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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 |
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INTRODUCTION |
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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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 
817, 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, 80, 118, and 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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–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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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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 N or C variants of Oct3/4 (27). We found that pLefty1-luc showed significantly weaker activity in N ES cells than in C ES cells (Fig. 2A). In contrast, the reporter Utf1tk-luc showed only slightly weaker activity in N ES cells than in 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 (Lefty1SacI), 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.
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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 C Oct3/4 could activate pLefty1-luc as well as wild-type Oct3/4. Interestingly, the DNA binding domain alone, designated NC, 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 (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.
Lefty1SacI 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.
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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 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 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.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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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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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 5 September 2006.
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REFERENCES |
|---|
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. Avilion, A. A., S. K. Nicolis, L. H. Pevny, L. Perez, N. Vivian, and R. Lovell-Badge. 2003. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17:126-140.
3. Ben-Shushan, E., J. R. Thompson, L. J. Gudas, and Y. Bergman. 1998. Rex-1, a gene encoding a transcription factor expressed in the early embryo, is regulated via Oct-3/4 and Oct-6 binding to an octamer site and a novel protein, Rox-1, binding to an adjacent site. Mol. Cell. Biol. 18:1866-1878.
4. Bergman, Y., D. Rice, R. Grosschedl, and D. Baltimore. 1984. Two regulatory elements for immunoglobulin kappa light chain gene expression. Proc. Natl. Acad. Sci. USA 81:7041-7045.
5. Bertolino, E., and H. Singh. 2002. POU/TBP cooperativity: a mechanism for enhancer action from a distance. Mol. Cell. 10:397-407.[CrossRef][Medline]
6. Besser, D. 2004. Expression of nodal, lefty-A, and lefty-B in undifferentiated human embryonic stem cells requires activation of Smad2/3. J. Biol. Chem. 279:45076-45084.
7. Botquin, V., H. Hess, G. Fuhrmann, C. Anastassiadis, M. K. Gross, G. Vriend, and H. R. Schöler. 1998. New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2. Genes Dev. 12:2073-2090.
8. Dang, D. T., W. Zhao, C. S. Mahatan, D. E. Geiman, and V. W. Yang. 2002. Opposing effects of Kruppel-like factor 4 (gut-enriched Kruppel-like factor) and Kruppel-like factor 5 (intestinal-enriched Kruppel-like factor) on the promoter of the Kruppel-like factor 4 gene. Nucleic Acids Res. 30:2736-2741.
9. Dierks, P., A. van Ooyen, M. D. Cochran, C. Dobkin, J. Reiser, and C. Weissmann. 1983. Three regions upstream from the cap site are required for efficient and accurate transcription of the rabbit beta-globin gene in mouse 3T6 cells. Cell 32:695-706.[CrossRef][Medline]
10. Geiman, D. E., H. Ton-That, J. M. Johnson, and V. W. Yang. 2000. Transactivation and growth suppression by the gut-enriched Kruppel-like factor (Kruppel-like factor 4) are dependent on acidic amino acid residues and protein-protein interaction. Nucleic Acids Res. 28:1106-1113.
11. Green, M. R., R. Treisman, and T. Maniatis. 1983. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products. Cell 35:137-148.[CrossRef][Medline]
12. Hen, R., P. Sassone-Corsi, J. Corden, M. P. Gaub, and P. Chambon. 1982. Sequences upstream from the T-A-T-A box are required in vivo and in vitro for efficient transcription from the adenovirus serotype 2 major late promoter. Proc. Natl. Acad. Sci. USA 79:7132-7136.
13. James, D., A. J. Levine, D. Besser, and A. Hemmati-Brivanlou. 2005. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132:1273-1282.
14. Kaczynski, J., T. Cook, and R. Urrutia. 2003. Sp1- and Kruppel-like transcription factors. Genome Biol. 4:206.[CrossRef][Medline]
15. Katz, J. P., N. Perreault, B. G. Goldstein, C. S. Lee, P. A. Labosky, V. W. Yang, and K. H. Kaestner. 2002. The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon. Development 129:2619-2628.
16. Kuo, C. T., M. L. Veselits, K. P. Barton, M. M. Lu, C. Clendenin, and J. M. Leiden. 1997. The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis. Genes Dev. 11:2996-3006.
17. Kuroda, T., M. Tada, H. Kubota, H. Kimura, S. Y. Hatano, H. Suemori, N. Nakatsuji, and T. Tada. 2005. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol. Cell. Biol. 25:2475-2485.
18. Li, Y., J. McClintick, L. Zhong, H. J. Edenberg, M. C. Yoder, and R. J. Chan. 2005. Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4. Blood 105:635-637.
19. Malik, S., and R. G. Roeder. 2005. Dynamic regulation of pol II transcription by the mammalian Mediator complex. Trends Biochem. Sci. 30:256-263.[CrossRef][Medline]
20. Meno, C., K. Gritsman, S. Ohishi, Y. Ohfuji, E. Heckscher, K. Mochida, A. Shimono, H. Kondoh, W. S. Talbot, E. J. Robertson, A. F. Schier, and H. Hamada. 1999. Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation. Mol. Cell 4:287-298.[CrossRef][Medline]
21. Meno, C., Y. Ito, Y. Saijoh, Y. Matsuda, K. Tashiro, S. Kuhara, and H. Hamada. 1997. Two closely-related left-right asymmetrically expressed genes, lefty-1 and lefty-2: their distinct expression domains, chromosomal linkage and direct neutralizing activity in Xenopus embryos. Genes Cells 2:513-524.[Abstract]
22. Meno, C., A. Shimono, Y. Saijoh, K. Yashiro, K. Mochida, S. Ohishi, S. Noji, H. Kondoh, and H. Hamada. 1998. lefty-1 is required for left-right determination as a regulator of lefty-2 and nodal. Cell 94:287-297.[CrossRef][Medline]
23. Nichols, J., B. Zevnik, K. Anastassiadis, H. Niwa, D. Klewe-Nebenius, I. Chambers, H. Schöler, and A. Smith. 1998. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95:379-391.[CrossRef][Medline]
24. Nishimoto, M., A. Fukushima, A. Okuda, and M. Muramatsu. 1999. The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2. Mol. Cell. Biol. 19:5453-5465.
25. Niswander, L., and G. R. Martin. 1992. Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114:755-768.[Abstract]
26. Niwa, H. 2001. Molecular mechanism to maintain stem cell renewal of ES cells. Cell Struct. Funct. 26:137-148.[CrossRef][Medline]
27. Niwa, H., S. Masui, I. Chambers, A. G. Smith, and J. Miyazaki. 2002. Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells. Mol. Cell. Biol. 22:1526-1536.
28. Niwa, H., J. Miyazaki, and A. G. Smith. 2000. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24:372-376.[CrossRef][Medline]
29. Niwa, H., Y. Toyooka, D. Shimosato, D. Strumpf, K. Takahashi, R. Yagi, and J. Rossant. 2005. Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell 123:917-929.[CrossRef][Medline]
30. Nowling, T. K., L. R. Johnson, M. S. Wiebe, and A. Rizzino. 2000. Identification of the transactivation domain of the transcription factor Sox-2 and an associated co-activator. J. Biol. Chem. 275:3810-3818.
31. Okuda, A., A. Fukushima, M. Nishimoto, A. Orimo, T. Yamagishi, Y. Nabeshima, M. Kuro-o, Y. Nabeshima, K. Boon, M. Keaveney, H. G. Stunnenberg, and M. Muramatsu. 1998. UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells. EMBO J. 17:2019-2032.[CrossRef][Medline]
32. Okumura-Nakanishi, S., M. Saito, H. Niwa, and F. Ishikawa. 2005. Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells. J. Biol. Chem. 280:5307-5317.
33. Oulad-Abdelghani, M., C. Chazaud, P. Bouillet, M. G. Mattei, P. Dolle, and P. Chambon. 1998. Stra3/lefty, a retinoic acid-inducible novel member of the transforming growth factor-beta superfamily. Int. J. Dev. Biol. 42:23-32.[Medline]
34. Roeder, R. G. 2005. Transcriptional regulation and the role of diverse coactivators in animal cells. FEBS Lett. 579:909-915.[CrossRef][Medline]
35. Saijoh, Y., H. Adachi, K. Mochida, S. Ohishi, A. Hirao, and H. Hamada. 1999. Distinct transcriptional regulatory mechanisms underlie left-right asymmetric expression of lefty-1 and lefty-2. Genes Dev. 13:259-269.
36. Sato, N., I. M. Sanjuan, M. Heke, M. Uchida, F. Naef, and A. H. Brivanlou. 2003. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev. Biol. 260:404-413.[CrossRef][Medline]
37. Schöler, H. R., G. R. Dressler, R. Balling, H. Rohdewohld, and P. Gruss. 1990. Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. EMBO J. 9:2185-2195.[Medline]
38. Segre, J. A., C. Bauer, and E. Fuchs. 1999. Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat. Genet. 22:356-360.[CrossRef][Medline]
39. Shields, J. M., R. J. Christy, and V. W. Yang. 1996. Identification and characterization of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J. Biol. Chem. 271:20009-20017.
40. Takaoka, K., M. Yamamoto, H. Shiratori, C. Meno, J. Rossant, Y. Saijoh, and H. Hamada. 2006. The mouse embryo autonomously acquires anterior-posterior polarity at implantation. Dev. Cell. 10:451-459.[Medline]
41. Tapscott, S. J. 2005. The circuitry of a master switch: Myod and the regulation of skeletal muscle gene transcription. Development 132:2685-2695.
42. Tokuzawa, Y., E. Kaiho, M. Maruyama, K. Takahashi, K. Mitsui, M. Maeda, H. Niwa, and S. Yamanaka. 2003. Fbx 15 is a novel target of Oct3/4 but dispensable for embryonic stem cell self-renewal and mouse development. Mol. Cell. Biol. 23:2699-2708.
43. Tomioka, M., M. Nishimoto, S. Miyagi, T. Katayanagi, N. Fukui, H. Niwa, M. Muramatsu, and A. Okuda. 2002. Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex. Nucleic Acids Res. 30:3202-3213.
44. Vallier, L., M. Alexander, and R. A. Pedersen. 2005. Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J. Cell Sci. 118:4495-4509.
45. Wei, C. L., T. Miura, P. Robson, S. K. Lim, X. Q. Xu, M. Y. Lee, S. Gupta, L. Stanton, Y. Luo, J. Schmitt, S. Thies, W. Wang, I. Khrebtukova, D. Zhou, E. T. Liu, Y. J. Ruan, M. Rao, and B. Lim. 2005. Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state. Stem Cells 23:166-185.
46. Xiao, L., X. Yuan, and S. J. Sharkis. 2006. Activin A maintains self-renewal and regulates FGF, Wnt and BMP pathways in human embryonic stem cells. Stem Cells 24:1476-1486.
47. Yashiro, K., Y. Saijoh, R. Sakuma, M. Tada, N. Tomita, K. Amano, Y. Matsuda, M. Monden, S. Okada, and H. Hamada. 2000. Distinct transcriptional regulation and phylogenetic divergence of human LEFTY genes. Genes Cells 5:343-357.[Abstract]
48. Yuan, H., N. Corbi, C. Basilico, and L. Dailey. 1995. Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3. Genes Dev. 9:2635-2645.
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