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a Laboratory of Molecular and Cellular Biology, Department of Life Science, Sogang University, Seoul, Korea;
b Laboratory of Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
Key Words. Oct-4 • Ewing’s sarcoma protein • Proto-oncogene • Transcriptional coactivator • Protein—protein interaction • Bacterial two-hybrid screening • Embryonic stem cells
Correspondence: Jungho Kim, Ph.D., Laboratory of Molecular and Cellular Biology, Department of Life Science, Sogang University, Seoul 121-742, Korea. Telephone: 82-2-705-8461; Fax: 82-2-716-2092; e-mail: jkim{at}sogang.ac.kr
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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r (platelet-derived growth factor receptor), and osteopontin [9–11]. In addition, genes, such as tau interferon (IFN-
) and the and ß subunits of chorionic gonadotropin (hCG), expressed in the trophectoderm but not in embryos before blastocyst formation may be targets for silencing by Oct-4 [12–14]. This suggests that Oct-4 functions as a master switch during differentiation by regulating cells that have pluripotent potential or can develop such potential [15, 16]. Oct-4 mRNA is normally found in the to tipotent and pluripotent stem cells of pregastrulation embryo, including oocytes, early cleavage-stage embryos, and the inner cell mass (ICM) of the blastocyst [1, 3, 17, 18]. In addition, the expression of this gene is downregulated during differentiation, suggesting that Oct-4 plays a pivotal role in the mammalian development [19]. Furthermore, knocking out the Oct-4 gene in mice causes early lethality due to lack of ICM formation [20], indicating critical function for self-renewal of ES cells [21]. During human development, expression of Oct-4 is found at least until the blastocyst stage, during which it is involved in gene expression regulation [22].
It was recently reported that Oct-4 is a key player in the genesis of human testicular germ cell tumors (TGCTs) [23, 24]. Human TGCTs are the most common malignancy in adolescent and young adult white males and are the cause of one in seven deaths in this group [25, 26]. The Oct-4 transcript can be consistently detected in a specific set of human TGCTs of adolescents and young adults: the seminomas and embryonal carcinomas [27]. In addition, the precursor lesions of human TGCT, known as CIS, also express Oct-4 [27]. The expression of Oct-4 has also been reported in human primary breast carcinomas, human breast cancer cell lines, and other types of carcinoma cell lines, suggesting that its expression may be implicated in tumorigenesis via upregulating its downstream target genes [28–30]. Consistent with these findings, Oct-4 expression in a heterologous cell system transforms nontumorigenic cells and endows tumorigenicity in the nude mouse, suggesting the possibility that aberrant expression of Oct-4 may contribute to the neoplastic process in cells [23].
The N- and C-termini of Oct-4 function as transactivation domains. Interestingly, although the N-terminus is active in various cultured cell types, the activity of the C-terminal domain depends on the cell type [31]. The POU domain of Oct-4 is a conserved DNA-binding domain that binds as a monomer to the octamer sequence motif, 5'-ATGCAAAT-3' [32]. This cis-acting element is important in determining the activity of many promoters and enhancers, including those of housekeeping and of cell type–specific genes [7]. In ES cells, the octamer sequence motif is active irrespective of the distance from its site of transcriptional initiation [33,34]. However, in differentiated cells, Oct-4 can transactivate only from an octamer motif at proximal positions [1, 31, 35]. To be active from distal sites, Oct-4 requires stem cell–specific bridging factors that link an Oct-4 molecule bound to a remote DNA region to the transcription initiation site [35]. To date, the only identified putative Oct-4 cofactors are the viral oncoproteins E1A and E7 that seem to mimic yet-to-be-defined, stem cell–specific coactivators [35, 36].
EWS was originally identified through its fusion with the Fli-1 gene, a member of the ETS transcription factor family, in Ewing’s sarcoma harboring at (11; 22) chromosomal translocation [37]. Subsequent studies indicated that other ETS transcription factor genes are also fused with the EWS gene and produce chimeric proteins in Ewing’s sarcoma. In addition, the EWS gene has been shown to form fusion proteins in other human cancers, including with ATF-1 in malignant melanoma of soft parts, WT1 in desmoplastic small round cell tumors, and orphan family nuclear receptor TEC in myxoid chondrosarcomas [38–40]. EWS has high homology to TLS, hTAFII68, and Drosophila protein SARFH [38–40]. Thus, these proteins are collectively called the TET family member [41].
EWS contains a transcriptional activation domain in its N-terminal domain (NTD) and an RNA recognition motif and RGG repeats, both of which are found in RNA-binding proteins, in its C-terminal domain [38–40]. Interestingly, EWS was found to associate with a subpopulation of the transcription factor IID (TFIID) complex in cells [41, 42]. Furthermore, SARFH, a Drosophila homologue of EWS, has been reported to colocalize with RNA polymerase II at active chromatin [43]. Recently, EWS was found associated with the transcriptional coactivator cyclic AMP-responsive element–binding (CREB) protein (CBP) and the hypophosphorylated RNA polymerase II, both of which are enriched in transcription preinitiation complexes [44, 45]. These interactions indicate that EWS may be involved in gene transcription, in turn suggesting that EWS may function as a coactivator of CBP-dependent transcription factors [45].
To identify cofactors that physically interact and potentially cooperate with Oct-4 in allowing cells to remain in the cycle of totipotency, we conducted a bacterial two-hybrid screen of an ES cell cDNA library using Oct-4 as bait. We found EWS to be a binding partner of Oct-4. We confirmed the interaction between EWS and Oct-4 in vitro using bacterially expressed fusion proteins and in vivo through immunoprecipitation/Western blot analyses. We also demonstrate that Oct-4 and EWS are coexpressed in the ES and carcinoma cells. In transient transfection assays, EWS activated Oct-4–dependent transactivation. These data indicate that transcriptional activity of Oct-4 is modulated by EWS.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P] dCTP (3,000 Ci/mmol) and [35S]-methionine (1,000 Ci/mmol) were obtained from PerkinElmer (Wellesley, MA) and GE Healthcare (Piscataway, NJ), respectively. Preparation of plasmid DNA, restriction enzyme digestion, agarose gel electrophoresis of DNA, DNA ligation, bacterial transformations, and SDS-polyacrylamide gel electrophoresis of proteins were carried out using standard methods [46].
Constructs
To generate pBT–Oct-4, full-length Oct-4 cDNA was amplified from pCEP4/Oct-4 (obtained from Dr. A. Okuda, Saitama Medical School, Saitama, Japan) by polymerase chain reaction (PCR) using primer 5'-Oct4-1 (5'-GATCGGATCCATGGCTGGACACCTGGCT-3') and primer 3'-Oct4-352 (5'-GATCCTCGAGCTATCAGTTTGAATGCATGGG-3'), digested with BamHI and XhoI, and cloned into the same sites of pBT bait vector (Stratagene, La Jolla, CA, http://www.stratagene.com) in frame with the bacteriophage 
cI gene. To construct pTRC·HisA-Oct-4, the BamHI-XhoI fragment of pBT–Oct-4 was subcloned into the same sites of pTRC·HisA vector (Invitrogen, Carlsbad, CA, http://www.invitrogen.com).
To isolate human Oct-4 (hOct-4), total RNA was prepared from human ES cells using RNeasy mini kit (Qiagen, Valencia, CA, http://www.qiagen.com) with on-column DNAse treatment. Then, the messenger RNAs were purified using an Oligodex-dT mRNA mini kit (Qiagen). cDNA was synthesized using Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) as instructed by the manufacturer. For pcDNA3–hOct-4, the human Oct-4 gene was amplified with the human Oct-4 primers 5'-GATCGGATCCATGGCGGGACACCTGGCT-3' and 5'-GATCGAATTCTCAGTTTGAATGCATGGG-3', digested with BamHI and EcoRI, and cloned into the same sites of pcDNA3.
GST-fusion Oct-4 plasmids GST–Oct-4, GST–Oct-4 (NTD), GST–Oct-4 (POU), and GST–Oct-4 (CTD) were generated through the following steps. For GST–Oct-4, plasmid pBT–Oct-4 was digested with BamHI and XhoI and cloned into the same sites of pGEX (4T-1). For GST–Oct-4 (NTD), the NTD of Oct-4 was amplified from pCEP4/Oct-4 by PCR using primers 5'-Oct4-1 and 3'-Oct4-131 (5'-GATCCTCGAGCTAGTCCTGGGACTCCTCGGG-3'), digested with BamHI and XhoI, and cloned into the same sites of pGEX (4T-1). For GST-Oct-4 (POU), the POU domain of Oct-4 was amplified from pCEP4/Oct-4 by PCR using primers 5'-Oct4-122 (5'-GATCGGATCCGTGGAACCAACTCCCGAG-3') and 3'-Oct4-287 (5'-GATCCTCGAGCTATTGGGAATACTCAATACT-3'), digested with BamHI and XhoI, and cloned into the same sites of pGEX (4T-1). For GST-Oct-4 (CTD), the CTD of Oct-4 was amplified from pCEP4/Oct-4 by PCR using primers 5'-Oct4-278 (5'-GATCGGATCCGGCAAAAGATCAAGTATT-3') and 3'-Oct4-352 (5'-GATCCTCGAGCTATCAGTTTGAATGCATGGG-3'), digested with BamHI and XhoI, and cloned into the same sites of pGEX (4T-1).
GST-fusion EWS plasmids were generated by the following steps. For GST-EWS, plasmid pSG5-Flag-EWS (kindly provided by Dr. L. Yang, University of Washington School of Medicine, Seattle) was digested with HindIII, blunted with Klenow fragment, and redigested with EcoRI. The excised fragment was ligated into the EcoRI and SmaI sites of pGEX (4T-1). For GST-EWS (NTD), the NTD of EWS was amplified from pSG5-Flag-EWS by the PCR using primers 5'-EWS-1 (5'-GATCGAATTCATGGCGTCCACGGATTAC-3') and 3'-EWS-295 (5'-GATCCTCGAGCTAACTCATGCTCCGGTTCTC-3'), digested with EcoRI and XhoI, and cloned into the same sites of pGEX (4T-1). For GST-EWS (GRPI), the glycine, arginine, proline-rich motif I of EWS was amplified from pSG5-Flag-EWS by the PC Rusing primers 5'-EWS-290 (5'-GATCGAATTCGAGAACCGGAGCATGAGT-3') and 3'-EWS-350 (5'-GATCCTCGAGCTATGGGCCTAGATCAAGATC-3'), digested with EcoRI and XhoI, and cloned into the same sites of pGEX (4T-1). For GST-EWS (RRM), the RNA recognition motif of EWS was amplified from pSG5-Flag-EWS by the PCR using primers 5'-EWS-352 (5'-GATCGAATTCGTAGATCCAGATGAAGAC-3') and 3'-EWS-463 (5'-GATCCTCGAGCTAGCCCTCACGGGGTGGCAG-3'), digested with EcoRI and XhoI, and cloned into the same sites of pGEX (4T-1). For GST-EWS (GRP II and III), plasmid pSG5-Flag-EWS was digested with MseI and HindIII, repaired with the Klenow fragment, and then ligated into the SmaI site of pGEX (4T-1). The descriptions of plasmids encoding GST-EWS (1–35), GST-EWS (70–163), and GST-EWS (192–265) have been previously described [47].
For expression in mammalian cells, the BamHI and XhoI fragment containing Oct-4 was isolated from pGEX (4T-1)–Oct-4 and subcloned into the same sites of pcDNA3 (Invitrogen) to generate pcDNA3/Oct-4.
To generate DsRed–Oct-4, Oct-4 was amplified with pcDNA3–Oct-4 by PCR using primers 5'-Oct4-1 and 3'-Oct4-351Age (5'-GATCACCGGTGCTCCGTTTGAATGCATGGG-3'), digested with BamHI and AgeI, and cloned into the same sites of pDsRed1-N1 (Clontech, Palo Alto, CA, http://www.clontech.com). For EGFP-EWS, the 5' portion of EWS was amplified from pSG5-Flag-EWS by PCR using primers 5'-EWS-1Hind (5'-GATCAAGCTTATGGCGTCCACGGATTAC-3') and 3'-EWS-250 (5'-AGCTTGGCTGTAGGATCC-3') and digested with HindIII and BamHI. The 3' portion of EWS was amplified from pSG5-Flag-EWS by PCR using primers 5'-EWS-241 (5'-CCACCCCAAACTGGATCC-3') and 3'-EWS-656Age (5'-GGCGACCGGTGGGTAGGGCCGATCTCTGCG-3') and digested with BamHI and AgeI. These excised products were directly ligated with the HindIII- and AgeI-digested pEGFP (N1) vector (Clontech) to generate pEGFP-EWS.
To generate GAL4-EWS (1–295), pGEX (4T-1)-EWS (NTD) was digested with XhoI, repaired with Klenow fragment, and redigested with EcoRI. The excised fragment was subcloned into the EcoRI and SmaI sites of pM vector (Clontech). To construct GAL4-EWS (290–656), the CTD of EWS was amplified by PCR using primer 5'EWS 290 and 3'EWS-656Age, digested with AgeI, and repaired with Klenow fragment. The excised fragment was redigested with EcoRI and subcloned into EcoRI and SmaI sites of pM vector.
The reporter plasmid, pOct-4 (10x) TATA luc, comprises 10 copies of the i-opn element that binds to Oct-4 [48], which was generated by annealing and multimerizing the oligonucleotides 5'-AAGTTAAAATCACATTTGAAATGCAAATGGAAAAGCAAGTTAAAATCACATTTG-3' and 3'-TTCAATTTTAGTGTAAACTTTACGTTTACCTTTTCGTTCAATTTTAGTGTAAAC-5' and precloned into TA vector (Invitrogen). Ten copies of this element were then digested with XhoI and HindIII and recloned into the same sites of the pGL3 Basicvector (Promega, Madison, WI, http://www.promega.com). To construct pOct-4 (10x) TATA luc, the TATA box was generated by annealing the synthetic oligonucleotides 5'TATA (5'-phospho-GATCTGAGGGTATATAATGGAA-3') and 3'TATA (5'-phospho-AGCTTTCCATTATATACCCTCA-3') and subcloned into the BamHI and HindIII sites of the pOct-4 (10x) luc, downstream from the Oct-4–binding sites. The human Rex-1 promoter, which contains an Oct-4 binding site (5'-ATTTGCAT-3') [49], was cloned into the XhoI-HindIII sites of the promoterless pGL3 Basic vector (Promega) by PCR amplification from genomic DNA. The amplification primers used for this purpose were 5'Rex (5'-GATCCTCGAGTCGGGATTCAGAAGAGGC-3') and 3'Rex (5'-GATCAAGCTTGGCGGCTAGGAGTTCAGC-3').
Cell Culture, Transfection, and Reporter Assays
Mouse and human ES cells were grown as previously described [50, 51]. P19, COS-7, HEK293T, or NIH3T3 cells were maintained in Dulbecco’s modified Eagle’s medium supplied with 10% heat-inactivated fetal calf serum (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com), penicillin, and streptomycin. The cells were transiently transfected with plasmids by electroporation using the Gene Pulser II RF module system (Bio-Rad, Hercules, CA, http://www.bio-rad.com) or by lipofection using FuGENE 6 transfection reagent (Roche Diagnostics, Mannheim, Germany, http://www.roche-applied-science.com) as instructed by the manufacturer. Luciferase assays were performed with the dual-Luciferase Assay System, in accordance with the supplier’s protocol (Promega). Renilla luciferase activities were used to normalize the transfection efficiency.
Bacterial Two-Hybrid Screening
To generate mouse ES cell–derived cDNA library, total RNA was extracted from mouse ES cells and cDNA library was constructed using StrataScript reverse transcriptase as instructed by the manufacturer (Stratagene). Also, following the instruction of the manufacturer, the BacterioMatch Two-Hybrid system reporter strain (Stratagene) was transformed sequentially with pBT-Oct-4 and plasmid pTRG-cDNA, which contains the amino-terminal domain of RNA polymerase subunit and a cDNA library from mouse ES cells subcloned into the multiple cloning site (Stratagene). Positive clones were screened for their potential to grow on selective medium and for the expression of a reporter enzyme, ß-galactosidase. After secondary screening assays eliminated false positives, plasmids containing potential Oct-4–binding partners were isolated and sequenced.
Northern Blot Analysis
For Northern blotting, total cellular RNAs were prepared using Trizol reagent (Invitogen), and aliquots (10 µg/lane) were separated on 1.5% agarose-formaldehyde gels. RNA was transferred to Hybond nylon membranes (Amersham) and crosslinked to the membrane in a GS Gene Linker UV Chamber (Bio-Rad). The PstI and XhoI (0.65 kb), HindIII and EcoRI (0.98 kb), or EcoRI and BamHI (0.73 kb)-digested DNA fragments from mouse Oct-4, human Oct-4, or EWS cDNAs, respectively, were gel purified and 32P-labeled using a Prime-It II Random Primer Labeling kit (Stratagene). Hybridizations, using the radiolabeled probe, were carried out overnight in Express Hyb Solution (Clontech) at 68°C. The blots were washed twice at 68°C with 2 x standard saline citrate/0.1% SDS, and radiolabeled bands were visualized by autoradiography.
Recombinant Six Histidine–Tagged Oct-4 Protein Purification
The recombinant pTRC·HisA-Oct-4 plasmid was transformed into Escherichia coli, the expression of (His)6-Oct-4 was induced by isopropyl-ß-D-thiogalactopyranoside, and the recombinant protein was purified by chromatography on Ni-nitrilotriacetic acid (NTA)–agarose in accordance with the supplier’s protocol (Qiagen). In brief, the cell pellet from a culture was resuspended in lysis/wash buffer (50 mM Tris·HCl [pH 8.0], 300 mM NaCl, 10 mM imidazole) and clarified by centrifugation (16,000 rpm for 15 minutes) at 4°C, and the supernatant was incubated with NTA-agarose resin for 1 hour at 4°C on a slowly rotating wheel. The Oct-4–bound NTA-agarose resin was then packed into a Micro Bio-Spin Chromatography Column (Bio-Rad) and washed extensively with lysis/wash buffer. The protein was eluted with elution buffer (50 mM Tris·HCl [pH 8.0], 300 mM NaCl, 20 mM imidazole). The elution profile of Oct-4 protein was monitored by Western blotting using monoclonal Xpress antibody (Invitrogen).
In Vitro Transcription and Translation
In vitro transcriptions and translations were performed in the presence of [35S]-methionine in rabbit reticulocyte lysates using the TNT T7–coupled reticulocyte system, as specifically instructed by the manufacturer (Promega).
GST Pull-Down Assays
GST pull-down assays were performed as described previously [52] using GST-fusion protein and [35S]-methionine–labeled EWS or recombinant (His)6-tagged Oct-4. Bound proteins were detected using autoradiography or immunoblotting as previously described [47].
Immunoprecipitation and Western Blot Analysis
For Oct-4/EWS immunoprecipitation, plasmid pSG5/Flag-EWS was cotransfected into COS-7 cells with either pcDNA3 alone or pcDNA3/Oct-4. After 48 hours, cell pellets were disrupted in ice-cold NP-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 x complete protease inhibitor cocktail solution [Roche Diagnostics]) on ice for 30 minutes. Supernatants were then collected by centrifugation at 16,000g for 15 minutes at 4°C and incubated for 1 hour with monoclonal antibody directed against Oct-4 (C-10, Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com).Complex precipitated with protein G-Sepharose (Pierce, Rockford, IL, http://www.piercenet.com) was washed four times with ice-cold NP-40 lysis buffer. The immunoprecipitated proteins were eluted in SDS loading buffer (62.5 mM Tris·HCl [pH 6.9], 10% glycerol, 2% SDS, 5% ß-mercaptoethanol), separated on 8% SDS-polyacrylamide gel, and transferred to polyvinylidene difluoride (PVDF) membrane. EWS was detected by immunoblotting using M2 anti-Flag monoclonal antibody (Sigma, St. Louis, http://www.sigmaaldrich.com), whereas Oct-4 was detected with the same antibody as described above. Detection was performed by chemiluminescence using Western Lightening (PerkinElmer Life Sciences, Boston, http://www.perkinelmer.com). Coimmuno-precipitation experiments of Oct-4 and EWS in P19 cells were performed as described in earlier reports [53, 54].
Subcellular Localization Experiment
COS-7 cells were plated on glass coverslips and transfected with the respective DNA plasmids using FuGENE 6 following the manufacturer’s instructions. After 48 hours of transfection, the cells were washed in phosphate-buffered saline (PBS) and fixed for 10 minutes at –20°C in a mixture of acetone and methanol (1:1, vol/vol). The coverslips were mounted with 50% glycerol/PBS. Green or red fluorescence was detected under a fluorescence microscope (IX51, Olympus, Tokyo, http://www.oylmpus.com) equipped with a CoolSNAP digital camera (Olympus).
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RESULTS |
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As depicted in Figure 1A
, the kanamycin-resistant reporter strain for the bacterial two-hybrid system was transformed with a series of plasmids containing Oct-4, EWS, or appropriate controls. All bacterial reporter cells harboring both pBT fusions (chloramphenicol-resistant) and pTRG fusions (tetracycline-resistant) were able to grow on Lurina-Bertani (LB) medium containing kanamycin (+Kan), chloramphenicol (+Chl), and tetracycline (+Tet), indicating that both plasmids are present (Fig. 1B). However, apart from the LGF2-Gal11 interaction, which serves as a positive control, we found that only the expression of both pBT–Oct-4 and pTRG-EWS in bacterial reporter cell line allowed growth on +Kan/+Chl/+Tet LB plates containing 0.47 or 0.59 mM carbenicillin, indicating a physical interaction between Oct-4 and EWS (Figs. 1C, 1D).
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, two upper panels).
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, third panel). The EWS gene is expressed in all cell lines investigated, including ES and EC cells. These results clearly show that Oct-4 and EWS are expressed in ES and EC cells. As a control for RNA loading, ethidium bromide staining of 28S rRNA indicated relatively equal amounts of total RNA present from the different cell lines analyzed (Fig. 2, lower gel).
Oct-4 Interacts with EWS In Vitro and In Vivo
To further define the specificity of the Oct-4/EWS interaction identified in the bacterial two-hybrid screen, we performed in vitro GST pull-down assays (Fig. 3A). Bacterially expressed GST or GST–Oct-4 fusion proteins immobilized on glutathione-Sepharose beads were mixed with in vitro–produced [35S]-methionine–labeled EWS protein. After extensive washing, the bound proteins were analyzed by 8% SDS-PAGE and autoradiography. As shown in Figure 3A, approximately 10% of input EWS protein was specifically retained on the Oct-4–conjugated Sepharose beads. Because EWS did not bind to GST alone (Fig. 3A, lane 2), the interaction was considered to be specific.
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, top panel) or EWS (bottom panel). After extensive washing of the beads, the bound proteins were eluted with sample buffer, separated by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane, and probed with anti-sera to EWS. GST–Oct-4 beads, but not control GST beads, efficiently retained endogenous EWS (Fig. 3B, top panel). In the reciprocal experiment, the GST-EWS fusion protein was also able to interact with endogenous Oct-4 (Fig. 3B, bottom panel). These results indicate that the proteins made in bacterial cells, synthesized by in vitro transcription/translation, and from cell lysates seem to be equivalent with respect to Oct-4–EWS interaction.
To determine whether the interaction between Oct-4 and EWS occurs in vivo, we performed immunoprecipitation experiments after transient transfection of COS-7 cells with expression vectors that expressed both proteins. Plasmid pcDNA3/Oct-4 or pcDNA3 was cotransfected with pSG5/Flag-EWS into COS-7 cells. Cells were lysed for 48 hours after the transfection and Oct-4 protein–immunoprecipitated with an -Oct-4 antibody (C-10, Santa Cruz Biotechnology). Immunoblotting was performed on eluents using an anti-Flag antibody (M2, Sigma) to detect the presence of EWS. EWS was found to specifically coprecipitate with Oct-4 (Fig. 3C, top panel). Probing for EWS (middle panel) or Oct-4 (lower panel) indicated the presence of the two proteins in the extracts from the transfected cells. To further examine whether endogenous Oct-4 and EWS associate in mammalian cells in vivo, immunoprecipitation experiments were also performed with cell extract from P19. Antibody against Oct-4 (C-10, Santa Cruz Biotechnology), and not control serum, coprecipitated EWS (Fig. 3D, top panel), whereas anti-EWS (C-19, Santa Cruz Biotechnology) coprecipitated Oct-4 (Fig. 3D, lower panel). These results suggest that Oct-4 and EWS can associate in vivo.
However, neither the in vivo interaction in bacterial (Fig. 1) or mammalian cells (Figs. 3C, 3D) nor the GST pull-down assay using in vitro–translated EWS protein (Fig. 3A) rules out the possibility that the observed interaction may occur through an intermediate bridging partner. Therefore, to address this, Oct-4 was expressed as a six histidine–containing fusion protein in E. coli and purified by Ni+2-NTA agarose resin. A GST pull-down assay was then performed using recombinant (His)6-Oct-4 with GST or GST-EWS fusion proteins, which had also been produced in E. coli. After extensive washing, the amount of Oct-4 retained was determined by SDS-PAGE and Western blotting with an -Xpress antibody (Clontech). As shown in Figure 3E, bacterially produced GST-EWS protein interacts with recombinant Oct-4, suggesting that the Oct-4-EWS interaction does not require an adaptor protein.
The POU Domain of Oct-4 Is Involved in EWS Interaction
To define the minimal region within Oct-4 required for binding to EWS, in vitro binding experiments were performed with truncation forms of Oct-4 functional domains. The structure of the Oct-4 deletion mutants used in the study is shown schematically in Figure 4A. First, we performed GST pull-down assays using in vitro translated [35S]-methionine–labeled EWS protein, together with deletion mutant Oct-4 fusion proteins. As shown in Figure 4B, EWS bound to GST-Oct-4 (POU) but did not interact with GST-Oct-4 (NTD) or GST-Oct-4 (CTD). The GST-Oct-4 (POU) showed similar binding affinity with full-length Oct-4 protein, indicating that the POU domain of Oct-4 contains the domains responsible for EWS interaction (J.L. and J.K., unpublished data). The same amounts of GST fusion proteins were used in these assays, as confirmed by fractionation on 15% SDS-PAGE (J.L. and J.K., data not shown).
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). To delineate the amino acids in EWS responsible for the interaction with Oct-4, we fused EWS (NTD) (amino acids 1 through 295), EWS (1–35) (amino acids 1 through 35), EWS (70–163) (amino acids 70 through 163), EWS (192–265) (amino acids 192 through 265), EWS (GRP I) (amino acids 290 through 350), EWS (RRM) (amino acids 352 through 463), or EWS (GRP II and III) (amino acids 441 through 656) to GST. These truncation mutants were then individually expressed as GST fusion proteins in E. coli and coupled to glutathione-Sepharose beads. After incubation with recombinant His6-tagged Oct-4 protein and after extensive washing, we found that four GST fusions containing the NTD (70–163), GRP I, and GRP II and III domains specifically retained Oct-4 protein (Fig. 5B), whereas Oct-4 did not interact with GST alone (Fig. 5B, lane 2), GST-EWS (1–35) (lane 4), or GST-EWS (RRM) (lane 8). This result suggests that EWS has at least three sites (amino acids 70 through 163, GRP I, and GRP II and III) that can bind to Oct-4 independently.
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). For this purpose, we constructed plasmids expressing fluorescent DsRed or GFP fusion proteins of Oct-4 or EWS, respectively. A pDsRed1–Oct-4 construct that expresses DsRed–Oct-4 protein was transiently transfected into COS-7 cells, and subcellular localization of the protein was detected by fluorescent microscopy for red fluorescence. In transiently transfected cells, the DsRed-tagged Oct-4 protein was clearly localized to the nucleus (Figs. 6Aa, 6Ab). In addition, we transfected enhanced green fluorescent protein (EGFP) fusion EWS construct into the same cell lines to determine its intracellular localization using green fluorescence. As shown in Figures 6Ac and 6Ad, COS-7 cells transiently transfected with EGFP-tagged EWS also contained EWS protein to the nucleus. To further examine a possible colocalization of Oct-4 and EWS proteins, we analyzed the simultaneous expression of these constructs in cotransfected cells. Both DsRed-tagged Oct-4 and GFP-tagged EWS proteins were distributed in a more or less fine punctate pattern in the nucleus (Figs. 6Ae–6Ah). Although they displayed a very similar pattern, Oct-4 seemed to be more evenly distributed. The overlay image indicated that Oct-4 and EWS in the nucleoplasm partially overlapped (Fig. 6Ag).
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-Oct-4 (C-10, Santa Cruz Biotechnology) or -EWS (C-19, Santa Cruz Biotechnology) antibodies. As shown in Figure 6B, both proteins are exclusively localized to the nucleus in P19 cells, suggesting that the nuclear localization of Oct-4 and EWS proteins was not affected by the nature of transient transfection or by the cell line studied. Therefore, we could conclude that Oct-4 and EWS proteins colocalize or are in close proximity in cells.
EWS Activates Oct-4–Mediated Transactivation
Given the suggested physical association and colocalization between Oct-4 and EWS in vitro and in vivo, we investigated the potential functional consequence of the interaction between Oct-4 and EWS. For these assays, we constructed an Oct-4 reporter plasmid, pOct-4 (10x) TATA luc, containing 10 copies of Oct-4–binding sites and a TATA box cloned upstream of the luciferase gene (Fig. 7A). The effect of EWS was examined on gene expression from this reporter plasmid by introducing pcDNA3–Oct-4 with or without pSG5-EWS in 293T cells. As shown in Figure 7A, Oct-4 activated gene expression from the pOct-4 (10x) TATA luc reporter by 18-fold (lane 3). However, cotransfection with the EWS expression construct led to a 53-fold increase in reporter expression (~300% augmentation by the effect of EWS) (lane 4), with no significant effect on the basal transcription level (lane 2). Similarly, EWS augmented Oct-4–mediated gene expression from human Rex-1 promoter containing one binding site for Oct-4 (Fig. 7B). These results strongly indicate that EWS potentiates Oct-4–mediated transactivation.
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). The EGFP expression plasmid served as an internal control for monitoring transfection efficiency (bottom panel). Fractionated cell extracts probed by anti–Oct-4 antibody demonstrated no increase in exogenously expressed Oct-4 protein (top panel).
To dissect how EWS activates Oct-4–mediated transcription, we created fusion proteins in which the GAL4 DNA-binding domain was fused to NTD (aa 1–295) or CTD (aa 290–656) domains of EWS, respectively (Fig. 7D). The pG5 luc reporter contains five GAL4 DNA-binding sites upstream of the TATA box and was used as a reporter in these experiments. An expression vector driving the synthesis of only the GAL4 DNA-binding domain, pcDNA3/GAL4, had no effect on the level of luciferase produced from pG5 luc when transfected into 293T cells (Fig. 7D, lane 1). Interestingly, pcDNA3/GAL4-EWS (1–295) strongly activated luciferase production from pG5 luc by 150-fold (lane 2), indicating that the NTD of EWS protein has intrinsic transcription activation property. On the other hand, EWS (290–656) (lane 3) was not a potent transactivator when fused to the DBD of GAL4. Furthermore, pcDNA3/EWS (1–295), which lacks a GAL4 DNA-binding domain, did not activate luciferase expression from pG5 luc (J.L. and J.K., data not shown). This suggests the need for the NTD of EWS to bind to the reporter construct to achieve activation of transcription. These results demonstrate that the NTD of EWS is capable of activating transcription. In sum, these results suggest that EWS specifically activates Oct-4–mediated transcriptional activation through its physical interaction with Oct-4.
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DISCUSSION |
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Oct-4 encodes a POU transcription factor that is expressed by all pluripotent cells during embryogenesis and is also abundantly expressed by ES, embryonic germ, and EC cell lines [17, 56, 59, 63, 64]. Differentiation of totipotent cells to somatic lineages occurs at the blastocyst stage and during gastrulation, coinciding with Oct-4 downregulation. The unique Oct-4 expression pattern in the mouse embryo led to the hypothesis on the totipotent cycle [65]. Consistent with this speculation, Oct-4–null embryos die at the time of implantation due to a failure to form the ICM [20]. These results demonstrate that Oct-4 is required for preventing somatic differentiation of the ICM and is important for maintaining the undifferentiated state during embryonic development. Despite the fact that Oct-4 function is critical for controlling totipotency of ES cells and promoting tumorigenesis in human tissues, there is still little known about the detailed molecular mechanisms and the proteins that regulate Oct-4 function.
Thus, in search of proteins that can regulate Oct-4 function, we set up a bacterial two-hybrid screening system [55]. Using a mouse ES cell cDNA library, five positive clones were isolated and further characterized by DNA sequencing. A BLAST search indicated that we isolated a cDNA encoding of EWS. EWS is a putative proto-oncogene [37], coactivator [44, 45], and RNA-binding protein [37] originally identified as a fusion protein with Fli-1 in Ewing’s sarcoma [37] and later found in several additional malignancies in which its NTD is fused to the DNA-binding domains of several DNA-binding proteins such as ERG, ETV1, E1A-F, FEV, WT1, ATF1, CHOP, and TEC [39]. The expression of both pBT–Oct-4 and pTRG-EWS in a bacterial two-hybrid system allowed growth on a +Kan/+Chl/+Tet LB plate containing carbenicillin, indicating a physical interaction between Oct-4 and EWS in bacterial cells (Fig. 1). In addition, several lines of evidence indicate that Oct-4 and EWS interact within the cell: Oct-4 mRNA is coexpressed with EWS in self-renewal ES and EC cells that we have examined (Fig. 2); Oct-4 associates with EWS in vitro, indicating that they can interact directly (Figs. 3A, 3B, 3E); and Oct-4 colocalizes with EWS in the nucleus (Fig. 6) and can be immunoprecipitated with EWS in vivo (Figs. 3C, 3D), suggesting that the interaction between Oct-4 and EWS occurs under physiological conditions.
EWS shares an extensive sequence similarity with a subgroup of TLS and hTAFII68, which are two human nuclear RNA-binding proto-oncoproteins that are products of genes commonly translocated in human sarcomas [39, 66]. These proteins have been found associated with TFIID complexes and copurified with the human RNA polymerase II, suggesting the involvement of transcriptional activation [66]. TFIID is a multiprotein complex composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs) and is the factor that nucleates preinitiation complex formation on protein-coding genes [67]. The TAFs in TFIID are essential for activator-dependent transcription.
The EWS NTD shows unique structural features. It contains large numbers of tyrosine, glutamine, alanine, serine, threonine, glycine, and proline residues, some of which are organized in a repeated and degenerated peptide motif with a frequently occurring serine-tyrosine dipeptide (NSYGQQS) [37]. A database search revealed that it shares homology with the CTD of the large subunit of eukaryotic RNA polymerase II [37]. This subunit was efficiently phosphorylated on tyrosine residues by c-Abl tyrosine kinase, and the sites of phosphorylation were found to be within the CTD of the RNA polymerase II [68]. The consequence of this phosphorylation event on RNA polymerase II is to convert transcriptionally paused complexes into elongation-competent molecules [69]. Thus, it will be important to assess the tyrosine phosphorylation status of EWS by c-Abl in ES and EC cells or human testicular germ cell tumors, because it shares homology to the C-terminal region of eukaryotic RNA polymerase II [37] and EWS/ WT1, one of EWS-fusion forms, is also a target for c-Abl [52].
We have mapped the interacting domains and demonstrated that Oct-4 binds with the EWS through its POU domain (Fig. 4). Additionally, three independent regions in the EWS, encompassing amino acids 70 through 163 in the NTD, amino acids 290 through 350, and amino acids 441 through 656 in the CTD of EWS, seem important for binding to Oct-4 (Fig. 5). There are several possible models suggested by these data. One is that single EWS binds to three Oct-4 molecules. Another is that three binding sites of EWS have different binding affinities, and there may be a dynamic interaction with Oct-4 in which Oct-4 binds to one site, after which it is transferred to another site during the activation. Lastly, Oct-4–related molecules can bind to some of the other sites through the POU domain. However, additional experiments will be required to distinguish between these possibilities.
Because EWS does not have a DNA-binding motif or significant DNA-binding activity to specific gene promoters, it is suggested that EWS acts as a coactivator of transcription if it is recruited to the vicinity of the promoter. Thus, to confirm this speculation, we investigated the functional consequences of the interaction between Oct-4 and EWS in transient transfection assays. As shown in Figures 7A and 7B, ectopic expression of EWS stimulates Oct-4–dependent transcriptional activation. Association of the EWS to Oct-4, mediated by their binding domains, may stably tether EWS to DNA and increase the activity of intrinsic activation domains within EWS. EWS and/or Oct-4 and Oct-4 and DNA interactions could also lead to reciprocal conformation changes that expose latent activation domains within EWS.
However, unlike intensively studied coactivators, such as p300 and CBP [70–72], the precise transactivation mechanism of EWS as a coactivator has yet to be demonstrated. In light of characterization of other coactivator proteins, there are several possible models for EWS. First, EWS may promote Oct-4–mediated gene expression by bridging between Oct-4 and the basal transcription machinery. It has been recently reported that EWS associates with important factors for transcription, such as a basal transcription factor TFIID [41, 42], a transcriptional coactivator CBP [44, 45], and the hypophosphorylated RNA polymerase II form [44]. Second, EWS may recruit transcription machinery–modifying enzymes necessary for transcription initiation or elongation steps. Protein tyrosine kinase c-Abl efficiently phosphorylates on tyrosine residues of the C-terminal domain of eukaryotic RNA polymerase II [68], and the consequence of this phosphorylation event on RNA polymerase II is to convert transcriptionally paused complexes into elongation-competent molecules [69]. Interestingly, the NTD of EWS contains two putative c-Abl SH3 domain binding sites [52]. Third, EWS may act as an adaptor molecule linking gene transcription and mRNA processing by interacting with splicing factors. It has been reported that transcriptional and post-transcriptional processing are closely coupled events in vivo [73], and, based on the structural features, it is likely that EWS is most likely to become involved in mRNA synthesis and splicing. Consistent with this speculation, EWS also interacts with the several splicing factors SF1 [74], U1C [75], TARS-1/TRSR-2 translocation liposarcoma protein–associated serine-arginine protein [76], and Y-box protein-1 [77].
Transcriptional coactivator proteins operate at the end points of a variety of signal transduction pathways, thereby modulating specific gene expression programs involved in cell growth, differentiation, homeostasis, and viral pathogenesis [78, 79]. We have reported that EWS/WT1 associates with several signaling proteins, such as SH3 domains of v-Src, c-Abl, and Crk (N) and SH2 domains of v-Src, Grb2, Fyn, and GAP (N+C) [47]. Although it is not clear whether EWS itself interacts with signal-dependent transcription factors, the ability of EWS-fusion protein to interact with several cell-signaling molecules and to modulate its function by signaling proteins (47, 52) suggests that EWS may function as a signal integrator by coordinating complex signal transduction events at the transcriptional level. In fact, CBP and p300 have been proposed to mediate transcription induction via intrinsic and associated histone acetytransferase activities, which may facilitate binding of nuclear factors to their target sites by destabilizing promoter-bound nucleosomes [80]. Thus, it would be valuable to test whether EWS also has these types of activity.
In conclusion, the findings of this study provide additional evidence that Oct-4 function can be modulated by EWS. Oct-4 likely contributes to maintaining cells in an undifferentiated, pluripotent, or totipotent state in two ways: by activating certain key genes [48, 81–83] and by silencing others [12, 14, 84]. This EWS-mediated Oct-4 regulation may be a key mechanism that can stimulate cells of the ICM from expressing products that prevent differentiation toward the trophectoderm. In addition, it would be interesting to determine whether EWS can collaborate with Oct-4 to transform cells in human testicular germ cell tumor.
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