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Mol Cell Biol. Aug 2009; 29(16): 4574–4583.
Published online Jun 15, 2009. doi:  10.1128/MCB.01863-08
PMCID: PMC2725734

Dax1 Binds to Oct3/4 and Inhibits Its Transcriptional Activity in Embryonic Stem Cells[down-pointing small open triangle]

Abstract

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Transcription factor Oct3/4 is an indispensable factor in the self-renewal of ES cells. In this study, we searched for a protein that would interact with Oct3/4 in ES cells and identified an orphan nuclear hormone receptor, Dax1. The association of Dax1 with Oct3/4 was mediated through the POU-specific domain of Oct3/4. Ectopic expression of Dax1 inhibited Oct3/4-mediated activation of an artificial Oct3/4-responsive promoter. Expression of Dax1 in ES cells also reduced the activities of Nanog and Rex1 promoters, while knockdown of Dax1 increased these activities. Pulldown and gel shift assays revealed that the interaction of Dax1 with Oct3/4 abolished the DNA binding activity of Oct3/4. Chromatin immunoprecipitation assay results showed that Dax1 inhibited Oct3/4 binding to the promoter/enhancer regions of Oct3/4 and Nanog. Furthermore, overexpression of Dax1 resulted in ES cell differentiation. Taken together, these data suggest that Dax1, a novel molecule interacting with Oct3/4, functions as a negative regulator of Oct3/4 in ES cells.

Mouse embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, can self-renew in the presence of leukemia inhibitory factor (LIF) and maintain their pluripotency, the ability to differentiate into all types of somatic and germ cells (6, 11). In the self-renewal of mouse ES cells, STAT3, Oct3/4, Sox2, and Nanog play important roles (23). STAT3 is a well-known transcription factor downstream of LIF, and expression of its dominant-negative mutant induces differentiation of ES cells (19). Artificial activation of STAT3 using STAT3ER, a fusion protein consisting of STAT3 and the ligand-binding domain of estrogen receptor, can maintain ES cell self-renewal in the absence of LIF (14). These observations indicate that STAT3 activation is essential and sufficient for the maintenance of self-renewal. Nanog is a homeobox transcription factor whose overexpression can bypass the requirement of LIF for self-renewal (3, 15). Since a recent report has demonstrated that this homeobox transcription factor is dispensable for ES cell self-renewal (4), Nanog seems to be a self-renewal-promoting factor.

Oct3/4 (encoded by pou5f1) belongs to the POU family of transcription factors and consists of three domains: the N-terminal, POU, and C-terminal domains (see Fig. Fig.1D).1D). The N- and C-terminal domains are transactivation domains with redundant functions (21), while the POU domain is a bipartite DNA-binding domain consisting of the POU-specific domain and the POU homeodomain. Although continuous expression of Oct3/4 fails to maintain the self-renewal of ES cells in the absence of LIF, targeted disruption of the pou5f1 gene results in loss of pluripotent inner cell mass, and conditional repression of this gene in ES cells leads to differentiation into trophectoderm, indicating that Oct3/4 is a central player in the self-renewal in ES cells (18, 20). Furthermore, recent findings that Oct3/4 is one of the four factors required for the production of induced pluripotent stem cells suggest the importance of Oct3/4 for acquisition of pluripotency (30). Interestingly, not only suppression but also overexpression of Oct3/4 induces ES cell differentiation (20), suggesting that the proper expression/activity level of Oct3/4 is required to maintain ES cell self-renewal.

FIG. 1.FIG. 1.
Association between Oct3/4 and Dax1. (A) Dax1 interacts with Oct3/4 in intact cells. (Left) HEK293 cells were cotransfected with Myc-Oct3/4 and Flag-Dax1 (+). Whole-cell lysates were subjected to immunoprecipitation (IP) with anti-Flag antibody, ...

In ES cells, the expression of Oct3/4 is regulated by several transcription factors (23). Previous studies have revealed that the upstream region of the pou5f1 gene contains two elements, proximal and distal enhancers, which regulate the stem cell-specific expression of Oct3/4 (36). An orphan nuclear receptor, liver receptor homolog 1 (LRH1, also known as Nr5a2), binds with the proximal enhancer (8). In LRH1-null ES cells, although Oct3/4 is still expressed, its downregulation during differentiation occurs more rapidly than in the wild-type cells, suggesting that LRH1 is involved in the maintenance of Oct3/4 expression (8). It is also documented that Oct3/4 itself associates with the distal enhancer to stimulate its expression (24). Several negative regulators of Oct3/4 expression have also been reported, including GCNF, Coup-tfs, and Cdx2, whose expression is induced upon ES cell differentiation (23). As for regulation of the transcriptional activity of Oct3/4, it is well-established that Sox2, an indispensable transcription factor for ES cell self-renewal (13), functions as a cofactor of Oct3/4 to synergistically stimulate the expression of self-renewal genes such as themselves and Nanog. β-Catenin has been reported to associate with Oct3/4 and to act as a coactivator for upregulation of Nanog (31).

Considering the importance of maintaining the optimal activity of Oct3/4, self-renewing ES cells may express a negative regulator that neutralizes the excess amount of Oct3/4. In the present study, therefore, we performed yeast two-hybrid screening and identified an orphan nuclear hormone receptor, Dax1 (also known as Nr0b1 and Ahch), as a novel Oct3/4-interacting protein. We found that association of Dax1 reduced the DNA binding activity of Oct3/4. Furthermore, Dax1-overexpressing ES cells exhibited a phenotype similar to that of Oct3/4-knocked down ES cells. These results suggest that, in self-renewing ES cells, Dax1 functions as a negative regulator of Oct3/4 to maintain proper Oct3/4 activity.

MATERIALS AND METHODS

Cell culture.

ES cell lines A3-1 (2), MGZ5 (7), ZHBTc4 (20), and E14 were cultured on gelatin-coated dishes with LIF-supplemented Dulbecco's modified Eagle's medium or Glasgow modified Eagle's medium as described previously (27, 37). A3/TRE-Biotin-Dax1 ES cells were established by introduction of pTRE-Biotin-Dax1 into A3-1 ES cells and cultured in the presence of 1 μg/ml of tetracycline and 0.5 μg/ml of puromycin (Nacalai Tesque, Kyoto, Japan). Human embryonal kidney (HEK) 293 and HeLa cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum.

Plasmid construction.

The coding regions of mouse Oct3/4 and Dax1, as well as their mutants, were amplified by PCR using primers listed in Table S1 in the supplemental material. Construction of mammalian expression vectors, pCMV5 and pCAG-IP, was described before (1, 21, 37). pEGFP-Dax1 was generated by inserting the Dax1 coding sequence into pEGFP-C1 (Clontech, Palo Alto, CA). pCAGIP-Myc-DsRed and pCMV-Flag-MBP were constructed by transferring the coding sequences of Discosoma red fluorescent protein (DsRed) from pDsRed Express-C1 (Clontech) and maltose-binding protein (MBP) from pMAL-c2 (New England Biolabs, Beverly, MA) into pCAGIP-Myc and pCMV5-Flag, respectively. Plasmids pCAGIP-Myc-Oct3/4, pCAGIP-Flag-Oct3/4, pCAGIP-Myc-Dax1, pCAGIP-Flag-Dax1, pCAGIP-Dax1, pCAGIP-Myc-DsRed-Oct3/4, pCMV-Flag-MBP-Oct3/4, pCMV-Flag-MBP-Dax1, and their derivatives were constructed by inserting corresponding coding sequences into expression vectors. To obtain pSi-H1p-Dax1, a target sequence for RNA interference of Dax1 (5′-TAGCGATGTCGTCACTGAA-3′) was introduced into a short hairpin RNA expression vector, pSilencer 3.1-H1 puro (Ambion, Austin, TX). Oct-797 was used as a small interfering RNA-siRNA-for Oct3/4 (33).

Two reporter plasmids, pOct2.2-luc and pOct-luc2, were constructed as described previously (24, 31). Mouse Nanog promoter (positions −332 to +50) (10, 31) and Rex1 promoter (positions −669 to +23) (9) were amplified by PCR from genomic DNA of A3-1 cells and inserted into the MluI and BglII sites of pGL2-basic (Promega, Madison, WI) to produce pGL2-Nanog (positions −332 to +50) and pGL2-Rex1 (positions −669 to +23). Two bacterial expression vectors, pMAL-c2-Flag-Oct3/4-His6 and pMAL-c2-Flag-Dax1-His6, were constructed by inserting coding sequences of Flag-Oct3/4-His6 (Flag-tagged Oct3/4 with a six-histidine tag) and Flag-Dax1-His6 (Flag-tagged Dax1 with a six-histidine tag) into pMAL-c2.

An inducible mammalian expression vector, pTRE-Biotin-Dax, was constructed by combining three gene cassettes, TRE promoter-Flag-biotin-Dax1-polyA, CAG promoter-tTA2-IRES-puromycin resistance-polyA, and CAG promoter-myc-BirA-polyA. Biotinylation tag (MAGGLNDIFEAQKIEWHEDTGGS) and biotin ligase BirA were transferred from pDW363, and the TRE promoter was obtained from pTRE-Myc (Clontech). pDW363 was obtained from the National BioResource Project (NIG, Japan).

Plasmid transfection.

Plasmids were introduced into cultured cells either by lipofection with Lipofectamine 2000 (Invitrogen, Carlsbad, CA), calcium phosphate-mediated transfection, or electroporation (200 V, 500 μF) using a Gene Pulser (Bio-Rad, Hercules, CA). One day after transfection, the medium was replaced with fresh medium. For transient-transfection experiments, samples were analyzed 2 days after transfection. To establish stable transfectants, ES cells were reseeded 2 days after transfection and treated with puromycin for another 7 days. Transfection of episomal expression vectors was performed as described previously (21).

Protein purification.

MBP-Flag-Dax1-His6 and MBP-Flag-Oct3/4-His6 were produced by Escherichia coli BL21(DE3) using pMAL-c2-Flag-Oct3/4-His6 and pMAL-c2-Flag-Dax1-His6, respectively, and purified with amylose resin (New England Biolabs). Partially purified proteins were then purified with Talon metal affinity resin (Clontech). To obtain Flag-Dax1-His6, MBP-Flag-Dax1-His6 purified with amylose resin was digested by factor Xa protease (New England Biolabs), followed by purification with Talon metal affinity resin.

Coimmunoprecipitation and pulldown assays.

For the coimmunoprecipitation assay, cell lysates were incubated overnight at 4°C with protein G-Sepharose 4 Fast Flow beads (Amersham Biosciences), mouse anti-Flag antibody (M2; Sigma), and rabbit anti-mouse immunoglobulin G antibody (ICN Biomedical, Irvine, CA). For pulldown assays using biotinylated Dax1, MBP-Oct3/4, and MBP-Dax1, cell lysates or purified proteins were incubated overnight at 4°C with streptavidin-agarose (Novagen, Darmstadt, Germany) and amylose resin, respectively. For the pulldown assay using oligonucleotide, 20 pmol of 3′-biotinylated oligonucleotide (5′-ACGCGTAGATGCATATATGCATTTTTGCATAGATCT-3′) was annealed with 5′-AGATCTATGCAAAAATGCATATATGCATCTACGCGT-3′ and incubated at 4°C for 2 to 4 h with cell lysates and/or purified proteins in the presence of streptavidin-agarose. In all experiments, the beads were washed three times with washing buffer (50 mM Tris-HCl [pH 7.5], 2 mM MgCl2, and 150 mM NaCl), and the bound proteins were eluted by boiling in 2× sodium dodecyl sulfate (SDS) sample buffer (2× SDS sample buffer is 20 mM Tris-HCl [pH 6.8], 12% glycerol, 4% SDS, 100 mM dithiothreitol, 4 mM EDTA, and 0.004% Coomassie brilliant blue R250) and subjected to Western blot analysis using anti-Myc antibody (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Flag antibody, anti-green fluorescent protein (anti-GFP) antibody (Molecular Probes, Eugene, OR), anti-Oct3/4 antibody (sc-9081; Santa Cruz Biotechnology), anti-Dax1 antibody (sc-13064; Santa Cruz Biotechnology), anti-α-tubulin antibody (MP Biomedicals, Solon, OH), horseradish peroxidase (HRP)-conjugated streptavidin (Zymed, San Francisco, CA), and HRP-conjugated anti-mouse or anti-rabbit immunoglobulin G antibody (Upstate Biotechnology, Charlottesville, VA). The blot was visualized by using enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA) with an LAS-1000 image analyzer (Fuji Film, Tokyo, Japan).

Reverse transcription-PCR (RT-PCR) analysis.

Total RNAs, isolated from ES cells with Trizol reagent (Invitrogen), were converted to cDNAs by Superscript III reverse transcriptase (Invitrogen) with oligo(dT)12-18 primers (Amersham Biosciences). Quantitative PCR was performed as described previously (22). Primers used are 5′-CCGTGTGAGGTGGAGTCTGGAGAC-3′ and 5′-CGCCGGTTACAGAACCATACTCG-3′ for Oct3/4, 5′-AGATGGAGAAAGCGGTCGTA-3′ and 5′-AAGCCAGTATGGAGCAGAGG-3′ for endogenous Dax1, and 5′-TCCTGTACCGCAGCTATGTG-3′ and 5′-ATCTGGAAGCAGGGCAAGTA-3′ for both endogenous and exogenous Dax1. Primers for other genes were described before (22, 26, 32).

Luciferase reporter assay.

Cell extracts were prepared 1 or 2 days after transfection, and luciferase activities in the extracts were measured by using a luciferase assay kit (Promega) with an AB-2200 (ATTO, Tokyo, Japan) or Centro LB960 (Berthold) luminometer.

EMSA.

Electrophoretic mobility shift assay (EMSA) analysis was performed as described previously (34). Briefly, purified recombinant proteins were incubated for 30 min at room temperature with poly(dI-dC) and 32P-labeled DNA probe (5′-ACGCGTAGATGCATATATGCATTTTTGCATAGATCT-3′ and 5′-AGATCTATGCAAAAATGCATATATGCATCTACGCGT-3′ for Oct3/4 probe 1; 5′-AGTAATGCACCCACACGCATGTACAATGCAAGGTGTGTAAA-3′ and 5′-TTTACACACCTTGCATTGTACATGCGTGTGGGTGCATTACT-3′ for Oct3/4 probe 2). Samples were separated on a 4% native polyacrylamide gel, and radiolabeled bands were visualized with BAS-2000 (Fuji Film).

ChIP assay.

An ES cell line with tetracycline-inducible Dax1 was established by using Rosa-Tet (Tet stands for tetracycline) system (12). Since exogenous Dax1 expression is induced after removal of tetracycline in this cell line, the cells were treated with tetracycline for 2 days or not treated with tetracycline and harvested for chromatin immunoprecipitation (ChIP) assay. Anti-Oct3/4 antibody (3 μg) (sc-8628; Santa Cruz Biotechnology) was added to the sheared chromatin derived from three 15-cm culture dishes, and chromatin-antibody complex was purified by a ChIP-IT express kit (Active Motif, Carlsbad, CA) according to the manufacturer's protocol. For the detection of precipitated genomic DNA, quantitative real-time PCR was performed using Sybr green PCR master mix and 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA). The sequences of primer sets were as follows: 5′-GAGGATGCCCCCTAAGCTTTCCCTCCC-3′ and 5′-CCTCCTACCCTACCCACCCCCTATTCTCCC-3′ for Nanog, 5′-GACGGCAGATGCATAACAAA-3′ and 5′-AATAAAGGCAGCGACTTGGA-3′ for Oct3/4, and 5′-GGAGTCCCCTAGGAAGGCATTAATAGTTT-3′ and 5′-GGATTCTCTCGGCTTCAGACAGACTTT-3′ for control.

For ChIP analysis for the eed and dax1 genes, A3-1 cells were transfected with Flag-Oct3/4 together with or without Myc-Dax1 and subjected to ChIP assay using anti-Flag antibody. Oct3/4-binding sites in the eed and dax1 genes were amplified using specific primers as described before (28, 34). In parallel, cDNA was synthesized from the transfected cells and mRNA expression of Flag-Oct3/4 and GAPDH was examined by RT-PCR. The primer set for Flag-Oct3/4 is 5′-ATGGACTACAAGGACGAC-3′ and 5′-CTGGGACTCCTCGGGAGTTG-3′, and that for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was described previously (28).

RESULTS

Identification of Dax1 as an Oct3/4-interacting molecule.

To search for Oct3/4-interacting proteins, we performed a yeast two-hybrid screening using a cDNA library of self-renewing ES cells and identified an orphan nuclear hormone receptor, Dax1. To confirm the interaction between Oct3/4 and Dax1 in mammalian cells, coimmunoprecipitation assay was performed using HEK293 cells expressing Myc epitope-tagged Oct3/4 (Myc-Oct3/4) and Flag epitope-tagged Dax1 (Flag-Dax1). As shown in Fig. Fig.1A,1A, Myc-Oct3/4 was detected in the immunoprecipitates obtained with anti-Flag antibody, while no precipitation was detected in the case of HEK293 cells expressing Myc-Oct3/4 alone. Similarly, when lysates from HEK293 cells expressing GFP-tagged Dax1 (GFP-Dax1) and Flag-Oct3/4 were subjected to immunoprecipitation with anti-Flag antibody, precipitation of GFP-Dax1 was observed. These results suggest that Dax1 binds to Oct3/4. To examine whether the interaction occurs at the endogenous level, we established A3/TRE-Biotin-Dax1 ES cells. In this cell line, expression of biotinylation-tagged Dax1 (biotin-Dax1) was induced by removal of tetracycline from the culture medium, and the expression level of induced biotin-Dax1 was lower than that of endogenous Dax1 (Fig. (Fig.1B).1B). When we performed a pulldown assay using A3/TRE-Biotin-Dax1 ES cells, endogenous Oct3/4 was coprecipitated with biotin-Dax1 by streptavidin-agarose, suggesting that Dax1 can interact with Oct3/4 in ES cells at the endogenous level. We further examined whether Dax1 binds directly to Oct3/4 using purified recombinant proteins. As shown in Fig. Fig.1C,1C, purified Flag-Dax1-His6 was coprecipitated with MBP-Flag-Oct3/4-His6, but not with MBP. To determine the Dax1-binding region of Oct3/4, we prepared several truncated mutants of Oct3/4. Dax1 was coprecipitated strongly with full-length Oct3/4, the POU domain, and the POU-specific domain, but weakly with the N-terminal domain, the C-terminal domain, and the POU homeodomain (Fig. (Fig.1D),1D), suggesting that Dax1 binds to the POU-specific domain of Oct3/4. To determine the Oct3/4-binding region of Dax1, we prepared two truncated mutants of Dax1 (Fig. (Fig.1E).1E). Oct3/4 was coprecipitated strongly with full-length Dax1 and Dax1 (residues 101 to 380) but weakly with Dax1 (residues 101 to 308) (Fig. (Fig.1E),1E), suggesting that Oct3/4 binds to an approximately 70-amino-acid region (residues 308 to 380) in the ligand-binding domain of Dax1.

We next examined the influence of Oct3/4 on cellular localization of Dax1. When GFP-Dax1 was overexpressed in HeLa cells, GFP-Dax1 was found in both the cytoplasm and nucleus (Fig. (Fig.2)2) (see Fig. S1 in the supplemental material). On the other hand, the distribution of GFP-Dax1 was restricted to the nucleus in the presence of Oct3/4, suggesting that the nuclear localization of Dax1 is enhanced by Oct3/4, probably through protein-protein interaction.

FIG. 2.
Nuclear localization of Dax1 is enhanced by Oct3/4. HeLa cells were transfected with GFP-Dax1 plus Myc-DsRed or GFP-Dax1 plus Myc-DsRed-Oct3/4. Cells were photographed 2 days after transfection. Bars, 50 μm.

Dax1 represses the transcriptional activity of Oct3/4.

To understand the biological significance of the interaction between Dax1 and Oct3/4, we examined the effect of Dax1 on the transcriptional activity of Oct3/4 using a reporter plasmid carrying five repeats of the Oct3/4-binding sequence. In HEK293 cells, expression of Oct3/4 stimulated strong induction of the reporter gene (Fig. (Fig.3A).3A). This induction was suppressed by the coexpression of Dax1. We also examined the effects of Dax1 on two natural promoters whose activities depend on Oct3/4 activity. When Dax1 was overexpressed in ES cells, a reduction of the Nanog promoter activity was observed (Fig. (Fig.3B)3B) (see Fig. S2 in the supplemental material). On the other hand, knockdown of Dax1 resulted in enhanced activity of the promoter. Similar effects were observed for the Rex1 promoter. These data indicate that Dax1 represses the transcriptional activity of Oct3/4.

FIG. 3.
Effect of Dax1 binding on Oct3/4 activity. (A) Dax1 represses transcriptional activity of Oct3/4. (Top) Schematic view of pOct-Luc2, which carries five repeats of the Oct3/4-binding sequence. PTAL, TATA-like promoter from the herpes simplex virus thymidine ...

Dax1 inhibits the DNA binding activity of Oct3/4.

It is well-known that Oct3/4 binds to DNA through its POU domain. Since Dax1 binds to this DNA-binding domain, it is possible that Dax1 represses the transcriptional activity of Oct3/4 by inhibiting the DNA binding activity of Oct3/4. To explore this possibility, we first performed a pulldown assay using biotinylated oligonucleotide carrying three Oct3/4-binding sites (Fig. (Fig.4A).4A). When the lysate from HEK293 cells expressing Oct3/4 was incubated with the biotinylated oligonucleotide and streptavidin-agarose, Oct3/4 was pulled down along with the oligonucleotide, suggesting that Oct3/4 binds to the oligonucleotide. This binding was competed by excess nonbiotinylated oligonucleotide of the same sequence, but not by mutated oligonucleotide (data not shown), confirming that the association between the oligonucleotide and Oct3/4 is sequence specific. When the same experiment was performed using HEK293 cells expressing both Oct3/4 and Dax1, the association of Oct3/4 with oligonucleotide was reduced (Fig. (Fig.4A).4A). Adding recombinant Dax1 to the lysate containing Oct3/4 also diminished the association between Oct3/4 and oligonucleotide (Fig. (Fig.4B).4B). These data suggest that Dax1 inhibits the DNA binding of Oct3/4.

FIG. 4.
Dax1 represses the DNA binding ability of Oct3/4. (A) Lysates from HEK293 cells expressing Flag-Oct3/4 and/or Flag-Dax1 were subjected to pulldown assay with biotinylated DNA. The precipitates were analyzed by immunoblotting with anti-Flag antibody. (Bottom) ...

To confirm the inhibitory effect of Dax1 on the DNA binding activity of Oct3/4, we next performed EMSA using purified recombinant proteins. When recombinant Oct3/4 protein was incubated with radiolabeled oligonucleotide carrying three Oct3/4-binding sites, the retardation of mobility was observed (Fig. (Fig.4C).4C). This retardation was inhibited by the presence of excess nonlabeled oligonucleotide (data not shown), indicating the specific binding of Oct3/4 to the oligonucleotide. Adding recombinant Dax1 protein to this reaction mixture resulted in inhibition of complex formation between DNA and Oct3/4 in a dose-dependent manner. The observed inhibition was not due to the competitive binding of Dax1 to DNA, since no complex formation was observed between Dax1 and the oligonucleotide. Similar results were obtained with the oligonucleotide encompassing an Oct3/4-binding site in the dax1 gene enhancer (28) (Fig. (Fig.4D4D).

We also examined the inhibitory function of Dax1 against the in vivo DNA binding of Oct3/4 by ChIP assay. ES cells with tetracycline-inducible Dax1 were treated with tetracycline or not treated with tetracycline and subjected to ChIP assay using anti-Oct3/4 antibody. Quantitative PCR analysis of immunoprecipitated DNA revealed that chromatin fragments containing Oct3/4-binding sites in the nanog and pou5f1 genes were precipitated by Oct3/4 antibody and that the amount of the precipitated chromatin decreased in the absence of tetracycline when exogenous Dax1 was overexpressed (Fig. (Fig.5A).5A). The observed reduction of Oct3/4 binding to the target genes was not a consequence of Dax1-induced downregulation of Oct3/4 expression, since the level of expression of Oct3/4 mRNA in Dax1-overexpressing cells is still comparable to that of control cells (Fig. (Fig.5B).5B). There is probably a time lag between the dissociation of Oct3/4 from the pou5f gene and the reduction of Oct3/4 expression.

FIG. 5.
Dax1 represses in vivo binding of Oct3/4 to the promoter/enhancer regions of nanog and pou5f1 genes. (A) ES cells with tetracycline-inducible Dax1 were cultured with (+) or without (−) tetracycline and subjected to ChIP assay. The amount ...

Similar results were obtained with ES cells transfected with Flag-Oct3/4 and Myc-Dax1: overexpression of Dax1 reduced the association of Oct3/4 with chromatin DNA containing Oct3/4-binding sites in other Oct3/4 target genes, eed and dax1 (see Fig. S3 in the supplemental material). Taken together, these data strongly indicate that Dax1 inhibits the DNA binding activity of Oct3/4.

Overexpression of Dax1 resulted in ES cell differentiation.

The present data suggest that Dax1 functions as a negative regulator of Oct3/4, a key player in ES cell self-renewal. If so, artificial up- and downregulation of Dax1 should affect the self-renewal of ES cells. In fact, knockdown or conditional knockout of Dax1 has been reported to induce the differentiation of ES cells (17, 35).

To examine the effect of artificial upregulation of Dax1 on ES cells, we used the episomal vector system for strong expression of the Dax1 transgene (19). When we introduced an episomal expression vector for Dax1 into ES cells, the majority of transfected cells showed differentiated morphologies compared with cells transfected with empty vector, although the number of drug-resistant colonies was not significantly different between Dax1-overexpressing cells and control cells (Fig. 6A and B). In addition, their morphologies resembled those of Oct3/4-knockdown ES cells (Fig. (Fig.6C).6C). A comparison of expression levels of several marker genes also indicated the similarity between Dax1 overexpression and Oct3/4 knockdown: self-renewal markers (Oct3/4, Nanog, and Sox2) were downregulated, and trophectodermal markers, Cdx2 and Rhox6, were strongly upregulated, while other lineage markers were slightly induced (Fig. (Fig.6D).6D). Immunostaining analysis confirmed the expression of Cdx2 in Dax1-overexpressing ES cells (Fig. (Fig.6E).6E). These results indicate that overexpression of Dax1 induces ES cell differentiation preferentially toward a trophectoderm lineage, mimicking the deficiency of Oct3/4.

FIG. 6.
Effect of Dax1 overexpression on ES cells. (A) Leischman staining of colonies generated by episomal transfection with Dax1 and empty expression vectors. Bar, 1 mm. (B) Numbers of stem and differentiated (dif) colonies were counted manually after Leischman ...

DISCUSSION

Dax1 (for DSS-AHC on X chromosome gene 1 where DSS stands for dosage-sensitive sex reversal and AHC stands for adrenal hypoplasia congenital), a member of the nuclear hormone receptor superfamily, has been shown to play an important role in the establishment and maintenance of steroid-producing tissues, such as testis and adrenal cortex (16). In humans, loss-of-function mutations in DAX1 result in adrenal hypoplasia congenital, while DAX1 duplications cause male-to-female sex reversal: dosage-sensitive sex reversal, indicating that DAX1 is involved in sex determination. The N-terminal domain of Dax1 contains three LXXLL motifs involved in protein-protein interaction. In the C terminus, Dax1 carries a ligand-binding domain that shares homology with other nuclear receptors, including steroidogenic factor 1. Although the role of Dax1 in steroidogenesis is well established, the previous studies showing the expression of Dax1 in inner cell mass and ES cells (5, 28) have suggested that Dax1 may play a certain role in ES cells. In fact, an attempt to establish Dax1-null ES cells resulted in failure (38). Furthermore, suppression of Dax1 expression either by knockdown or the inducible knockout method induced ES cell differentiation (17, 35), indicating that Dax1 plays an important role in the self-renewal of ES cells. The mechanism behind Dax1-mediated ES cell self-renewal, however, is unknown.

Previous works have shown that Dax1 binds with several transcription factors and inhibits their transcriptional activity, such as steroidogenic factor 1, LRH1, Nur77, and androgen receptor (16). In this study, we demonstrated that Dax1 binds with Oct3/4, a transcription factor that functions as a gatekeeper to prevent ES cell differentiation. Interaction of Dax1 with Oct3/4 is mediated through the DNA-binding domain of Oct3/4, which probably leads to the inhibition of the DNA binding activity of Oct3/4. Furthermore, we demonstrated the similarity between Dax1-overexpressing ES cells and Oct3/4-downregulated ES cells. These results indicate that Dax1 is a negative regulator of Oct3/4 in ES cells.

The present study demonstrated that Dax1 negatively regulates Oct3/4 by interfering with the transcriptional activity of Oct3/4 protein. In addition, we presume that Dax1 may negatively regulate expression of Oct3/4 mRNA, since we noticed that expression of Oct3/4 mRNA is downregulated by Dax1 (Fig. (Fig.6D).6D). Although we cannot exclude the possibility that the Oct3/4 downregulation might be a result of ES cell differentiation, it is also possible that Dax1 directly inhibits Oct3/4 expression. It has been reported that the expression of Oct3/4 is positively regulated by Oct3/4 itself and LRH-1 (8, 24). Since Dax1 suppresses the transcriptional activity of Oct3/4, Dax1 has the potential ability to inhibit the Oct3/4-mediated Oct3/4 induction. Similarly, it is also possible that Dax1 represses Oct3/4 expression by interfering with the LRH1-dependent Oct3/4 induction, since Dax1 is a negative regulator of LRH1 (25, 29). Support for this possibility is provided by our observation that Dax1 inhibits the LRH1-mediated activation of the Oct3/4 promoter (Y. Nakatake et al., unpublished data).

We previously demonstrated that Oct3/4 binds to the enhancer region of the dax1 gene and positively regulates Dax1 expression (28). Combining these results with the present findings, it is possible that a regulatory circuit may exist between Dax1 and Oct3/4 in self-renewing ES cells. If so, when an excess amount of Oct3/4 exists in ES cells, it would trigger induction of Dax1 protein, which in turn suppresses the activity of Oct3/4. On the other hand, when Oct3/4 is slightly downregulated, the expression level of its negative regulator, Dax1, may also decrease, which allows Oct3/4 to maintain its activity at the normal level. In this way, suppression of Oct3/4 by Dax1 may always depend on the activity of Oct3/4, and this dependency may enable Dax1 to act as a “fine tuner” that maintains the Oct3/4 activity at an appropriate level in ES cells. These possibilities will be examined in future studies.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported in part by the Naito Foundation, Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the Intramural Research Program of the NIH, National Institute on Aging.

Footnotes

[down-pointing small open triangle]Published ahead of print on 15 June 2009.

Supplemental material for this article may be found at http://mcb.asm.org/.

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