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Laboratory of Animal Molecular Technology, Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara 630-0192,1 First Department of Anatomy, Osaka City University Medical School, Osaka 545-8585,2 Laboratory of Pluripotent Cell Studies, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan3
Received 8 August 2002/ Returned for modification 1 November 2002/ Accepted 19 December 2002
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ABSTRACT |
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INTRODUCTION |
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POU transcription factor Oct3/4 is expressed specifically in pluripotent cells, including ES cells, early embryos, and germ cells (27, 31). Targeted disruption of the Oct3/4 gene in the mouse results in early embryonic lethality (21). The inner cellular mass of Oct3/4-null blastocysts differentiates exclusively into trophoblasts. Furthermore, conditional deletion of Oct3/4 in ES cells leads to spontaneous differentiation into trophectoderm (25), demonstrating that Oct3/4 is essential for self-renewal of ES cells and mouse early development.
Only a few Oct3/4 target genes have been identified. These include FGF-4 (4) and Rex-1 (2), in which Oct3/4 binds to an octamer motif, ATT(T/A)GCAT, located in regulatory elements. In FGF-4, SRY-related transcription factor Sox2 binds to a motif adjacent to the octamer sequence and synergistically activates transcription (5). In Rex-1, hypothetical factor ROX1 functions in a similar manner (2). It is not clear whether synergetic interaction with other transcription factors is common among target genes. Even consensus nucleotide sequences of Oct3/4-binding sites have not been fully determined. For example, the Oct3/4-binding site in UTF1 is one nucleotide different from the octamer sequence (22). Furthermore, it remains largely unknown how Oct3/4 maintains self-renewal of ES cells. Identification of novel Oct3/4 target genes is crucial to answering these questions.
In this study, we utilized expression analyses, reporter gene analyses, and a gel mobility shift assay to demonstrate that Fbx15, which encodes an F-box-containing protein (35), is a novel target of Oct3/4. We also performed gene-targeting experiments to study physiological functions of Fbx15 in self-renewal of ES cells, mouse development, and fertility.
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MATERIALS AND METHODS |
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Cell culture. The RF8 (16), JI (13), CGR8 (20), and MG1.19 (9) ES cell lines were cultured as previously described. Differentiation of ES cells was induced with retinoic acid as previously described (36). NIH 3T3 cells were cultured with Dulbecco's modified Eagle medium (Sigma) containing 10% fetal bovine serum (Sanko Junyaku, Tokyo, Japan) and maintained at 37°C with 5% CO2.
Northern blot analyses. Total RNAs were prepared from RF8 and MG1.19 ES cells with Trizol (Invitrogen). Total RNAs from untreated and tetracycline-treated ZHBTc4 ES cells were prepared as previously described (23). Total RNAs from 12 adult mouse tissues were purchased from Sawady Technology (Tokyo, Japan) and Funakoshi (Tokyo, Japan). Northern blot analyses were performed with formalin agarose gels as previously described (36, 37). As a probe, we used a NotI/SalI fragment of an EST clone (accession number AA571680) containing Fbx15 cDNA.
Reverse transcription (RT)-PCR.
First-strand cDNA was synthesized from total RNA with ReverTra Ace-
(Toyobo). Fbx15, Oct3/4, and NAT1 were amplified with primers U1110 and L1431, Oct3-U474 and Oct3-L935, and NAT1-U10 and NAT1-L21, respectively. The sequences of the primers used in this study are available upon request.
Western blot analyses. ES cell extracts were prepared with M-Per (Pierce), separated on sodium dodecyl sulfate (SDS)-14% polyacrylamide gels, and transferred to nitrocellulose membranes (Millipore). The primary antibodies used were anti-Skp1 (SC1568, 1/600 dilution; Santa Cruz), anti-hemagglutinin (anti-HA; SC1568, 1/600; Roche), anti-myc (SC789, 1/600; Santa Cruz), and anti-GST (SC459, 1/2,000; Santa Cruz). The secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (SC2030, 1/5,000; Santa Cruz) and horseradish peroxidase-conjugated anti-rat immunoglobulin G (SC2006, 1/5,000; Santa Cruz).
5' RACE. Fbx15 transcription initiation sites in ES cells were determined with the 5' rapid amplification of cDNA ends (5' RACE) system, version 2.0 (Invitrogen). The primers used were L1102 for cDNA synthesis and L782 and L350 for PCR.
Determination of the nucleotide sequence of the 5' flanking region. A bacterial artificial chromosome (BAC) clone containing the Fbx15 gene was identified from mouse BAC library DNA pools (Research Genetics) by PCR-based screening with primers U889 and L1102. BAC DNA was directly sequenced with primers Fbx-AS1, Fbx-AS2, Fbx-AS3, and Fbx-AS4 as previously described (37).
Construction of luciferase reporter plasmids. A DNA fragment (-526 to +145) was amplified from the BAC clone by PCR with primers mFbx-pro.U637 and mFbx-pro.L1337 and cloned into pCR2.1 (Invitrogen). An EcoRI/MunI fragment of this was cloned into the EcoRI site of pGV-BM2, which we made from pGV-B (Promega) by inserting an EcoRI/XbaI linker into the KpnI site. The resulting plasmid was designated p(-526/+114)LUC and used to construct reporter plasmids with longer fragments as follows. PCR products amplified from the BAC clone with the primers described below were cloned into the pCR2.1 vector. DNA inserts were then isolated by EcoRI digestion and cloned into the EcoRI site of p(-526/+114)LUC. The upper primers used were Fbx-pro.U95 and Fbx-pro.U 573 for DNA fragments starting at positions -1120 and -640. The common lower primer used was mFbx-pro.L688, whose 5' end was at position -504.
Construction of reporter plasmids with Fbx15 enhancer and minimum thymidine kinase promoter. A PCR product amplified from the BAC clone with primers Fbx-pro.U573 and mFbx-pro.L688 was cloned into pCR2.1. An EcoRI fragment of the resulting plasmid, which contained the Fbx15 enhancer (-640 to -527), was blunt end ligated into the BglII site of pTA-Luc (Clontech). We constructed reporter plasmids with deleted or mutated sequences by inserting various linkers into the MluI/BglII site of pTA-Luc. The linker sequences are available upon request.
Construction of entry vectors for the Gateway cloning system. We utilized Gateway cloning technology (Invitrogen) to construct expression vectors for various genes. In this method, genes of interest were first cloned into pDONR201 to construct entry vectors, which were then recombined with destination vectors to construct expression vectors. Coding regions of Fbx15, Skp1, and Cul1 were amplified by PCR from EST clones (accession numbers: AA571680 for Fbx15, AA08144 for Skp1, and BE533604 for Cul1). The primers used were fbx-gw-S and fbx-gw-AS for Fbx15, fbxC-gw-S and fbxC-gw-AS for the C-terminal portion of Fbx15 (amino acids 297 to 473), Skp1-gw-S and Skp1-gw-AS for Skp1, and Cul1-gw-S and Cul1-gw-AS for Cul1. Sox2 cDNA was amplified by RT-PCR from mouse ES cell total RNA with primers Sox-2-gw-S and Sox-2-gw-AS. The PCR products were reamplified with primers attB1-AD and attB2-AD and cloned into pDONR201 to construct pDONR-Fbx15, pDONR-Fbx15C, pDONR-Skp1, pDONR-CulI, and pDONR-Sox2.
Construction of destination vectors for the Gateway cloning system. An NdeI/EcoRI fragment of pCX-EGFP (enhanced green fluorescent protein) containing the CAG promoter (26) was inserted into the same site of pIRES-neo (Clontech) to construct pCAG-IRES-neo. the Gateway refA cassette was blunt end ligated into the EcoRI site to construct pCAG-gw-IRES-neo. The Gateway refC cassette was blunt end ligated into the EcoRI/XbaI site of pCS2+MT (17) to construct pMyc-gw. An NheI/BamHI fragment of pEGFP-C2 (Clontech) was inserted into the same site of pcDNA3.1 (Clontech) to construct pcDNA3.1-EGFP. The Gateway refC cassette was blunt end ligated into the XhoI site of pcDNA3.1-EGFP to construct pcDNA3.1-EGFP-gw. pDEST17 was purchased from Invitrogen.
Construction of expression vectors. The expression vectors constructed by a recombination reaction between the entry vectors and destination vectors were pMyc-Fbx15, pMyc-Skp1, pCAG-EGFP-Skp1, pDEST17-Fbx15C, and pCAG-Sox2-IRES-neo. pCAG-Oct3/4-IRES-neo was constructed as follows. An EcoRI/BclI fragment of the plasmid containing Oct3/4 cDNA (27) was inserted into the EcoRI/BamHI site of pIRES-neo (Clontech) to construct pCMV-Oct3/4-IRES-Neo. An NdeI/EcoRI fragment of pCAG-IRES-Neo containing the CAG promoter was inserted into the same site of pCMV-Oct3/4-IRES-Neo to construct pCAG-Oct3/4-IRES-Neo.
Construction of episomal expression plasmids. BamHI/NotI fragments of pMyc-Fbx15 and pMyc-Skp1 and an Eco47III/XbaI fragment of pCAG-EGFP-Skp1 were blunt end ligated into the XhoI site of pCAGIP (24) to construct pCAGIP-Myc-Fbx15, pCAGIP-Myc-Skp1, and pCAGIP-EGFP-Skp1. pCAGIP-HA-gw (K. Takahashi et al., unpublished data) was recombined with pDONR-Cul1 and pDONR-Fbx15 by LR reaction to construct pCAGIP-HA-Cul1 and pCAGIP-HA-Fbx15.
Luciferase reporter assay. DNA transfection into ES and NIH 3T3 cells was performed with Lipofectamine 2000 (Invitrogen) in accordance with the manufacture's protocol. After 24 h, cell lysates were collected and luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega) as described elsewhere (37).
Immunoprecipitation. Twenty-microgram samples of expression vectors were introduced into MG1.19 ES cells grown in 10-cm-diameter dishes. After 24 h, cell lysates were corrected with 500 µl of M-PER (Pierce) supplemented with 10 µl of protease inhibitor cocktail (Nacalai Tesque). Myc-tagged proteins were precipitated with agarose-conjugated anti-myc antibody sc40AC (Santa Cruz), separated by SDS-polyacrylamide gel electrophoresis (PAGE), and analyzed by Western blotting.
Generation of anti-Fbx15 polyclonal antibody. pDEST17-Fbx15C was transformed into BL21-SI (Invitrogen). Recombinant protein was induced in accordance with the manufacture's protocol. Purification of histidine-tagged Fbx15C was performed with Ni-nitrilotriacetic acid agarose (Qiagen) under denaturing conditions. In brief, cells were lysed with buffer A and incubated with Ni-nitrilotriacetic acid resin. After washing with buffers C and D, histidine-tagged protein was eluted with buffer E. Purified proteins were dialyzed in phosphate-buffered saline containing 6 M urea overnight at 4°C. New Zealand White rabbits were immunized with this recombinant protein to generate anti-Fbx15 serum.
Gel mobility shift assay. pCAGIP-HA-Oct3/4 and pCAGIP-HA-Sox2 (M. Maruyama et al., unpublished data) were introduced into Cos7 cells with Fugene 6 (Roche). Cell lysates were collected with extraction buffer (20 mM HEPES [pH 7.8], 450 mM NaCl, 0.4 mM EDTA, 0.5 mM dithiothreitol, 25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitor cocktail and then dialyzed to remove salt. F9 embryonic carcinoma cell extract was purchased from Funakoshi. The DNA mobility shift assay was performed as described by Dent and Latchman (7), except that 0.5x Tris-borate-EDTA buffer was used for electrophoresis, gels were prerun for 10 min, and poly(dG-dC) was used instead of poly(dI-dC).
Targeted disruption of the mouse Fbx15 gene. A targeting vector with promoter trap selection was designed to replace exons 3 to 7 of the mouse Fbx15 gene, which contain the F-box domain, with an IRES (internal ribosome entry site)-ß-geo (fusion of ß-galactosidase [ß-gal]- and neomycin resistance-encoding genes) cassette (19). A 1.4-kbp fragment containing intron 1 to exon 3 and a 3.5-kbp fragment containing exons 7 and 8 were amplified from the BAC clone and used as the 5' and 3' homologous regions of the targeting vector. The 5' arm was amplified by using a TOPO Walker Kit (Invitrogen) with primer L289 for extension and nested primer L255 for PCR. The 3' arm was amplified by PCR with primers U939 and L1102. An IRES-ß-geo cassette was ligated between the two DNA fragments. The resulting targeting vector was linearized by NotI digestion and introduced into RF8 ES cells by electroporation (16). Genomic DNAs from G418-resistant colonies were screened for homologous recombination by Southern blot analyses. For 5' recombination, genomic DNA was digested with HindIII, separated on a 0.8% agarose gel, and transferred to a nylon membrane. Hybridization with a 600-bp probe from intron 1 produced a 10-kbp band from the wild-type locus and an 8-kbp band from the targeted locus. For 3' recombination, the same membrane was hybridized with a 300-bp probe from intron 8, which produced a 9-kbp band from the wild-type locus and a 10-kbp band from the targeted locus.
Genotyping of mice and ES cells. After identifying ES cell clones that were correctly targeted, we determined genotypes of mice and ES cells with a three-primer PCR. The first sense primer, -/-screeningS2, was designed from intron 6 to amplify the wild-type locus. The second sense primer, ß-geo screening1, was designed from the ß-geo cassette to amplify the targeted locus. A single antisense primer, -/-screeningAS2, was designed from intron 7 to amplify both the wild-type and targeted loci. PCR with these primers produces a 280-bp fragment from the wild-type locus and a 725-bp fragment from the targeted locus. PCR was performed with the Expand Long Template PCR system (Roche) in accordance with the manufacturer's protocol. The PCR program consisted of initial denaturation at 94°C for 2 min; 35 cycles of 94°C for 30 s, 53°C for 30 s, and 68°C for 1 min; and a final extension at 68°C for 7 min.
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RESULTS |
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To determine whether Fbx15 is indeed expressed predominantly in ES cells, we studied its expression level by RT-PCR analyses. We found a high level of Fbx15 expression in MG1.19 ES cells (16) (Fig. 1A). Upon differentiation induced with retinoic acid, Fbx15 expression was repressed. Testis tissue also expressed Fbx15 but at a level lower than that at which undifferentiated ES cells did. There was faint expression in ovary tissue. No expression was detected in 10 other tissue types from adult mice. This Fbx15 expression pattern was nearly identical to that of Oct3/4 (Fig. 1A).
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To study a putative role of Oct3/4 in the ES cell-specific expression of Fbx15, we utilized ZHBTc4 ES cells, in which both copies of the Oct3/4 gene were deleted while self-renewal was maintained by tetracycline-controlled expression of Oct3/4 (25). Because of a cryptic polyadenylation site, the transgene produced two transcripts that both encoded functional Oct3/4 protein (Fig. 2). Upon addition of tetracycline to the culture medium, the longer and shorter transcripts disappeared within 24 and 72 h, respectively. Expression of Fbx15 also disappeared within 72 h after tetracycline addition. This result suggests that Oct3/4 controls the expression of Fbx15 in ES cells.
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To study the roles of the octamer-like sequence and the Sox-binding motif, we constructed luciferase reporter vectors with DNA fragments that started at positions -1120, -640, and -526. These constructs were introduced into undifferentiated ES cells. The DNA fragment starting at position -640 showed marked enhancement in a luciferase activity compared to that starting at -526 (Fig. 3). Only a small enhancement was observed with the fragment starting at -1120. All constructs were inactive in differentiated ES cells and NIH 3T3 cells (data not shown). These data indicate that the DNA fragment (-640 to -527) containing the octamer-like motif and the Sox-binding site is essential for ES cell-specific expression of Fbx15.
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We also examined the function of Fbx15 in ES cells by a gain-of-function approach. We generated ES cells constitutively expressing Fbx15 cDNA from the CAG promoter. These cells expressed Fbx15 even after LIF removal or retinoic acid treatment (not shown). However, those cells differentiated normally when subjected to these treatments. They also formed normal embryoid bodies and teratomas (not shown). These findings indicate that Fbx15 alone is not capable of maintaining the undifferentiated state of ES cells.
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DISCUSSION |
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The first evidence supporting the notion that Fbx15 is an Oct3/4 target is the high degree of similarity in expression profiles. Oct3/4 expression is restricted in pluripotent cells, including ES cells and germ cells (30). In unfertilized eggs and one-cell stage embryos, Oct3/4 is expressed at a low level (28). Its expression level becomes higher at the four-cell stage and reaches a maximum at the eight-cell and morula stages. Our Northern blot and RT-PCR analyses showed that Fbx15 expression was highest in ES cells, intermediate in testis tissue, and low in ovary tissue. No expression was detected in any of the other adult tissue types examined. X-Gal staining of ß-gal knock-in mice demonstrated that Fbx15 expression was undetectable in unfertilized eggs and one-cell stage embryos. It was activated at the two-cell stage and was highest at the eight-cell stage. Thus, Fbx15 expression is restricted to cells and tissues where Oct3/4 is expressed at a high level, suggesting that Oct3/4 may control Fbx15 gene expression.
Additional data supporting the notion that Fbx15 is an Oct3/4 target came from ZHBTc4 ES cells, in which both copies of the Oct3/4 gene had been deleted by gene targeting (25). Self-renewal of these cells was maintained by transgene expression of Oct3/4 cDNA under control of the tetracycline-responsive element. Addition of tetracycline to culture medium resulted in rapid inactivation of the Oct3/4 transgene. Oct3/4 target genes, such as Rex-1, were rapidly repressed after transgene inactivation. Our study showed that Fbx15 expression disappeared within 72 h after tetracycline addition. Since inactivation of Oct3/4 led to spontaneous differentiation of ES cells, the decline in Fbx15 may be an indirect effect. However, the rapid reduction in Fbx15 argues that Oct3/4 controls Fbx15 expression.
Analyses of regulatory regions further support the notion that Fbx15 is a novel Oct3/4 target. Reporter gene analyses showed that the ES cell-specific enhancer (TTTATCATAACAAT) of the Fbx15 gene contained a sequence similar to the octamer motif (ATT[A/T]GCAT) (29) and an adjacent Sox-binding sequence (AACAAT) (11). Deletion or point mutation of either motif abolished the enhancer activity. The enhancer was inactive in NIH 3T3 cells but became active when Oct3/4 and Sox2 expression vectors were introduced. Furthermore, a gel mobility shift assay demonstrated direct binding of Oct3/4 and Sox2 to the enhancer. These data indicate that Oct3/4, together with Sox2, controls the Fbx15 enhancer.
The putative Oct3/4-binding site (TTTATCAT) in Fbx15 differs from the canonical octamer motif (ATT[A/T]GCAT) by two nucleotides (the mutations are underlined). Such variations have been documented in other cases. For example, although the octamer motif was first identified in and highly conserved among immunoglobulin genes, the human h100 gene has a motif, ATTTTCAT, that differs from the consensus by one nucleotide (29). The Oct3/4-binding site in UTF1 (ATCTGCAT) also differs from the consensus by one nucleotide (22). It is likely that these differences reduce affinity for Oct3/4. In consistent with this, our gel mobility shift assay suggested that Oct3/4 requires the cooperation of Sox2 for stable binding to the octamer-like sequence. This may explain why Fbx15 is undetectable in cells expressing low levels of Oct3/4, such as unfertilized eggs and one-cell stage embryos.
Fbx15 was identified as a novel F-box-containing protein. The F box was first defined in Skp2 and cyclin F as a Skp1-binding site (1). F-box-containing proteins bind Skp1 and Cul1 to form the SCF complexes that function as E3 ubiquitin ligases. In general, C-terminal portions of F-box proteins bind to substrate proteins, thus promoting their ubiquitin-dependent proteolysis. Our study showed that Fbx15 formed an SCF complex when Fbx15, Skp1, and Cul1 were overexpressed, suggesting its involvement in the ubiquitin-proteasome pathway. Fbx15 may play a role in determining the expression levels of regulatory proteins necessary for pluripotency. In B cells, the transcription coactivator OBF-1, which binds to Oct-1, Oct-2, and the octamer sequence, is regulated by ubiquitin proteasome-mediated degradation (34). A similar mechanism may operate in ES cells. However, whether endogenous Fbx15 forms the SCF complex has not been determined. Furthermore, Fbx15 lacks known domains for interaction with substrate proteins, such as leucine-rich and WD40 motifs. Recently, Fbx2 (38) and Fbx32 (3, 10), which both lack leucine-rich and WD40 motifs, were shown to function as ubiquitin ligases, suggesting the existence of novel substrate-interacting domains. Fbx15 may also bind to substrates via a novel interacting motif.
To understand the in vivo functions of Fbx15, we deleted its gene by homologous recombination in ES cells and mice. Despite its specific expression, Fbx15-null ES cells were normal in morphology, proliferation, and pluripotency. Mutant mice showed no gross abnormalities. Both male and female homozygous mice were fertile. These data suggest that Fbx15 does not play important roles. However, it is more likely that other genes play redundant roles, thus eliminating obvious phenotypes in Fbx15-null cells and mice. It is known that multiple ubiquitin ligases have the same substrates. For example, p27Kip1 is still degraded in Skp2-/- cells (12). An EST database search showed that ES cells express various F-box proteins, including Skp2, Fbl6, and Fbl12. Some of these may compensate Fbx15 functions.
In summary, we have demonstrated three lines of evidence supporting the idea that Fbx15 is a novel target of Oct3/4. We also generated ES cells and mice deficient in Fbx15. Lack of obvious phenotypes in these cells and mice suggested that other ubiquitin ligases compensate Fbx15. Identification of Fbx15 substrates and other ubiquitin ligases that use the same substrates and simultaneous gene knockout are required to understand the roles of the ubiquitin-proteasome pathway in self-renewal of ES cells, early mouse development, and fertility.
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ACKNOWLEDGMENTS |
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We are grateful to T. Ichisaka and Y. Samitsu for blastocyst microinjection. We thank C. Takigawa and J. Iida for technical assistance and R. Sasaki for plasmid construction.
This work was supported in part by grants from The Sumitomo Foundation, the Uehara Memorial Foundation, and the Japanese Ministry of Education, Culture, Sports, Science and Technology.
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FOOTNOTES |
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