 10% acrylamide gels by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer of the proteins onto polyvinylidene difluoride membranes for detection with relevant antibodies. The antibodies used were anti-Oct4 antibodies (ab27985; Abcam, Boston MA, USA), anti-S antibodies (sc-802; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-6xHIS monoclonal antibody horseradish peroxidase (HRP) conjugate (631210; Clontech, Mountain View, CA, USA), anti-CBP (sc-33000; Santa Cruz Biotechnology) and anti-FLAG M2 monoclonal antibodies (F3165 or F1804; Sigma). Unlabeled primary antibodies were detected by using HRP-linked antirabbit antibody (NA934; GE Healthcare Life Sciences, GE Healthcare Pte Ltd., Life Sciences Consumables, Singapore, Singapore), HRP-linked antimouse antibody (NXA931; GE Healthcare Life Sciences) or HRP-linked antigoat antibody (sc-2020; Santa Cruz Biotechnology). BAP tag was detected by using streptavidin-HRP (NEL750; PerkinElmer, Waltham Massachusetts, USA).
Proteins eluted by affinity purification were resolved by SDS-PAGE and Coomassie-stained with SimplyBlue SafeStain (Invitrogen, Carlsbad, California, USA). Each gel lane was cut into five sections. Gel pieces were subjected to in-gel tryptic digestion. Each section was submitted as an individual sample for liquid chromatography-mass spectrometry (LC-MS/MS) analysis at the Genome Institute of Singapore proteomic facility. Samples were injected using a nano-LC pump to a reversed-phase column and analyzed by using an LTQ-MS/MS spectrometer (ThermoFinnigan, San Jose, CA, USA). LC-MS/MS spectra from each purification were identified using both SEQUEST and X!Tandem search engine algorithms. The results were then loaded onto the Scaffold Proteome Software platform (Proteome Software, Inc., Portland, OR, USA), and the parameters of 95% probability of correct protein and peptide identification were set as filters.
Endogenous tagged Oct4 cell lines express tagged Oct4 at physiological levels and remain pluripotent
Expression of the tags was confirmed by using anti-S antibody (Figure 2C) and anti-FLAG antibody (Figure 2D) for the NSC-Oct4- and NFH-Oct4-expressing cell lines, respectively. For the NBH-Oct4 expressing cell line, detection of the BAP tag was verified by using streptavidin (Figure 2E).
To evaluate the effect of each of these tags on ES cell character, we stained each of the four cell lines for alkaline phosphatase activity. All tagged cell lines stained similarly to the wild type (Figure 2F) and exhibited morphology and passage times similar to those of wild-type ES cells, suggesting that endogenous tagging does not affect ES cells.
NTAP-Oct4 cell line produces a lethal phenotype
Number and percentage of pups belonging to each genotype as a result of mating heterozygotesa
Wild-type pups (Oct4WT/WT)
Heterozygous pups (Oct4WT/TAG)
Homozygous pups (Oct4TAG/TAG)
Identification of Oct4 interacting proteins
In contrast to researchers in other studies, we identified Oct4 interacting proteins using four different affinity-tag purification approaches. The advantage of this method is that the protein interactors discovered are not limited by the conditions of one approach. Western blot analysis of the purification was used to detect the tagged Oct4 following purification from Oct WT/TAG or from wild-type ES cells as starting material. An example of purification for the S tag (Figure 2G) shows that Oct4 is enriched only in the tagged ES cell line, but not in the wild-type ES cell line. Detection using anti-S antibody confirmed the presence of the tag in the enriched Oct4, while detection for tubulin suggested a depletion of background protein after purification amid the enrichment of NSC-Oct4. Following affinity purification, eluates were separated by gel electrophoresis. Whole lanes were excised into multiple gel bands and subjected to further tryptic digestion and peptide identification by LC-MS/MS. Raw MS/MS data were subjected to protein identification by searches using the mouse International Protein Index database (European Bioinformatics Institute, Cambridge, UK). Only proteins identified in the overlap of two separate algorithm searches (Sequest and X!Tandem) were considered confident identifications and pursued for further analysis.
Oct4-associated proteins using four different affinity tag approachesa
Entrez Gene ID
Catalytic activity based on PANTHER
POU domain, class 5, transcription factor 1
Cell cycle associated
Cyclin-dependent kinase 1
Structural maintenance of chromosome 2
RAD50 homolog (Saccharomyces cerevisiae)
Thyroid hormone receptor interactor 12
DEAD (Asp-Glu-Ala-Asp) box polypeptide 1
Helicase, translation initiation
DEAH (Asp-Glu-Ala-His) box polypeptide 15
Nucleolar protein 5A
RNA and protein transport and localization
Karyopherin (importin) α2
Nucleolar protein 5
Regulator of chromosome condensation 1
Ligase, small GTPase regulator, guanyl-nucleotide exchange factor
THO complex 4
Lamin B receptor
Tight junction protein 2
Exportin 1, CRM1 homolog (yeast)
Elongation factor Tu GTP binding domain containing 2
Nucleotidyltransferase, GTPase, translation initiation and elongation
U2 small nuclear ribonucleoprotein auxiliary factor 2
Synaptotagmin binding, cytoplasmic RNA interacting protein
RNA splicing factor, transesterification mechanism
Ras suppressor protein 1
SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 1
Far upstream element (FUSE) binding protein 3
Fusion, derived from t(12;16) malignant liposarcoma
RNA splicing factor, transesterification mechanism
Proline, glutamic acid and leucine rich protein 1
PC4 and SFRS1 interacting protein 1
TAR DNA binding protein
RNA splicing factor, transesterification mechanism
Topoisomerase (DNA) IIα
Insulin-like growth factor 2 mRNA binding protein 1
RNA splicing factor, transesterification mechanism
Oct4 interactors indicate Oct4 engagements with multiple cellular mechanisms
Phenotypes for loss of function of Oct4-associated transcriptional regulators
Transcription factor activity (PANTHER)
Perinatal death (survivors show reduced fertility)
Transcription factor activity (PANTHER)
Embryonic lethality before somite formation with impaired inner cell mass proliferation
Positive regulation of transcription, DNA-dependent (DAVID), transcription factor activity (PANTHER)
High neonatal mortality, and male sterility associated with lack of chromosomal pairing
Positive regulation of transcription, DNA-dependent (DAVID)
Positive regulation of transcription, DNA-dependent (DAVID)
Phenotypes for loss of function of Oct4-associated proteins that are not known to be transcriptional regulators
Death prior to embryonic day 1.5
Increased neonatal lethality associated with multiple abnormalities
Impaired growth and skin defects
Embryonic lethality associated with atrial fibrillation
Embryonic death. Hypomorphic mutant shows predisposition toward cancer and loss of spermatogenic and hematopoietic stem cells, leading to death.
Embryonic lethality associated with gastrulation defect
Novel transcriptional regulators coenriched with Oct4
As Oct4 is a transcription factor expected to interact with other transcription factors in a modular fashion to effect transcription regulation, we were most interested in the proteins with a role in transcription regulation. Five proteins, Fubp3, Fus, Psip1, Tardbp and Top2a, were annotated by the DAVID and/or PANTHER databases as proteins with a role in transcription regulation (Table 4). Of these five proteins, Top2a has been reported to be an Oct4 interactor , while the other four proteins have yet to be reported.
Understanding the transcriptional regulatory role of Oct4 allows for the control of embryonic or induced pluripotent stem cell applications . As a master regulator, Oct4 is already present in the unfertilized egg via maternal transcripts to modulate gene expression from the earliest stages of embryonic development. To coordinate gene regulation both positively and negatively in the dynamic and temporal stages of development, Oct4 presumably must interact with multiple functional modules involved in different areas of cellular regulation. Insights into such regulatory mechanisms of Oct4 can come from understanding the Oct4 protein interaction network.
Previous studies on Oct4 have employed transgenic methods that introduced a tagged Oct4 into ES cells. While care has been taken to ensure that the level of extra Oct4 does not exceed 50% of the endogenous level, an increase in Oct4 is unavoidable. Therefore, all previous work raises the concern that these changes would affect ES cells. Hence, there is a dilemma with regard to keeping the exogenous tagged Oct4 as low as possible to avoid changing cell fate and making it high so that the purification yield is better. Since our strategy does not change the endogenous level of Oct4, we can have all the Oct4 present physiologically contribute to the purification yield. Indeed, we can get detectable Oct4 by using LC-MS/MS with a low starting material level of 400 μg of nuclear extract to get a signal of 11 spectra for Oct4 in the tag purification and no signal in the wild-type control. This is a significant reduction compared to what is required (50 to 100 mg) when tagged Oct4 is expressed as a low percentage of total Oct4 [36, 37]. Also, since the endogenous Oct4 is modified, the presence of untagged Oct4 acting as a competitor for interactors is reduced.
While keeping Oct4 to its endogenous level is important, no previous study has addressed a separate concern that the tags used may impede the function of Oct4. We tested two different tags in animals and found that the classically used NTAP tag [38, 39], comprising two protein A and one calmodulin-binding protein, prevents Oct4 from driving embryonic development normally. This information is especially useful for future work involving gene tagging in both in vitro and in vivo studies.
Three of four previous reports on Oct4 protein interaction network used the FLAG tag in their approach. The protein interactors found showed overlaps, lending confidence to what are identified as true Oct4 interactors. However, the use of a similar tag means that common contaminants raised from a specific affinity purification will also be repeatedly identified. Although tandem affinity tags have previously been used [26, 28], the number of proteins found was lower than when single purifications are used, suggesting that the inclusion of different tags can produce a low overlap. One problem with tandem affinity purification is the loss of yield as the number of steps and experimentation time increase. To overcome this, we simply performed the purifications using different tags on fresh ES cell samples and performed in silico tandem affinity purification instead. This method allowed us to discover a total of 33 proteins using a low amount of starting material (400 μg of nuclear extract) per purification.
In the online discussion by Pardo et al. following his publications on Oct4 interactors, this study group suggested that heterogeneity in data sets can arise from the cell line, the tagging strategy and particularly the purification procedure used. Therefore, to expand the list of interactors from the previous data sets, we tested whether we could find novel interactors that bind to Oct4 under different purification procedures using different tags from different cell lines. Although our approach for protein extraction is similar to that of van den Berg et al., our purification procedure following protein extraction is different from those used in studies by both van den Berg et al. and Pardo et al.. Additionally, we included a different combination of tags for analysis. Using purification buffers for each affinity tag that differ by parameters including ionic charge, pH and the use of detergents, we intended to identify a group of Oct4 interactors that were robustly identified across varying conditions. These would be representative of stalwart Oct4 interactions that could occur despite the microenvironments that might arise in a cell. Also, by considering only interactors that remained bound to Oct4 under at least two different purification procedures, we ensured that these interactions were not an artefact of a specific purification procedure alone.
The focus of Oct4 interaction has been on chromatin modifiers and transcriptional factors. With the use of different approaches of purification, the majority of our proteins showed other catalytic activities in addition to transcriptional regulation. These include helicases, ligases and RNA processing. Oct4 may recruit these enzymes to modify itself or its associated proteins as an additional means of regulation. Consistent with this hypothesis is a report that Oct4 associates with the glycosylating enzyme Ogt , suggesting that posttranslational and splicing activities should not be overlooked in considering Oct4 function. Indeed, Oct4 and other transcription factors have been shown to regulate their activity via O-GlcNAc modification [40–42]. Sumoylation of Oct4 has also been reported to enhance its stability, DNA binding and transactivation . In a separate study, Oct4 was reported to be ubiquitinated by Wwp2, an E3 ubiquitin ligase . Beyond modification of Oct4 activity levels, association with enzymes could give Oct4 the ability to modify DNA or other proteins. Potentially, Oct4-associated helicases could be recruited to Oct4-mediated transcriptional sites to keep the genome stable. In view of the fact that ES cells and embryos are both systems that require rapid DNA replication and transcription , there is a need for helicases to keep the genome stable when replication and transcriptional complexes collide .
In addition to transcription-related activities, Oct4 also affiliates with proteins involved in cell cycle regulation. Cdk1 has previously been established in an Oct4 interaction network  and is critical for the self-renewal of ES cells . Because of the unique cell cycle phasing of ES cells with a short G1/S phase that promotes rapid proliferation [48–50], the coupling of Oct4 activity with cell cycle kinases such as Cdk1 may be necessary for rapid and direct coordination of genomic activity with cell division, failing which apoptosis may occur, as shown by ES cells depleted of Cdk1 . A separate Oct4 interactor discovered in our study, Smc2, forms part of the condensins I and II protein complexes required for proper DNA compaction during the interphase [51, 52]. An RNA interference screen in ES cells identified Smc2 as a protein essential for proper chromosomal compaction in ES cells, and a deficiency in Smc2 results in metaphase arrest in these cells . As ES cells are known to maintain much of their chromatin in a heterochromatin state , there is a significant role for Oct4 in mediating both epigenetic machinery and condensin complexes to enable the removal of activating histone modifications that can perturb proper compaction for mitosis.
Cul4b has also previously been identified as an Oct4 interactor  and is an E3 ubiquitin ligase . Interestingly, like Oct4, Cul4b is involved in Wnt signaling through its repression of nuclear β-catenin levels that can otherwise serve as a positive factor for differentiation . Separately, Oct4 and Cul4b have also been shown to interact with β-catenin in immunoprecipitated complexes [56–58].
In addition to the role of Oct4 in cell cycle regulation and inhibition of differentiation through the Wnt pathway, it appears that Oct4 also associates with a number of proteins involved in nuclear transport. While Kpna2 is a previously known Oct4 interactor  itself, Kpna2 and another novel Oct4 interactor, Rcc1, are both specifically involved in the nuclear import of proteins . In our study, various nucleoporins were also found to associate with Oct4, which suggests that a complex comprising nuclear pore proteins supporting factors such as Kpna2 and Rcc1 work in tandem with Oct4, although their imported cargo is as yet unclear. Given the known link between Rcc1 and chromatin , it is highly plausible that Oct4 utilizes Rcc1 as an intermediary between the current chromatin state and the transport of necessary proteins for gene expression from their cytosolic compartment.
The diversity in function of novel and known Oct4 interactors identified in our work clearly highlights the need for a multifaceted approach for the completion of the Oct4 interaction network. We believe that our use of endogenous tagging methods and a combined in silico analysis of identified proteins from different purification conditions serves as a resource for research in this direction. While future work should include validation of the interactors and a detailed investigation into the molecular mechanism of each interactor, we are confident that our present findings are of great value in expanding our framework for understanding Oct4 interactions beyond transcriptional control alone.
In summary, we have used endogenously tagged Oct4 to study its interaction under physiological Oct4 levels. The use of multitag purification platforms allowed for a wide scope in the discovery of interactors. The proteins identified in this study include novel transcription factor interactions and a demonstrated role for Oct4 in ES cells that involves catalytic activities other than transcriptional regulation.
N-terminal biotin acceptor peptide HIS
N-terminal FLAG HIS
N-terminal S peptide calmodulin-binding peptide
N-terminal tandem affinity purification.
We thank Stephanie Lim Wai Lin for technical assistance in performing mass spectrometry and Chan Hsiao-Yun, Geraldine Leong and Petra Kraus for animal work done within the GIS-GAP facility. This work was supported by the Agency for Science, Technology and Research (Singapore).
- Schöler HR, Dressler GR, Balling R, Rohdewohld H, Gruss P: Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. EMBO J. 1990, 9: 2185-2195.PubMed CentralPubMedGoogle Scholar
- Okamoto K, Okazawa H, Okuda A, Sakai M, Muramatsu M, Hamada H: A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell. 1990, 60: 461-472. 10.1016/0092-8674(90)90597-8.View ArticlePubMedGoogle Scholar
- Schöler HR, Hatzopoulos AK, Balling R, Suzuki N, Gruss P: A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor. EMBO J. 1989, 8: 2543-2550.PubMed CentralPubMedGoogle Scholar
- Boiani M, Schöler HR: Regulatory networks in embryo-derived pluripotent stem cells. Nat Rev Mol Cell Biol. 2005, 6: 872-884. 10.1038/nrm1744.View ArticlePubMedGoogle Scholar
- Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Schöler H, Smith A: Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell. 1998, 95: 379-391. 10.1016/S0092-8674(00)81769-9.View ArticlePubMedGoogle Scholar
- Niwa H, Miyazaki J, Smith AG: Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet. 2000, 24: 372-376. 10.1038/74199.View ArticlePubMedGoogle Scholar
- Shimozaki K, Nakashima K, Niwa H, Taga T: Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures. Development. 2003, 130: 2505-2512. 10.1242/dev.00476.View ArticlePubMedGoogle Scholar
- Feng B, Jiang J, Kraus P, Ng JH, Heng JC, Chan YS, Yaw LP, Zhang W, Loh YH, Han J, Vega B, Cacheux-Rataboul V, Lim B, Lufkin T, Ng HH: Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb. Nat Cell Biol. 2009, 11: 197-203. 10.1038/ncb1827.View ArticlePubMedGoogle Scholar
- Chang CW, Lai YS, Pawlik KM, Liu K, Sun CW, Li C, Schoeb TR, Townes TM: Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells. Stem Cells. 2009, 27: 1042-1049. 10.1002/stem.39.View ArticlePubMedGoogle Scholar
- Takahashi K, Yamanaka S: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006, 126: 663-676. 10.1016/j.cell.2006.07.024.View ArticlePubMedGoogle Scholar
- Niwa H: How is pluripotency determined and maintained?. Development. 2007, 134: 635-646. 10.1242/dev.02787.View ArticlePubMedGoogle Scholar
- Smith AG: Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol. 2001, 17: 435-462. 10.1146/annurev.cellbio.17.1.435.View ArticlePubMedGoogle Scholar
- Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S: Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. 2008, 26: 101-106. 10.1038/nbt1374.View ArticlePubMedGoogle Scholar
- Yamanaka S: A fresh look at iPS cells. Cell. 2009, 137: 13-17. 10.1016/j.cell.2009.03.034.View ArticlePubMedGoogle Scholar
- Okita K, Ichisaka T, Yamanaka S: Generation of germline-competent induced pluripotent stem cells. Nature. 2007, 448: 313-317. 10.1038/nature05934.View ArticlePubMedGoogle Scholar
- Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R: In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007, 448: 318-324. 10.1038/nature05944.View ArticlePubMedGoogle Scholar
- Hochedlinger K, Plath K: Epigenetic reprogramming and induced pluripotency. Development. 2009, 136: 509-523. 10.1242/dev.020867.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang W, Jiang J, Loh YH, Yeo HC, Yeo ZX, Narang V, Govindarajan KR, Leong B, Shahab A, Ruan Y, Bourque G, Sung WK, Clarke ND, Wei CL, Ng HH: Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell. 2008, 133: 1106-1117. 10.1016/j.cell.2008.04.043.View ArticlePubMedGoogle Scholar
- Sharov AA, Masui S, Sharova LV, Piao Y, Aiba K, Matoba R, Xin L, Niwa H, Ko MS: Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data. BMC Genomics. 2008, 9: 269-10.1186/1471-2164-9-269.PubMed CentralView ArticlePubMedGoogle Scholar
- Sridharan R, Tchieu J, Mason MJ, Yachechko R, Kuoy E, Horvath S, Zhou Q, Plath K: Role of the murine reprogramming factors in the induction of pluripotency. Cell. 2009, 136: 364-377. 10.1016/j.cell.2009.01.001.PubMed CentralView ArticlePubMedGoogle Scholar
- Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, Chen X, Bourque G, George J, Leong B, Liu J, Wong KY, Sung KW, Lee CW, Zhao XD, Chiu KP, Lipovich L, Kuznetsov VA, Robson P, Stanton LW, Wei CL, Ruan Y, Lim B, Ng HH: The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet. 2006, 38: 431-440. 10.1038/ng1760.View ArticlePubMedGoogle Scholar
- Wang ZX, Teh CH, Kueh JL, Lufkin T, Robson P, Stanton LW: Oct4 and Sox2 directly regulate expression of another pluripotency transcription factor, Zfp206, in embryonic stem cells. J Biol Chem. 2007, 282: 12822-12830. 10.1074/jbc.M611814200.View ArticlePubMedGoogle Scholar
- Okumura-Nakanishi S, Saito M, Niwa H, Ishikawa F: Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells. J Biol Chem. 2005, 280: 5307-5317.View ArticlePubMedGoogle Scholar
- Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, Robson P: Transcriptional regulation of Nanog by OCT4 and SOX2. J Biol Chem. 2005, 280: 24731-24737. 10.1074/jbc.M502573200.View ArticlePubMedGoogle Scholar
- Liang J, Wan M, Zhang Y, Gu P, Xin H, Jung SY, Qin J, Wong J, Cooney AJ, Liu D, Songyang Z: Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells. Nat Cell Biol. 2008, 10: 731-739. 10.1038/ncb1736.View ArticlePubMedGoogle Scholar
- Pardo M, Lang B, Yu L, Prosser H, Bradley A, Babu MM, Choudhary J: An expanded Oct4 interaction network: implications for stem cell biology, development, and disease. Cell Stem Cell. 2010, 6: 382-395. 10.1016/j.stem.2010.03.004.PubMed CentralView ArticlePubMedGoogle Scholar
- van den Berg DL, Snoek T, Mullin NP, Yates A, Bezstarosti K, Demmers J, Chambers I, Poot RA: An Oct4-centered protein interaction network in embryonic stem cells. Cell Stem Cell. 2010, 6: 369-381. 10.1016/j.stem.2010.02.014.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang J, Rao S, Chu J, Shen X, Levasseur DN, Theunissen TW, Orkin SH: A protein interaction network for pluripotency of embryonic stem cells. Nature. 2006, 444: 364-368. 10.1038/nature05284.View ArticlePubMedGoogle Scholar
- Lemischka IR: Hooking up with Oct4. Cell Stem Cell. 2010, 6: 291-292. 10.1016/j.stem.2010.03.011.View ArticlePubMedGoogle Scholar
- Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M, Rideout WM, Yanagimachi R, Jaenisch R: Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci USA. 2001, 98: 6209-6214. 10.1073/pnas.101118898.PubMed CentralView ArticlePubMedGoogle Scholar
- Lufkin T, Dierich A, LeMeur M, Mark M, Chambon P: Disruption of the Hox-1.6 homeobox gene results in defects in a region corresponding to its rostral domain of expression. Cell. 1991, 66: 1105-1119. 10.1016/0092-8674(91)90034-V.View ArticlePubMedGoogle Scholar
- Thomas PD, Campbell MJ, Kejariwal A, Mi H, Karlak B, Daverman R, Diemer K, Muruganujan A, Narechania A: PANTHER: a library of protein families and subfamilies indexed by function. Genome Res. 2003, 13: 2129-2141. 10.1101/gr.772403.PubMed CentralView ArticlePubMedGoogle Scholar
- Hall J, Guo G, Wray J, Eyres I, Nichols J, Grotewold L, Morfopoulou S, Humphreys P, Mansfield W, Walker R, Tomlinson S, Smith A: Oct4 and LIF/Stat3 additively induce Krüppel factors to sustain embryonic stem cell self-renewal. Cell Stem Cell. 2009, 5: 597-609. 10.1016/j.stem.2009.11.003.View ArticlePubMedGoogle Scholar
- Okamura D, Tokitake Y, Niwa H, Matsui Y: Requirement of Oct3/4 function for germ cell specification. Dev Biol. 2008, 317: 576-584. 10.1016/j.ydbio.2008.03.002.View ArticlePubMedGoogle Scholar
- Amabile G, Meissner A: Induced pluripotent stem cells: current progress and potential for regenerative medicine. Trends Mol Med. 2009, 15: 59-68. 10.1016/j.molmed.2008.12.003.View ArticlePubMedGoogle Scholar
- Wang J, Cantor AB, Orkin SH: Tandem affinity purification of protein complexes in mouse embryonic stem cells using in vivo biotinylation. Curr Protoc Stem Cell Biol. 2009, Chapter 1: Unit1B.5-PubMedGoogle Scholar
- Kim J, Cantor AB, Orkin SH, Wang J: Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells. Nat Protoc. 2009, 4: 506-517. 10.1038/nprot.2009.23.View ArticlePubMedGoogle Scholar
- Li Y: Commonly used tag combinations for tandem affinity purification. Biotechnol Appl Biochem. 2010, 55: 73-83. 10.1042/BA20090273.View ArticlePubMedGoogle Scholar
- Xu X, Song Y, Li Y, Chang J, Zhang H, An L: The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification. Protein Expr Purif. 2010, 72: 149-156. 10.1016/j.pep.2010.04.009.View ArticlePubMedGoogle Scholar
- Kuo M, Zilberfarb V, Gangneux N, Christeff N, Issad T: O-GlcNAc modification of FoxO1 increases its transcriptional activity: a role in the glucotoxicity phenomenon?. Biochimie. 2008, 90: 679-685. 10.1016/j.biochi.2008.03.005.View ArticlePubMedGoogle Scholar
- Issad T, Kuo M: O-GlcNAc modification of transcription factors, glucose sensing and glucotoxicity. Trends Endocrinol Metab. 2008, 19: 380-389. 10.1016/j.tem.2008.09.001.View ArticlePubMedGoogle Scholar
- Webster DM, Teo CF, Sun Y, Wloga D, Gay S, Klonowski KD, Wells L, Dougan ST: O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development. BMC Dev Biol. 2009, 9: 28-10.1186/1471-213X-9-28.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei F, Schöler HR, Atchison ML: Sumoylation of Oct4 enhances its stability, DNA binding, and transactivation. J Biol Chem. 2007, 282: 21551-21560. 10.1074/jbc.M611041200.View ArticlePubMedGoogle Scholar
- Xu HM, Liao B, Zhang QJ, Wang BB, Li H, Zhong XM, Sheng HZ, Zhao YX, Zhao YM, Jin Y: Wwp2, an E3 ubiquitin ligase that targets transcription factor Oct-4 for ubiquitination. J Biol Chem. 2004, 279: 23495-23503. 10.1074/jbc.M400516200.View ArticlePubMedGoogle Scholar
- Koledova Z, Kafkova LR, Calabkova L, Krystof V, Dolezel P, Divoky V: Cdk2 inhibition prolongs G1 phase progression in mouse embryonic stem cells. Stem Cells Dev. 2010, 19: 181-194. 10.1089/scd.2009.0065.View ArticlePubMedGoogle Scholar
- Pomerantz RT, O'Donnell M: What happens when replication and transcription complexes collide?. Cell Cycle. 2010, 9: 2535-2541.View ArticleGoogle Scholar
- Zhang WW, Zhang XJ, Liu HX, Chen J, Ren YH, Huang DG, Zou XH, Xiao W: Cdk1 is required for the self-renewal of mouse embryonic stem cells. J Cell Biochem. 2011, 112: 942-948. 10.1002/jcb.23010.View ArticlePubMedGoogle Scholar
- Burdon T, Smith A, Savatier P: Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol. 2002, 12: 432-438. 10.1016/S0962-8924(02)02352-8.View ArticlePubMedGoogle Scholar
- Fluckiger AC, Marcy G, Marchand M, Négre D, Cosset FL, Mitalipov S, Wolf D, Savatier P, Dehay C: Cell cycle features of primate embryonic stem cells. Stem Cells. 2006, 24: 547-556. 10.1634/stemcells.2005-0194.PubMed CentralView ArticlePubMedGoogle Scholar
- Becker KA, Ghule PN, Therrien JA, Lian JB, Stein JL, van Wijnen AJ, Stein GS: Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J Cell Physiol. 2006, 209: 883-893. 10.1002/jcp.20776.View ArticlePubMedGoogle Scholar
- Losada A, Hirano T: Dynamic molecular linkers of the genome: the first decade of SMC proteins. Genes Dev. 2005, 19: 1269-1287. 10.1101/gad.1320505.View ArticlePubMedGoogle Scholar
- Belmont AS: Mitotic chromosome structure and condensation. Curr Opin Cell Biol. 2006, 18: 632-638. 10.1016/j.ceb.2006.09.007.View ArticlePubMedGoogle Scholar
- Fazzio TG, Panning B: Condensin complexes regulate mitotic progression and interphase chromatin structure in embryonic stem cells. J Cell Biol. 2010, 188: 491-503. 10.1083/jcb.200908026.PubMed CentralView ArticlePubMedGoogle Scholar
- Mattout A, Meshorer E: Chromatin plasticity and genome organization in pluripotent embryonic stem cells. Curr Opin Cell Biol. 2010, 22: 334-341. 10.1016/j.ceb.2010.02.001.View ArticlePubMedGoogle Scholar
- Jackson S, Xiong Y: CRL4s: the CUL4-RING E3 ubiquitin ligases. Trends Biochem Sci. 2009, 34: 562-570. 10.1016/j.tibs.2009.07.002.PubMed CentralView ArticlePubMedGoogle Scholar
- Tripathi R, Kota SK, Srinivas UK: Cullin4B/E3-ubiquitin ligase negatively regulates β-catenin. J Biosci. 2007, 32: 1133-1138. 10.1007/s12038-007-0114-0.View ArticlePubMedGoogle Scholar
- Takao Y, Yokota T, Koide H: β-catenin up-regulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells. Biochem Biophys Res Commun. 2007, 353: 699-705. 10.1016/j.bbrc.2006.12.072.View ArticlePubMedGoogle Scholar
- Abu-Remaileh M, Gerson A, Farago M, Nathan G, Alkalay I, Zins Rousso S, Gur M, Fainsod A, Bergman Y: Oct-3/4 regulates stem cell identity and cell fate decisions by modulating Wnt/β-catenin signalling. EMBO J. 2010, 29: 3236-3248. 10.1038/emboj.2010.200.PubMed CentralView ArticlePubMedGoogle Scholar
- Li X, Sun L, Jin Y: Identification of karyopherin-α 2 as an Oct4 associated protein. J Genet Genomics. 2008, 35: 723-728. 10.1016/S1673-8527(08)60227-1.View ArticlePubMedGoogle Scholar
- Richards SA, Carey KL, Macara IG: Requirement of guanosine triphosphate-bound ran for signal-mediated nuclear protein export. Science. 1997, 276: 1842-1844. 10.1126/science.276.5320.1842.View ArticlePubMedGoogle Scholar
- England JR, Huang J, Jennings MJ, Makde RD, Tan S: RCC1 uses a conformationally diverse loop region to interact with the nucleosome: a model for the RCC1-nucleosome complex. J Mol Biol. 2010, 398: 518-529. 10.1016/j.jmb.2010.03.037.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.