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Molecular and Cellular Biology, October 2006, p. 7772-7782, Vol. 26, No. 20
0270-7306/06/$08.00+0     doi:10.1128/MCB.00468-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Klf4 Cooperates with Oct3/4 and Sox2 To Activate the Lefty1 Core Promoter in Embryonic Stem Cells{triangledown}

Yuhki Nakatake,1 Nobutaka Fukui,1,2,3 Yuko Iwamatsu,1 Shinji Masui,1 Kadue Takahashi,1 Rika Yagi,1 Kiyohito Yagi,2 Jun-ichi Miyazaki,3 Ryo Matoba,4 Minoru S. H. Ko,4 and Hitoshi Niwa1,5*

Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology (CDB), Minatojima-Minamimachi 2-2-3, Chuo-ku, Kobe 650-0047, Japan,1 Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka 1-6, Suita C., Osaka 565-0871, Japan,2 Stem Cell Regulation Research, Area of Molecular Therapeutics, Course of Advanced Medicine, Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita C., Osaka 565-0871, Japan,3 Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224,4 Laboratory for Development and Regenerative Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunokicho, Chu-o-ku, Kobe, Hyogo 650-0017, Japan5

Received 17 March 2006/ Returned for modification 2 May 2006/ Accepted 15 August 2006


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ABSTRACT
 
Although the POU transcription factor Oct3/4 is pivotal in maintaining self renewal of embryonic stem (ES) cells, little is known of its molecular mechanisms. We previously reported that the N-terminal transactivation domain of Oct3/4 is required for activation of Lefty1 expression (H. Niwa, S. Masui, I. Chambers, A. G. Smith, and J. Miyazaki, Mol. Cell. Biol. 22:1526-1536, 2002). Here we test whether Lefty1 is a direct target of Oct3/4. We identified an ES cell-specific enhancer upstream of the Lefty1 promoter that contains binding sites for Oct3/4 and Sox2. Unlike other known Oct3/4-Sox2-dependent enhancers, however, this enhancer element could not be activated by Oct3/4 and Sox2 in differentiated cells. By functional screening of ES-specific transcription factors, we found that Krüppel-like factor 4 (Klf4) cooperates with Oct3/4 and Sox2 to activate Lefty1 expression, and that Klf4 acts as a mediating factor that specifically binds to the proximal element of the Lefty1 promoter. DNA microarray analysis revealed that a subset of putative Oct3/4 target genes may be regulated in the same manner. Our findings shed light on a novel function of Oct3/4 in ES cells.


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INTRODUCTION
 
Tissue-specific transcription factors are pivotal for determining cellular phenotypes during development. These factors bind to target DNA in a sequence-specific manner up- or downstream of the promoter element (19, 34). Subsequently, the activator domains of these transcription factors bind directly to TFIIA and TFIIB and facilitate their assembly with TFIID on the promoter (5). RNA polymerase II is then placed at the promoter to initiate transcription. Finally, differential gene transcription is responsible for the generation of different cell types.

Among the interactions between tissue-specific transcription factors are those that occur during myocyte development, in which the basic helix-loop-helix transcription factor MyoD is key in directing differentiation. Although MyoD can bind to the target sequence by cooperation with its partner E12/47 and recruit TFIID with its C-terminal domain, interactions with molecules such as MEF2, Sp1, and p300 are required for activation of target gene expression (41). Overexpression of MyoD can induce differentiation of myocytes only in particular cell types, due to the requirement of cellular factors for MyoD activity as well as the accessibility of the target sequence in chromatin.

In pluripotent stem cells, the undifferentiated phenotype is maintained by a combination of specific transcription factors. We previously reported that the POU family transcription factor Oct3/4 (encoded by Pou5f1) is necessary to maintain self renewal of pluripotent embryonic stem (ES) cells (28). In ES cells, artificial repression of Oct3/4 induces differentiation toward the trophectoderm, whereas overexpression of Oct3/4 induces differentiation mainly to extraembryonic endoderm, indicating that an appropriate level of Oct3/4 expression is required for continuous propagation of ES cells. We hypothesized that overexpression of Oct3/4 resulted in partial interference with its function by a squelching mechanism in cooperation with its partner molecule (26). To date, however, the only molecule functionally identified as an Oct3/4 partner is the Sry-related HMG box transcription factor Sox2. Cooperation of Oct3/4 and Sox2 was initially detected on the Fibroblast growth factor 4 (Fgf4) enhancer (48) and subsequently observed on several other genes, including Opn (7), Utf1 (24), Fbox15 (42), and Nanog (17). Moreover, we found that both Oct3/4 and Sox2 were regulated in the same manner (32, 43), suggesting the general importance of this partnership in ES cells. Indeed, Sox2 function was also necessary for the establishment of ES cell lines from the inner cell mass (ICM) of blastocyst-stage embryos (2).

Although Sox2 is an important partner of Oct3/4, there is evidence suggesting that Oct3/4 functions differently in ES cells. For example, Zfp42/Rex1, which encodes a zinc finger transcription factor, was identified as a target of Oct3/4, but the partner of the latter was not identified (3). In addition, we recently reported that Oct3/4 variants maintained expression of Lefty1 in ES cells (27). In ES cells expressing the Oct3/4 variant lacking the N-terminal domain (NTD), all known Sox2-dependent target genes as well as Zfp42/Rex1 were expressed normally, but Lefty1 expression was dramatically reduced, suggesting that the mechanism of Oct3/4 regulation of Lefty1 expression is different from the mechanism by which it regulates expression of other genes. We therefore sought to determine the molecular mechanism by which Lefty1 gene expression is activated in ES cells. We found that the 1.3-kb genomic DNA fragment containing the promoter element of the Lefty1 gene was responsible for Oct3/4-dependent ES-specific transcriptional activity. We also found that Oct3/4 activates the ES-specific enhancer element in this fragment by cooperation with Sox2. Unlike their other known targets, however, the combination of Oct3/4 and Sox2 was not sufficient for activation of the Lefty1 promoter. Rather, a Krüppel-type zinc finger transcription factor, Klf4, was identified as a mediating factor that binds to the proximal element and cooperates with Oct3/4 and Sox2 on the distal enhancer in activating the Lefty1 promoter. Repression of Klf4 by RNA interference in ES cells resulted in downregulation of Lefty1 expression, and cooperation of Klf4 with Oct3/4 required the N-terminal domain of Oct3/4. We also found that Klf4 has physiological significance in activating a subset of Oct3/4 target genes in ES cells.


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MATERIALS AND METHODS
 
Plasmid construction. The mouse Lefty1 genomic DNA containing the promoter element was PCR amplified from mouse ZHBTc4 ES cell genomic DNA (28) using the primers 5'-CCTTTACACTGGTCTCGAGCC-3' and 5'-ATAAGCTTGTCCGGGAAGAGGAGCCTTG-3' and high-fidelity Pfx DNA polymerase (Invitrogen, Groningen, The Netherlands). The amplified fragment was digested with XhoI and HindIII and subcloned into the XhoI and HindIII sites of the pGL3-Basic luciferase reporter (Promega, Madison, Wis.). The resulting plasmid, designated pLefty1-luc, contains the genomic sequence 182321131 to 182322504 on chromosome 1 in the Ensembl mouse genome database. Large deletions were generated by digestion of pLefty1-luc with XhoI and either ApaI, PvuII, or SacII, followed by T4 DNA polymerase treatment and self ligation. The small deletion series shown in Fig. 2B and 3A and the mutated reporters in Fig. 3B were generated by PCR using Pfx DNA polymerase.


Figure 2
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  		FIG. 2. Activation of Lefty1 promoter by Oct3/4 and Sox2. (A) Activities of pLefty1-luc in ES cells maintained by mutant forms of Oct3/4. Relative expression levels of plefty1-luc (filled) and Fgf4tkluc (hatched) are shown. Fgf4tkluc has a tk minimal promoter ligated with the enhancer elements in which Oct3/4 and Sox2 can associate. The luciferase activity in ES cells expressing wild-type (wt) Oct3/4 was set at 1.0. (B) Relative activation of reporters by Oct3/4 and Sox2 in HeLa cells. The reporter plasmids were cotransfected with Oct3/4 and/or Sox2 expression vectors. The luciferase activity of reporter alone was set at 1.0. (C) Reactivation of reporters by Oct3/4 in Oct3/4-depleted ES cells. Reporter plasmids were transfected into ZHBTc4 ES cells, with or without Oct3/4 expression vector, and the cells were cultured with or without Tc. Resulting luciferase activities, relative to that in ES cells without Tc and Oct3/4 expression vector, are shown.


Figure 3
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  		FIG. 3. Activation of the Lefty1 reporter by Klf4. (A) Activation of pLefty1-luc by various ES-specific transcription factors with (open) or without (filled) Oct3/4 and Sox2 expression vectors in HeLa cells. Induction of luciferase activity was measured relative to that of empty vector. (B) Activation of various reporters by Klf4 in HeLa cells. Reporter plasmids were cotransfected with empty or Klf4 expression vector into HeLa cells and the luciferase activities, relative to that of empty vector, were measured. (C) Activation of pLefty1-luc by Klf family members. pLefty1-luc was cotransfected with empty or Klf family expression vectors into HeLa cells and luciferase activities, relative to that of empty vector, were measured.

The reporter plasmids ptk-luc, Fgf4tk-luc, and 6Wtk-luc have been described previously (27), as has been Oct3/4-luc, which carries the 4.8-kb Oct3/4 promoter (32). The mouse Utf1 promoter was PCR amplified using the primers 5'-AACTCGAGAATAAGCAAGGCACAGGCCAAG-3' and 5'-AAAGCTTGAGCCAGGTAGAGGTGCGTG-3', followed by digestion with XhoI and HindIII and insertion into the XhoI and HindIII sites of the pGL3-Basic luciferase reporter, generating Utf1p-luc. Utf1tk-luc and Utf1-luc were generated by introduction of the 1,040-bp SalI-BamHI fragment carrying the mouse Utf1 enhancer (24) into the SalI-BamHI sites of ptk-luc and pUtf1p-luc, respectively. The mouse 1200015N20Rik promoter was PCR amplified using the primers 5'-AAGTGGACAGGGTATACACAAACAT-3' and 5'-GGCATTGCCAGGCAAGTCTTAGGGC-3' and inserted into the SmaI site of the pGL3-Basic luciferase reporter, generating 1200015N20Rik-luc.

The entire open reading frames of cDNAs encoding transcription factors were subcloned into the pCAG-IP expression vector (27). Deletion mutants of Oct3/4 were made as described previously. Sox2 cDNA and the primers 5'-AACTCGAGCGCCCGCATGTATAACATGATG-3' (sense) and 5'-TTGCGGCCGCTAACCCAGGCCGGCGCCCACC-3' as well as 5'-TTGCGGCCGCCTACTCCTGCATCATGCTGTAG-3' and 5'-TTGCGGCCGCCTACATGTGCGACAGGGGCAG-3' (antisense) were used to generate {Delta}817, {Delta}895, and wild-type Sox-2, respectively. Klf4 cDNA and the primers of 5'-CCTCGAGGACCTTCTGGGCCCCCACATTAATG-3', 5'-CCTCGAGCCACCATGGCTTGCAGCAGTAACAACCC-3', 5'-CCTCGAGCCACCATGGCCGCCACCGTGACCACCTC-3', 5'-CCTCGAGCCACCATGGCGGTCCCGTGGTGCACGG-3' (sense), and 5'-CCGCGGCCGCACTACGTGGGATTTAAAAGTG-3' (antisense) for were used to generate wild-type, {Delta}80, {Delta}118, and {Delta}278 Klf4, respectively. All of these PCR products were digested with XhoI and NotI and inserted into the XhoI and NotI sites of pCAG-IP.

Cell culture, transfection, and luciferase assay. ZHBTc4 ES cells were maintained as described previously (28). For transfection of reporter plasmids, 3 x 104 cells were seeded in each well of a 24-well plate and incubated with 2 µg reporter plasmid and 0.02 µg of the internal control plasmid pRL-CMV, together with Lipofectamine 2000 (Invitrogen), following the manufacturer's protocol. Luciferase assays were performed 24 h later using a dual-luciferase assay kit (Promega). The ES cell lines maintained by mutant forms of Oct3/4 were made as described previously (27).

EMSA. Probe DNA sequences are shown in Tables 1 . The Cy-5-labeled oligonucleotides were annealed and 2 as double-stranded probes. Electrophoretic mobility shift assay (EMSA) was performed essentially as described previously (3). The whole-cell extracts (10 µg) were incubated on ice for 15 min with DNA probes containing the regulatory element of the Lefty1 gene. In competition assays, the whole-cell extracts were preincubated for 10 min with a 50-fold molar excess of unlabeled oligonucleotide and protein extracts prior to the addition of the probe mixture. For supershift experiments, protein extracts were preincubated for 30 min on ice with 0.2 µg of antibody to Klf4 (AB4138; Chemicon), Gata4 (sc-9053; Santa Cruz Biotechnology), Oct3/4, or Sox2 (AB5603; Chemicon). All resulting gels were scanned with a Fluorimager (Typhoon 8600; GE Healthcare, Little Chalfont, United Kingdom).


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  		TABLE 1. EMSA probes for Oct3/4 and Sox2

ChIP assay. Chromatin immunoprecipitation (ChIP) was performed with a ChIP-IT kit (Active Motif) according to the manufacturer's protocol. Genomic DNA was extracted from the precipitates and amplified by QPCR with the following primers: 5' control region, 5'-TCTGGAAGTCCCCTCTGCACT-3' and 5'-CAGGCTAGCTCCTGGTGTCT-3' (–1726 ~ –1548); Oct-Sox binding enhancer region, 5'-AAGCTGCAGACTTCATTCCA-3' and 5'-CGGGGGATAGATGAAGAAAC-3' (–1264 ~ –1060); inner control region, 5'-ATAGCCACACACCCTTGTCC-3' and 5'-CAGACGAGGGGCAACATAGT-3'; Klf4 binding region, 5'-GTCCAGACAGGCTTTTGTGT-3' and 5'-AGTCTGCGGAGGAATGGTA-3' (–147 ~ +26); 3' control region, 5'-GACTCCTGTTCCACTGAACG-3' and 5'-AGGCCCTGAATGCTAACTCT-3' (+1105 ~ +1343).

Microarray analysis. DNA microarray analyses were performed as described previously (1), using an NIA Mouse 22K Microarray 2.0 (Dev2; Agilent Technologies), which contained the genes listed at the National Institute of Aging mouse cDNA project web site (http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html). Briefly, 5 µg total RNA was transcribed into double-stranded T7 RNA polymerase-tagged cDNA and amplified into single-stranded, fluorescence-tagged cRNA by T7 polymerase. The samples for siKlf4 and mock transfectants were hybridized against a common reference pool of RNA at 60°C on the DNA microarrays. After washing, microarrays were scanned with an Agilent DNA Microarray Scanner. Complete array data will be available on the GEO (NCBI) website.

Knock-down analysis and QPCR. Each Klf4 short interfering RNA (5'-GACAUCGCCGGUUUAUAUUGA-3' and 5'-AAUAUAAACCGGCGAUGUCUU-3') was annealed and transfected into ES cells by use of the manufacturer's protocol (RNAi Co., Ltd., Tokyo, Japan). Total RNA was isolated from transfected cells, and the resulting cDNA was used in quantitative PCR (QPCR) with primers for Lefty1 (5'-TGTGTGTGCTCTTTGCTTCC-3' and 5'-GGGGATTCTGTCCTTGGTTT-3'), Klf4 (5'-CAAGTCCCCTCTCTCCATTATCAAGAG-3' and 5'-CCACTACGTGGGATTTAAAAGTGCCTC-3'), Oct3/4 (5'-CACGAGTGGAAAGCAACTCA-3' and 5'-AGATGGTGGTCTGGCTGAAC-3'), Sox2 (5'-ACATGTGAGGGCTGGACTGCGAAC-3' and 5'-GAAGCGCCTAACGTACCACTAGAAC-3'), Fgf4 (5'-GGGAGGCTACAGACAGCAAG-3' and 5'-CTGTGAGCCACCAGACAGAA-3'), Utf1 (5'-ACGTGGAGCATCTACGAGGT-3' and 5'-TAGACTGGGGGTCGTTTCTG-3'), Nanog (5'-ACCTGAGCTATAAGCAGGTTAAGAC-3' and 5'-GTGCTGAGCCCTTCTGAATCAGAC-3'), Fbx15 (5'-TCGCCTGCTTCCACTTACTT-3' and 5'-CATGCTGCTTCGTGACAGAT-3'), Rex1 (5'-GAGTTCGTCCATCTAAAAAGGGAGG-3' and 5'-TCTTAGCTGCTTCCTTGAACAATGCC-3'), and GAPDH (5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3').


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RESULTS
 
Sequences upstream of the Lefty1 gene are sufficient to promote Oct3/4-dependent expression of a reporter gene. Using genomic DNA and primers designed from the sequence of the Lefty1 gene (GenBank accession number AJ000083), we amplified its 1.3-kb upstream region, containing a promoter with a TATA box (33), and subcloned it into the luciferase (luc) reporter plasmid pGL3-Basic, generating pLefty1-luc (Fig. 1A). This fragment also contains the bilateral neural plate-specific enhancer (NPE) active in the prospective floor plate (35) and the retinoic acid-responsive element (33) of Lefty1. In undifferentiated ES cells, this DNA fragment had significant transcriptional activity comparable to that of other reporters activated by Oct3/4 (data not shown). We therefore tested the Oct3/4-dependent activity of this reporter construct in ZHBTc4 ES cells, which harbor the tetracycline (Tc)-dependent transactivator tTA and the Oct3/4 transgene under the control of the tTA-dependent promoter, and in which both endogenous alleles of Oct3/4 have been inactivated by gene targeting (28). ZHBTc4 cells can be propagated as ES cells in the absence of Tc when the Oct3/4 transgene is active but not in the presence of Tc, which represses the transgene. We previously showed that the activities of all reporter constructs activated by Oct3/4 and its cofactors were downregulated when Oct3/4 was repressed in ZHBTc4 ES cells, indicating that assays in the presence or absence of Tc can be used to test the Oct3/4 dependency of the reporter constructs (27). We found that pLefty1-luc activity was downregulated by addition of Tc, similar to the activity of the Oct3/4-dependent reporter Utf1tk-luc, which contains the Utf1 enhancer element with Oct3/4 and Sox2 binding sites (24), and the activity of the minimal promoter of the herpes simplex virus thymidine kinase (tk) gene (Fig. 1B). These results indicated that the 1.3-kb upstream region of Lefty1 contains an Oct3/4-dependent regulatory element.


Figure 1
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  		FIG. 1. Identification of an Oct3/4-dependent enhancer in the Lefty1 gene. (A) Isolation of the mouse Lefty1 promoter. A 1,379-bp genomic DNA fragment, from –1297 to +82 relative to the transcription start site, was subcloned by PCR. Ch. 1, chromosome 1. (B) Oct3/4-dependent promoter activity of pLefty1-luc in ES cells. Reporter plasmids were transfected into ZHBTc4 ES cells, followed by culture with or without Tc. The ratios (–Tc/+Tc) of the resulting luciferase activities are shown. (C and D) Oct3/4-dependent activities of deletion mutants of pLefty1-luc. (E and F) Enhancer activity of the element containing the putative Oct-Sox binding site. The activities of small deletion mutants (E) and pLefty1-luc with mutations in the Oct or Sox element (F) were assayed in ZHBTc4 ES cells (F). (G) EMSA of the Oct-Sox element. Wild-type (wt) Oct-Sox elements were incubated with ES nuclear extracts, with or without excess cold competitors (lanes 1 to 7), or incubated with antibodies to Oct3/4, Sox2, or Gata4 (lanes 8 to 10), as indicated, and subjected to EMSA. The sequences of the probes are shown in Table 1. Note that the supershifted bands (ss) and reduction of signal were obtained with each antibody.

Oct3/4-dependent expression is controlled by ESE. To locate cis elements responsible for the Oct3/4-dependent expression of Lefty1, we tested various restriction fragments derived from the 1.3-kb upstream region for their ability to confer Oct3/4-dependent expression on the luc reporter gene. Although pLefty1-luc and pDelApaI-luc retained this ability, Oct3/4-dependent expression was not apparent with pDelPvuII-luc or pDelSacI-luc, suggesting the presence of an Oct3/4-dependent distal enhancer in the 375-bp fragment between –1239 and –865 (Fig. 1C). We also found that pDelSacI-luc showed significant luciferase activity, indicating that the DNA fragment between –76 and +82 contains a minimal promoter of Lefty1 (Fig. 1C). When we tested a series of deletion mutants within the 1.3-kb upstream region of pLefty-luc in ZHBTc4 ES cells (Fig. 1D), we found that deletions between –1273 and –1068, between –1165 and –955, and between –1165 and –1068 removed the Oct3/4-dependent activity, indicating that ES-specific enhancer (ESE) activity is present in the 98-bp region between –1165 and –1068.

The Oct3/4-dependent enhancer contains Oct3/4 and Sox2 binding sites. Within the 98-bp region carrying ESE, we found an atypical putative Oct3/4 binding sequence (TTCTGCAT) flanked by a typical Sox2 binding sequence (AACAAAG) but without a spacer between them (Fig. 1E). Similar combinations of these binding sites are found in several Oct3/4-dependent enhancers (Table 3). This region, however, has been reported to contain the NPE with an atypical FAST binding sequence between –1097 and –1081 (GTCTTACAATCCACTA) (35). Precise deletion analysis in this region revealed that the 28-bp region between –1163 and –1136, which contains the putative Oct3/4 and Sox2 binding sites, also contains the ESE activity (Fig. 1E). These data also indicated that ESE is separate from NPE.


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  		TABLE 3. Sequences of Oct3/4-Sox2 binding sitesa

To determine whether these putative Oct3/4 and Sox2 binding sites are responsible for the observed enhancer activity of the Lefty1 gene, we tested the effect of mutations in each element on ESE activity. We found that mutation of either the Oct3/4 or Sox2 binding site markedly decreased the level of expression in ES cells, indicating that both sites are required to support Lefty1 ESE activity (Fig. 1F).

To identify the complexes containing Oct3/4 and/or Sox2, we incubated the DNA fragment containing the Lefty1 ESE that was used with an ES cell extract in the presence or absence of cold probes or specific antibodies (Fig. 1G). These EMSA experiments showed that the upper complex was supershifted by either anti-Oct3/4 or anti-Sox2 antibodies, indicating that it contained both Oct3/4 and Sox2. In contrast, the lower complex was supershifted by anti-Sox2 antibody but not by anti-Oct3/4 antibody, indicating that it contained only Sox2. The inability to detect Oct3/4 complex suggests that Sox2 may bind to this binding site with higher affinity than Oct3/4.

The Oct3/4 and Sox2 binding motifs are conserved in the human LEFTY-B gene. Since functionally important regulatory sequences should be conserved in evolution, we compared the sequence of the human ortholog of Lefty1, LEFTY-B (47), with that of mouse Lefty1. We found that the Oct3/4 and Sox2 binding motifs were highly conserved between species (Table 3). Indeed, LEFTY-B is expressed in undifferentiated but not in differentiated human ES cells (6). A comparison of the Oct-Sox binding sites of the Fgf4, Utf1, Fbx15, Sox2, Oct3/4, and Nanog genes with that of Lefty1 found that they were highly conserved (Table 3). From these comparisons, we deduced that the consensus sequence of the Oct3/4-Sox2 binding sites was WWWWGCATWACAAWG (W = A or T).

The N-terminal domain of Oct3/4 is required for efficient activation of pLefty1-luc. We have shown that Lefty1 expression is absent in ES cells maintained by an Oct3/4 variant lacking the N-terminal domain (NTD) (27). To test whether the regulatory element in pLefty1-luc is responsible for this phenomenon, we introduced pLefty1-luc into ES cell lines maintained by {Delta}N or {Delta}C variants of Oct3/4 (27). We found that pLefty1-luc showed significantly weaker activity in {Delta}N ES cells than in {Delta}C ES cells (Fig. 2A). In contrast, the reporter Utf1tk-luc showed only slightly weaker activity in {Delta}N ES cells than in {Delta}C ES cells, indicating that Oct3/4 activation of Lefty1 expression is dependent on the NTD of Oct3/4.

Oct3/4 and Sox2 are insufficient to activate pLefty1-luc in differentiated cells. The reporter assays in ES cells showed that pLefty1-luc expression was dependent on Oct3/4. To confirm the role of Oct3/4 and Sox2 in the activation of pLefty1-luc, we cotransfected Oct3/4 and Sox2 expression vectors, together with pLefty1-luc, into HeLa cells, but we observed no activation of pLefty1-luc expression (Fig. 2B). In contrast, both Fgf4tk-luc and Utf1tk-luc were efficiently activated under the same conditions (Fig. 2B), suggesting that an additional factor(s) in ES cells was required to activate pLefty1-luc.

Addition of Tc to ZHBTc4 ES cells has been found to completely deplete Oct3/4 within 24 h, repressing expression from all Oct3/4-dependent reporters (27). Since expression of endogenous Sox2 is maintained, activities of Fgf4tk-luc and Utf1tk-luc were restored by cotransfection of Oct3/4 expression vector alone (Fig. 2C). In contrast, pLefty1-luc was not activated under the same conditions (Fig. 2C), suggesting that the additional factor(s) required for activation of pLefty1-luc was downregulated by repression of Oct3/4.

Functional screening of an ES-specific factor for activation of pLefty1-luc. Since our results suggested that an unknown factor(s) expressed in undifferentiated ES cells is required to activate the Lefty1 promoter, we surveyed candidate transcription factors for their ability to activate pLefty1-luc in HeLa cells. Microarray analysis identified 11 candidate genes by their expression pattern after Oct3/4 repression (R. Matoba, H. Niwa, S. Masui, S. Ohtsuka, M. G. Carter, A. A. Sharov, and M. S. H. Ko, unpublished data). When we cotransfected expression vectors for these genes and pLefty1-luc with or without expression vectors for Oct3/4 and Sox2, we found that only one gene, Klf4, significantly activated pLefty1-luc. The Klf4 gene, which encodes a Krüppel-like zinc finger transcription factor, showed 8.8-fold activation of pLefty1-luc in the absence of Oct3/4 and Sox2 and 25.7-fold activation in their presence (Fig. 3A), indicating that it may be the missing factor.

Nonspecific activation may be observed under these artificial heterologous assay conditions. To exclude this possibility, we tested the specificity of pLefty1-luc activation by Klf4. Transfection of the Klf4 expression vector, together with reporters containing promoters for the Lefty1-luc, Oct3/4, Zfp42/Rex1, Utf1, and Tcl1 genes into HeLa cells, showed efficient activation of only pLefty1-luc (Fig. 3B). Significant activation was observed on the element at base pair –76 of Lefty1 (Lefty1{Delta}SacI), suggesting that Klf4 specifically activates the Lefty1 promoter via promoter-proximal elements.

Functional redundancy in Klf family members expressed in ES cells. Our microarray analysis showed that several Klf family members (14) are expressed in ES cells, with Klf2, Klf3, Klf4, and Klf9 showing stem cell-specific expression patterns (Matoba et al., unpublished), which was confirmed by quantitative PCR (QPCR) (data not shown). To test whether these four genes share redundant functions, we tested the ability of each to activate pLefty1-luc in HeLa cells; we also tested Klf5 because of its ability to compete with Klf4 (8). Of these five genes, only Klf2 showed significant ability to transactivate pLefty1-luc, although its activity was less than that of Klf4, suggesting that the function of these two genes overlaps in ES cells (Fig. 3C).

Klf4 acts as a cofactor of the Oct3/4-Sox2 complex. To determine the mechanism by which Klf4 cooperates with Oct3/4 and Sox2 to activate pLefty1-luc, we tested the ability of various combinations of these three gene products to transactivate pLefty1-luc. Although Oct3/4, Sox2, or the two together cannot activate pLefty1-luc, Klf4 alone significantly activated pLefty1-luc in HeLa cells (Fig. 4A). Addition of Oct3/4 or Sox2 to Klf4 enhanced this activation, and a combination of all three factors showed the highest activation of pLefty1-luc, indicating that the action of the distal enhancer activated by Oct3/4 and Sox2 is mediated by Klf4 binding to the proximal element to activate the Lefty1 core promoter.


Figure 4
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  		FIG. 4. Cooperation of Oct3/4, Sox2, and Klf4 in activating the Lefty1 reporter. (A) Cooperative activation of the Lefty1 reporter by Oct3/4, Sox2, and Klf4. pLefty1-luc was cotransfected with various combinations of empty, Oct3/4, Sox2, and Klf4 expression vectors into HeLa cells, and luciferase activities were measured relative to that of empty vector. (B) Activation of the Lefty1 reporter by Oct3/4 variants. pLefty1-luc was cotransfected with expression vectors of Oct3/4 variants, Sox2, and Klf4 into HeLa cells, and luciferase activities were measured relative to that of empty vector. (C) Activation of the Lefty1 reporter by Sox2 variants. pLefty1-luc was cotransfected with expression vectors of Sox2 variants, Oct3/4, and Klf4 into HeLa cells, and luciferase activities were measured relative to that of empty vector. (D and E) Activation of the Lefty1 reporter by Klf4 variants. pLefty1-luc was cotransfected with expression vectors of Klf4 variants alone (D) or Klf4 variants plus Oct3/4 and Sox2 (E) into HeLa cells, and luciferase activities were measured relative to that of empty vector. wt, wild type.

As shown above, pLefty1-luc cannot be activated efficiently by an Oct3/4 variant lacking the NTD (Fig. 2A). To confirm the requirement for the NTD to cooperate with Sox2 and Klf4, we transfected expression vectors for Oct3/4 variants (27), together with pLefty1-luc, Sox2, and Klf4 expression vectors, into HeLa cells (Fig. 4B). Activation of pLefty1-luc was not observed when {Delta}N was transfected, indicating its inability to cooperate with Sox2 and Klf4, nor was pLefty1-luc activated when an Oct3/4 mutant lacking DNA binding ability (267V/P) (27) was transfected. In contrast, transfection of {Delta}C Oct3/4 could activate pLefty1-luc as well as wild-type Oct3/4. Interestingly, the DNA binding domain alone, designated {Delta}N{Delta}C, was sufficient for cooperation with Sox2 and Klf4. These data indicated that the heterologous transactivation system in HeLa cells mimics the situation in ES cells, that pLefty1-luc activation was dependent on the NTD of Oct3/4, and that the C-terminal domain might have an inhibitory effect in the synergic action on the Lefty1 transactivation.

We also tested the contribution of the two transactivation domains of Sox2, R1 and R3 (30). When Sox2 variants lacking R1 ({Delta}R1) were cotransfected with pLefty1-luc and expression vectors for Oct3/4 and Klf4, activation of pLefty1-luc was inhibited (Fig. 4C). Further deletion of R3, however, did not further reduce pLefty1-luc transactivation, suggesting that the R1 transactivation domain of Sox2 cooperates with Oct3/4 and Klf4 in activating pLefty1-luc.

Deletion of the transactivation domain of Klf4, between amino acids 80 and 118 (10), reduced pLefty1-luc activation (Fig. 4D) as well as its ability to cooperate with Oct3/4 and Sox2 (Fig. 4E), indicating that the transactivation domain of Klf4 is required for proper activation of the Lefty1 reporter.

Identification of Klf4 binding elements proximal to the Lefty1 promoter. Lefty1{Delta}SacI consists of a 76-bp sequence upstream of the transcription start site. This reporter contains two putative Klf4 binding sites, K1 and K2 (Fig. 5A), that match the consensus sequence (RRGGYGY) (39) and are conserved in human LEFTY-B. Using EMSA, we tested the ability of Klf4 to bind to these sites (Fig. 5B). Using a probe containing both putative sites, we observed binding of recombinant Klf4 in COS cells. Of the complexes observed in ES cells, one showed the same mobility as that observed in COS cells. Complex formation was strongly inhibited by competition with cold wild-type K1 and weakly inhibited by competition with cold wild-type K2, suggesting that Klf4 binds to these sites with different affinities.


Figure 5
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  		FIG. 5. Identification of Klf4 binding sites proximal to the Lefty1 core promoter. (A) Sequence around the Lefty1 core promoter in pLefty1{Delta}SacI-luc. Two putative Klf4 binding sites (K1 and K2) were identified proximal to the core promoter, both of which are conserved in human LEFTYB. Mutated sequences for K1m and K2m are also shown. (B) Contribution of the putative Klf4 binding sites to the Klf4-dependent activity of pLefty1{Delta}SacI-luc. pLefty1{Delta}SacI-luc derivatives with mutations in K1 and/or K2 or deletion of both sites were cotransfected with Klf4 or empty expression vector into HeLa cells, and luciferase activities were determined relative to that of empty vector. (C) Cell lysate of ES or Cos-7 cells transfected with Klf4 or empty expression vector was subjected to EMSA with or without competitor. All probes are described in Table 2. (D) ChIP assay of the Lefty1 distal enhancer, proximal element, and core promoter. Primers for QPCR were designed to detect the distal enhancer bound by Oct3/4 and Sox2 (region b), the proximal element bound by Klf4 and the core promoter (region d), and their flanking regions (a, c, and e) as controls. Chromatin samples derived from undifferentiated (ZHBTc4 Tc–), differentiated (ZHBTc4 Tc+), and {Delta}N ES cells were immunoprecipitated with the indicated antibody, followed by QPCR with normalization by input DNA. The amounts of region a were set at 1.0 in each set of analyses, and the relative signal intensities for regions b, c, d, and e were calculated.

The functions of these promoter-proximal elements were assayed using reporter constructs carrying mutations in one or both elements (Fig. 5C). A mutation in K1 did not affect Klf4-dependent reporter activity, whereas K2 mutation reduced this activity. Mutations in both sites showed a reduction in reporter activity comparable to that observed when the short sequence with both sites was deleted, suggesting that both of these sites function as targets of Klf4.

To investigate the dynamics of recruitment of Klf4 at the proximal element and Oct3/4-Sox2 at the distal enhancer in vivo, ChIP analysis was performed in ES cells (Fig. 5D). Chromatin samples were prepared from undifferentiated (ZHBTc4 without Tc), differentiated (ZHBTc4 72 h after addition of Tc as a negative control), and {Delta}N ES cells, in which Lefty1 is activated, repressed, and inefficiently activated, respectively. When these chromatin samples were immunoprecipitated with either anti-Oct3/4, anti-Sox2, or anti-Klf4, significant accumulation of both the distal enhancer element (region b) and the region including the proximal element and the core promoter (region d) were observed from a chromatin sample of undifferentiated ES cells but not from that of differentiated ES cells. These data clearly demonstrate interaction between Oct3/4 and Sox2 on the distal enhancer and Klf4 on the proximal element to activate the Lefty1 core promoter in vivo. In {Delta}N ES cells, accumulation of region d by anti-Oct3/4 or anti-Sox2 was not observed, whereas weak enrichment of this region by anti-Klf4 was still observed. These data indicate that deletion of the N terminus of Oct3/4 caused its inability to form a stable regulatory complex on the distal enhancer with Sox2, resulting in inefficient recruitment of Klf4 on the proximal element and weak activation of the core promoter.

Physiological role of Klf4 in the activation of Oct3/4 targets including Lefty1. To confirm the physiological role of Klf4 on the expression of Lefty1 in ES cells, we analyzed gene expression patterns in ES cells following reduction of expression of Klf4. When we introduced short interfering RNA specific for Klf4 (siKlf4) into EB5 ES cells, we found that although these transfectants showed no morphological changes, QPCR analysis showed that the level of expression of Klf4 was reduced to 50% of that observed in mock transfectants, confirming the expected effect of siKlf4 in ES cells (Fig. 6A). When we assayed the level of expression of Oct3/4, Sox2 and their known target genes, including Lefty1, we found that only Lefty1 showed significantly reduced expression (Fig. 6A). Using a DNA microarray, we found that many genes were downregulated after repression of Klf4 (Fig. 6B). After normalization to mock transfectants, we identified 226 candidate Klf4 target genes. When we compared these genes with the 307 putative target genes positively regulated by Oct3/4 (Matoba et al., unpublished), we found 28 that overlapped, including Lefty1 (Fig. 6C). These results indicate that there is a set of target genes in ES cells whose expression may be regulated by cooperation of Klf4 and Oct3/4.


Figure 6
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  		FIG. 6. Klf4 target genes in ES cells. (A) Expression of stem cell marker genes in ES cells treated with siKlf4. Relative expression levels of each gene in ES cells treated with mock transfectant (filled; set at 1.0) or siKlf4 (hatched) was estimated by QPCR. (B) DNA microarray analyses of ES cells treated with siKlf4. Log ratios were plotted for genes with relative expression levels. The plot for Lefty1 is indicated. (C) Identification of putative target genes regulated by both Klf4 and Oct3/4. We identified 28 genes whose expression was dependent on both Klf4 and Oct3/4. (D) Activation of the 1200015N20Rik promoter by Klf4. 1200015N20Rik-luc, 6Wtk-luc, or tk-luc was cotransfected with empty or Klf4 expression vector into HeLa cells, and the luciferase activities relative to empty vector were determined. (E) Oct3/4 reactivation of reporters in Oct3/4-depleted ES cells. Reporter plasmids were transfected into ZHBTc4 ES cells with or without Oct3/4 expression vector, the cells were cultured with or without Tc, and luciferase activities relative to that in ES cells without Tc and Oct3/4 expression vector were determined.

1200015N20Rik is a putative target of Klf4 and Oct3/4 in ES cells. To confirm the accuracy of our microarray screening, we tested the expression of 14 candidate genes in ES cells treated with siKlf4. Using QPCR, we found that 1200015N20Rik showed differential expression (data not shown). Following isolation and subcloning of the 3.8-kb promoter region of 1200015N20Rik into pGL3-Basic, it was found that this reporter was activated by Klf4 in HeLa cells (Fig. 6D) and showed Oct3/4-dependent activity in ZHBTc4 ES cells (Fig. 6E). Interestingly, similar to pLefty1-luc, the reduction of reporter activity by incubation with Tc, which depletes Oct3/4, was not restored by cotransfection of Oct3/4, suggesting that 1200015N20Rik and pLefty1-luc are regulated similarly in ES cells.


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DISCUSSION
 
Synergistic action of tissue-specific transcription factors is a pivotal mechanism for determining cellular phenotypes. In mouse ES cells, the pluripotent phenotype depends on the function of Oct3/4 and Nanog. In addition, Sox2 may be important because it cooperates with Oct3/4 to activate the three genes. Since only a few of their target genes have been identified, none of which has a critical function in the self-renewal process, it is still unclear how these transcription factors maintain pluripotency. We recently reported that Oct3/4 maintains pluripotency, at least in part, by interference with the function of Cdx2, which acts as a trigger for trophectoderm differentiation (29). Here we describe a previously unknown function of Oct3/4 in cooperation with a new partner, Klf4, in ES cells.

The ability of Oct3/4 to cooperate with Sox2 was first identified on the Fgf4 enhancer and then found to be a common mechanism of Oct3/4 action (26). Activation by Oct3/4-Sox2 has been confirmed for various ES-specific enhancers, including those of Oct3/4 and Sox2 themselves (32, 43). We have shown here that Lefty1 can be added to this list. We previously reported that Lefty1 expression dramatically decreased in ES cells expressing an Oct3/4 variant lacking the NTD, whereas expression of all other known target genes was not affected (27 and data not shown), indicating that NTD dependency is unique to Lefty1. In addition, we found that pLefty1-luc is hardly activated by Oct3/4 and Sox2 in HeLa cells and shows poor reactivation by Oct3/4 in Oct3/4-depleted ES cells, whereas other Oct3/4-Sox2-dependent reporters are activated under these conditions. These observations are consistent with the specific recruitment of Klf4 to the proximal element of the Lefty1 core promoter. Indeed, all known Oct3/4-Sox2 binding elements shown above are distal enhancers located far from the core promoters, which require one or more promoter-proximal elements situated within 100 to 200 bp of the transcription start sites (4, 5, 9, 11, 12). We have shown here that Klf4 cooperates with Oct3/4 in an NTD-dependent manner and is expressed in ES cells in an Oct3/4-dependent manner. These findings indicate that the target genes of the Oct3/4-Sox2 complex can be categorized into different classes by their different dependencies on the cofactors recruited on the promoter-proximal elements. Our DNA microarray analysis revealed that there are only a few Klf4-dependent Oct3/4 target genes, suggesting the presence of a yet-unidentified partner molecule(s) (Fig. 7A).


Figure 7
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  		FIG. 7. Role of Klf4 in the activation of the Lefty1 promoter. (A) Differential promoter factor requirement for activation by Oct3/4 and Sox2. The Lefty1 promoter is activated by recruitment of Klf4 as a promoter factor, but requirements for the Utf1 and Oct3/4 promoters have not yet been identified. (B) Regulation of Lefty1 by ES-specific transcription factors. Oct3/4 and Sox2 are activated by themselves, and Klf4 is under the control of Oct3/4.

One possible explanation for the variety of cofactors recruited by Oct3/4-Sox2 is that this mechanism ensures the precise differential expression pattern of target genes along the developmental time course in pluripotent cells. While Fgf4 (25) and Utf1 (31) are expressed in primitive ectoderm of the egg cylinder-stage embryo, Lefty1 is not (20). It was recently reported that Lefty1 expression begins randomly in ICM and is regionalized to one side of the tilted ICM shortly after implantation (40). Although Klf4 expression has not yet been assayed in this stage, the differential expression of Oct3/4-Sox2 target genes may be due to the differential expression of these cofactors. During early embryogenesis, pluripotent cells change character, from ICM to epiblast to primitive ectoderm, while expressing Oct3/4 (37) and Sox2 (2). Thus, regulation of cofactor expression may be involved in this transition. The rapid downregulation of Klf4 expression following repression of Oct3/4 in ES cells suggests that Klf expression is under the control of Oct3/4 (Matoba et al., unpublished). Therefore, Lefty1 expression is regulated by Oct3/4, both directly and indirectly via Klf4 (Fig. 7B), and Klf4 itself should have a unique mode of regulation by Oct3/4.

Although stem cell-specific expression of Lefty1/LEFTYB has been observed in human ES cells (6, 36, 45), its functional significance has not yet been demonstrated. Activin/Nodal signaling through Smad2/3 activation was recently reported to be necessary to maintain the pluripotent status of human ES cells (13, 44, 46). Interestingly, both human and mouse ES cells produce Nodal and Cripto, a component of the Nodal receptor, in stem cell-specific manners (6, 36). Since Lefty1 acts as an inhibitor of Activin/Nodal, overexpression of Lefty1 in human ES cells blocked this autocrine loop and the induction of differentiation (44). These findings suggest that Lefty1/LEFTYB may control the capacity for self renewal by competing with the Activin/Nodal autocrine loop. We demonstrated here that the ES-specific enhancer contributed to the activation of Lefty1 expression in a stem cell-specific manner, providing the opportunity to control self renewal by the stem cell-specific transcription factor network. Although it is not clear whether Lefty1 functions during embryogenesis in pluripotent cell populations, Lefty1-null embryos showed defective left-right axis determination (22). In contrast, Lefty2-null embryos died earlier, during the process of gastrulation (20), but they passed the early developmental stage without defects in pluripotent cell populations. Whether Lefty1 and Lefty2 have functional redundancy may be revealed by assaying double mutants, but this may be difficult to generate due to their proximity on mouse chromosome 1 (21).

Klf4 was initially identified as a Klf family member expressed in the gut (39). Klf4-null mice die within 15 h after birth due to a skin barrier defect caused by a perturbation in the differentiation of epidermis (38). A differentiation defect is also observed in the goblet cells of the colon in these knockout mice (15), indicating the indispensable function of Klf4 in the proper development of these organs. In contrast to Lefty1-null embryos, however, Klf4-null mice have no abnormalities in their pluripotent cell population during embryogenesis, indicating that Klf4 function is dispensable in the establishment and maintenance of pluripotency. In contrast, overexpression of Klf4 in mouse ES cells was found to prevent differentiation in embryoid bodies formed in suspension culture, suggesting that Klf4 contributes to ES self renewal (18). We also found that Klf4 overexpression can support leukemia inhibitory factor-independent self renewal of mouse ES cells (unpublished data). This discrepancy between loss- and gain-of-function phenotypes suggests that the former is masked by redundancy between closely related genes. In this case, Klf2 is a good candidate for a functionally overlapping gene, because we found it could activate the Lefty1 promoter and support leukemia inhibitory factor-independent self renewal. Klf2-null embryos died between 12.5 and 14.5 days postcoitum due to severe hemorrhaging (16). Analysis of Klf2/Klf4 double-null embryos may reveal their functions in pluripotent cells in embryos.

Since Oct3/4 is pivotal for maintaining pluripotency in ES cells (28) and during embryogenesis (23), analysis of its function is important for understanding the molecular mechanism governing pluripotency. Although cooperation with Sox2 is important for Oct3/4 function, this complex still requires additional partners to acquire physiological function. While our findings show that Klf4 is one of these partners, Klf4 is involved in the activation of only a subset of Oct3/4 target genes. However, the strategy we used to identify Klf4 should be a powerful tool to identify other partner molecules.


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  		TABLE 2. EMSA probes for Klf4


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ACKNOWLEDGMENTS
 
We thank Yulan Piao for technical assistance in microarray analysis. This research was supported by a RIKEN grant and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, including that for the 21st Century COE Program "Center of Excellence for Signal Transduction Disease: Diabetes Mellitus as Model" and the Leading Project (to H.N.), as well as in part by the Intramural Research Program of the National Institute on Aging, NIH (to M.S.H.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology, Minatojima-Minamimachi 2-2-3, Chu-o-ku, Kobe 650-0047, Japan. Phone: 81-78-306-1927. Fax: 81-78-306-1929. E-mail: niwa{at}cdb.riken.jp. Back

{triangledown} Published ahead of print on 5 September 2006. Back


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Molecular and Cellular Biology, October 2006, p. 7772-7782, Vol. 26, No. 20
0270-7306/06/$08.00+0     doi:10.1128/MCB.00468-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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