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Department of Development and Differentiation,1 Department of Molecular and Cellular Biology,3 Laboratory of Embryonic Stem Cell Research,4 Laboratory of Stem Cell Engineering, Institute for Frontier Medical Sciences, Kyoto University, Shogoin, Sakyo-ku, Kyoto,5 ReproCELL Inc., Chiyoda-ku, Tokyo, Japan2
Received 14 July 2004/ Returned for modification 26 August 2004/ Accepted 7 December 2004
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ABSTRACT |
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INTRODUCTION |
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The pluripotential state of cells is maintained under the regulation of some key genes whose expression is specific to pluripotential cells. The Oct4 gene, which is a member of the mammalian POU family of transcriptional factor genes, functions as a key regulator of the pluripotential state (16, 20). Sox2, known to act cooperatively at promoters with Oct4, activates transcription of the Fgf4, Utf1, Sox2, and Fbx15 genes (17, 33, 34, 42). Furthermore, the genes transcribed in the trophoectodermal lineage, Cdx-2 and Hand-1, are negatively regulated by Oct4 (20).
Another key molecule involved in the signaling pathway for maintaining the capacity for the self-renewal and pluripotency of mouse ES cells is leukemia inhibitory factor (LIF) (26, 38). LIF directs the activation of transcription factor STAT3 by phosphorylation through binding to the heterodimer of the LIF receptor and gp130 (6). Recently, it was also shown that the LIF signal is not sufficient to support the self-renewal of mouse ES cells under culture conditions in the absence of serum and feeder cells. An additional signal provided by bone morphogenetic proteins is required and induces the activation of the inhibitor of differentiation (Id) genes, which repress differentiation into the neuroectodermal lineage (41). In addition to the roles of these genes, it was demonstrated that Ezh2, a mammalian homologue of the Polycomb-group gene Enhancer of zeste in Drosophila (12), forms a complex with Eed (embryonic ectoderm development). This complex plays an important role in maintaining the pluripotency of ES cells and blastocyst inner cell mass cells through histone H3 lysine 27 methylation-based repression of specific homeotic genes (4, 7). Null mutation of the Oct4, Sox2, or Ezh2 gene results in early embryonic lethality (1, 16, 21); interestingly, however, loss of the Bmp4, Lif, Lif receptor, or Stat3 gene induces no obvious defect, at least in mouse preimplantation development (28, 31, 36, 39). It is known that LIF is dispensable for supporting the self-renewal and pluripotency of monkey and human ES cells (32).
NANOG is a newly identified homeodomain-bearing protein that may act as a transcription factor and that is transcribed specifically in pluripotential cells in mouse preimplantation embryos, ES cells, and EG cells (3, 15, 35) and monkey and human ES cells (8, 9). A critical requirement for Nanog in the maintenance of pluripotency has been suggested by the loss of pluripotency in Nanog-deficient ES cells and in Nanog-null embryos shortly after implantation (15). In addition, Nanog overexpression leads to the clonal expansion of ES cells by bypassing regulation by LIF-STAT3 signaling and maintenance of OCT4 levels (3). Thus, Nanog is an important regulator of pluripotency and self-renewal of ES cells and early embryonic cells. However, it remains largely unknown how the pluripotential cell-specific expression of Nanog is controlled and how the other stem cell-specific genes are implicated in Nanog expression.
To address the molecular mechanisms of pluripotential cell-specific expression, we investigated the regulatory elements that are involved in the control of Nanog transcription. We show that the undifferentiated state-specific expression of a green fluorescence protein (GFP) reporter gene in mouse ES cells can be induced by the addition of a 2.5-kb 5'-flanking region of Nanog, indicating that transcriptional cis regulatory elements exist in this region. Luciferase assays with deletion constructs of the 5'-flanking region revealed that the –332-bp fragment (–332 fragment) containing a pair of adjacent Octamer and Sox elements plays a crucial role in directing transcriptional up-regulation. Consistent with these results, we found that transcription was down-regulated by the introduction of sequence mutations in the Octamer and/or Sox elements. In nuclear extracts from F9 EC cells, specific binding of OCT4 to the Octamer element and of SOX2 to the Sox element was detected. Similar results were seen in EG cell extracts. In ES cell extracts, however, a complex of OCT4 and a novel pluripotential cell-specific Sox element-binding protein (PSBP) preferentially bound to the Octamer/Sox element. Nanog transcription is therefore regulated differently in ES, EG, and EC cells, and a novel factor (PSBP) may be involved in maintaining the ES cell-specific undifferentiated state.
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MATERIALS AND METHODS |
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Transgenic cell lines. The LR/Nanog-GFP transgene was constructed with a GFP-internal ribosome entry site (IRES)-puro-pA reporter cassette and with a 2.5-kb 5'-end genomic fragment and a 3.9-kb 3'-end genomic fragment of Nanog in vector pGEM-T Easy (Promega). After linearization, the transgene was electroporated into 107 R1 ES cells at 250 V and 500 µF with a Gene Pulser (Bio-Rad). Genomic DNA obtained from puromycin-resistant clones was screened by Southern blot hybridization analysis. A stable transformant ES cell line, –332-GFP TG, was obtained as a G418-resistant clone after cotransfection of vector p–332-pEGFP-N1 (Clontech) and vector pPgk-neo(TAKARA) into R1 ES cells.
Southern blot hybridization analysis. Genomic DNA was digested with restriction enzymes, electrophoresed through 1.0% agarose, and transferred to Hybond N+ nylon membranes (Amersham) by alkali blotting. Membranes were hybridized at 42°C overnight with either a 5' probe (500 bp) or a 3' probe (750 bp) labeled with 32P-dCTP by using a Megaprime DNA labeling system (Amersham). Membranes then were washed with 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS) at 65°C for 30 min and with 0.1x SSC-0.1% SDS at 65°C for 15 min.
Construction of reporter and expression vectors. Deletion fragments of the mouse Nanog promoter were PCR amplified from the mouse genome with a common antisense primer that spans an XhoI restriction site (+50 bp from the transcriptional start site, 5'-CTACTCGAGCGCAGCCTTCCCACAGAAA-3') and various sense primers into which an XhoI restriction site was introduced (–2,342 bp, 5'-CTACTCGAGTGGTGTAAACAGTGGGTCTG-3'; –332 bp, 5'-CTACTCGAGATCGCCAGGGTCTGGA-3'; and –153 bp, 5'-CTACTCGAGCCTGCAGGTGGGATTAACT-3'). The PCR products were digested with XhoI and ligated into the XhoI site of pGL3-Basic (Promega) or cytomegalovirus promoterless vector pEGFP-N1 (Clontech) (–m332 or –m153). PCR fragments of the human Nanog promoter were amplified from human ES cell DNA with a sense primer (–380 bp from the transcriptional start site) having the sequence 5'-GCTGGTTTCAAACTCCTGACTTC-3' and an antisense primer (+24 bp) having the sequence 5'-TCCTGGAGTCTCTAGATT-3' and ligated into vector pGEM-T Easy. NotI-NotI (–380 to +24 bp), NotI-PstI (–123 to +24 bp), and NotI-StyI (–101 to +24 bp) fragments were blunt ended and recloned into the SmaI site of cytomegalovirus promoterless vector pEGFP-N1. Oligonucleotide-directed mutations were introduced into the Octamer and/or Sox elements by PCR with primers having nucleotide replacements.
Oct4 and Sox2 open reading frames (ORFs) amplified by reverse transcription-PCR with primers EcoRI-Oct4-ORF-F (5'-CCGAATTCGGATGGCTGGACACCTGGCTTCAG-3'), BglII-Oct4-ORF-R (5'-AGAGATCTTTAACCCCAAAGCTCCAGGTTC-3'), EcoRI-Sox2-ORF-F (5'-CCGAATTCGGATGTATAACATGATGGAGACGG-3'), and BglII-Sox2-ORF-R (5'-AGAGATCTTCACATGTGCGACAGGGGCAGT-3') were subcloned into vector pGEM-T Easy. EcoRI-BglII fragments of Oct4 and Sox2 were ligated into expression vectors pCMV-Myc and pCMV-HA (Promega), respectively.
For cotransfection reporter assays, three tandem repeats of the Octamer and Sox elements, which were produced by ligation of synthetic oligonucleotides (Nanog-O/S-F, 5'-GATCCTTACAGCTTCTTTTGCATTACAATGTCCATGGTGGA-3'; and Nanog-O/S-R, 5'-GATCTCCACCATGGACATTGTAATGCAAAAGAAGCTGTAAG-3'), were cloned into vector pTK-Luc (Clontech) to produce pTAL-Luc.
Transient expression assays were performed with Lipofectamine 2000 (Invitrogen). Samples were analyzed 2 days after transfection.
Luciferase reporter assays. ES (5.0 x 105), F9 (5.0 x 105), and NIH 3T3 (2.5 x 105) cells were incubated in six-well tissue culture plates for 24 h. Each reporter construct (1.25 pmol) was cotransfected with vector phRL-TK (0.125 pmol) (Promega) as an internal control by using Lipofectamine 2000. Cell extracts were prepared 48 h after transfection, and luciferase activities were evaluated by using a dual-luciferase assay system (Promega). The luciferase activity of each construct was calculated relative to that of control vector pGL3-Basic. All transfection experiments were repeated in triplicate. For cotransfection reporter assays, construct 3xOct/Sox-pTK-Luc (0.06 pmol) was cotransfected with vectors pCMV-Myc-Oct4 (0.6 pmol), pCMV-HA-Sox2 (0.6 pmol), and phRL-TK (0.006 pmol) into NIH 3T3 cells. The promoter activities are reported as means ± standard errors.
Western blot hybridization analysis. Cell extracts (20 µg/lane) were separated through 12% polyacrylamide by SDS-polyacrylamide gel electrophoresis and transferred to Protran nitrocellulose membranes (Schleicher & Schuell). After blocking was done with 3% skim milk in phosphate-buffered saline for 1 h, the membranes were incubated with anti-NANOG (1:1,000 dilution) (9), anti-OCT4 (1:500) (Santa Cruz), anti-SOX2 (1:500) (Chemicon), anti-GFP (1:1,000) (Clontech), anti-Myc (1:1,000) (Covance), anti-hemagglutinin (HA) (1:1,000) (Covance), anti-histone H3 (1:3,000) (AbCam), or anti-ß-actin (1:3,000) (AbCam) antibody overnight at 4°C. After washing was done with 0.1% Triton X-100 in phosphate-buffered saline, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:3,000) (Amersham) for 90 min. Bands were detected with an ECL Western blotting detection kit (Amersham).
Electrophoretic mobility shift assays (EMSAs). Expression vectors pCMV-Myc-Oct4 and pCMV-HA-Sox2 were transfected into COS-1 cells by using Lipofectoamine 2000. Whole-cell lysates were collected 30 h after transfection (11). Nuclear extracts of F9 EC, TMA-58G EG, and R1 ES cells were prepared as reported previously (22). Double-stranded synthetic oligonucleotide probes were labeled with 32P-dCTP. Whole-cell extracts (10 µg) and nuclear extracts (10 µg) were preincubated for 10 min on ice in the presence of 2 µg of poly(dG-dC) (Amersham) in 20 µl of reaction buffer (10 mM Tris-HCl [pH 7.5], 1 mM MgCl2, 0.5 mM dithiothreitol, 10% glycerol) and then incubated with 0.1 ng of radiolabeled probes for 30 min. Competition or supershift assays were performed by adding 1- to 300-fold excess cold competitors or 1 µg of rabbit polyclonal anti-OCT4, goat polyclonal anti-SOX2, or rabbit polyclonal anti-SOX2 antibody (each from Santa Cruz) prior to treatment with radiolabeled probes. Probe DNA-protein complexes were separated by electrophoresis at 150 V through 4% polyacrylamide in 0.25x Tris-borate-EDTA buffer at 4°C for 135 min and visualized by autoradiography.
Chromatin immunoprecipitation assays. Chromatin immunoprecipitation was performed as described previously (10). Protein and DNA were cross-linked by incubation in 1% formaldehyde. The chromatin then was sonicated to an average DNA fragment length of 200 to 1,000 bp. Soluble chromatin reacted with or without 2 µg of rabbit polyclonal anti-OCT4 or goat polyclonal anti-SOX2 antibody (each from Santa Cruz) was purified and collected in elution buffer (0.1 M NaHCO3, 1% SDS). Cross-linking then was reversed with elution buffer containing RNase A (0.03 mg/ml) and NaCl (0.3 M) by incubation for 4 h at 65°C. Supernatant obtained without antibody was used as an input control. Following treatment with proteinase K for 1 h at 45°C, the DNA was purified and analyzed by PCR with the following primers: Nanog-O/S-ChIP-F, 5'-GTCTTTAGATCAGAGGATGCCCC-3'; Nanog-O/S-ChIP-R, 5'-CTACCCACCCCCTATTCTCCCA-3'; Fgf4-O/S-ChIP-F, 5'-AGACTTCTGAGCAACCTCCCGAA-3'; and Fgf4-O/S-ChIP-R, 5'-CAACTGTCTTCTCCCCAACACTCT-3'.
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RESULTS |
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Octamer and Sox elements are required for Nanog expression. To further characterize the region required for Nanog expression, two additional GFP reporter transgenes (carrying a –2342 or –332 5'-end genomic fragment but no 3'-end fragment) were constructed and transfected into ES cells. In addition, the constructs were transfected into NIH 3T3 fibroblast cells, which do not express endogenous Nanog. A pCMV-GFP construct was used in parallel to control for transfection efficiency. With both experimental constructs, GFP was highly expressed in ES cells but not in fibroblasts (Fig. 2A), while the control construct was highly expressed in both cell types (data not shown). These results suggested that regulatory elements essential for pluripotential state-specific expression are located in the 382-bp region immediately upstream of Nanog. To confirm this notion, stably transformed –332-GFP TG ES cells were generated and differentiated in vitro through RA treatment for 5 days. GFP expression that was observed in undifferentiated –332-GFP TG ES cells was completely suppressed on differentiation.
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Sequence analysis demonstrated the presence of conserved Octamer (TTTTGCAT) and Sox (TACAATG) elements between positions –166 and –180 (Fig. 2C). To examine the functions of these elements, triple point mutations were introduced by replacement of DNA residues in each element (Fig. 2D). Luciferase assays with –332 fragments carrying mutated elements clearly showed a dramatic reduction in luciferase activities in all cases, to levels similar to that found with the –153 fragment (Fig. 2E). Thus, factors that interact with the Octamer and Sox elements are likely to play a crucial role in regulating the expression of Nanog in a pluripotential state-specific manner.
Octamer and Sox elements regulate expression in human ES cells. Nanog has also been identified in humans and monkeys, in which specific expression in the nuclei of undifferentiated ES cells has been shown by immunocytochemical analysis with anti-NANOG antibody (9). Comparative DNA sequence analysis of the region spanning from –300 bp to the translational start site revealed a high degree of conservation between humans and monkeys (93.2%) but not between humans and mice (54.1%) or between monkeys and mice (51.7%). However, the Octamer and Sox elements present in both humans and monkeys showed 100% identity to the mouse sequence (Fig. 3A).
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OCT4 and SOX2 bind to the Octamer and Sox elements. OCT4 and SOX2 are expressed in pluripotential cells and are well characterized as factors that bind to the Octamer and Sox elements, respectively. Indeed, it has been shown that they bind to these elements in regions proximal to the pluripotential cell-specific Fgf4, Utf1, Sox2, and Fbx15 genes, where they act synergistically to activate transcription (17, 33, 34, 42). We therefore investigated the abilities of OCT4 and SOX2 to bind to the Nanog Octamer and Sox elements by EMSAs. Exogenous Myc-tagged Oct4 (Myc-Oct4) and HA-tagged Sox2 (HA-Sox2) were transfected into COS-1 cells, in which endogenous OCT4 and SOX2 are repressed. Western blot hybridization analyses with antibodies against OCT4, SOX2, Myc, and HA showed that exogenous OCT4 and SOX2 were abundant in whole extracts from COS-1 cells carrying the transgene(s) (Fig. 4A).
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To examine whether OCT4 and SOX2 activate Nanog expression by binding to their respective elements, reporter assays were performed with NIH 3T3 cells and with a pTK-Luc construct into which three tandem repeats of the Octamer/Sox element had been introduced (Fig. 4D). Cotransfection of this construct with Myc-Oct4 and HA-Sox2 led to an 
3.5-fold increase in luciferase activity. Taken together, our data clearly show that the Nanog Octamer and Sox elements are able to recruit OCT4 and SOX2, respectively, leading to the up-regulation of Nanog gene expression.
Binding of OCT4 and SOX2 to Octamer and Sox elements in EC cell extracts. F9 cells are EC cells that have defective pluripotency and that were derived from embryonic day 6.0 (E6.0) embryos through carcinogenesis (27). As in R1 ES cells, OCT4, SOX2, and NANOG are expressed in the nuclei of F9 EC cells, as shown by Western blotting hybridization analyses (Fig. 5A). To examine the abilities of endogenous OCT4 and SOX2 to bind to the Nanog Octamer and Sox elements, nuclear extracts from F9 cells were prepared for EMSAs. Incubation with the radiolabeled Nanog probe resulted in a prominent band at the position of the OCT4/SOX2 complex, while an independent SOX2-specific band was barely detectable (Fig. 5B). The complex mobilities correlated well with those found when COS-1 cell extracts containing exogenous Myc-OCT4 and HA-SOX2 were used.
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While we were unable to demonstrate specificity for SOX2 in these initial experiments, further analysis by supershift assays clearly demonstrated that both OCT4 and SOX2 are involved, since the inclusion of anti-OCT4 and anti-SOX2 antibodies resulted in reduced complex mobility (supershift), while the inclusion of rabbit and goat control IgGs had no effect (Fig. 5D). Thus, endogenous OCT4 and SOX2 form ternary protein-DNA complexes with the Nanog Octamer and Sox elements in F9 EC cell extracts.
We next examined the in vivo potential for the binding of OCT4 and SOX2 to the Nanog elements in ES and EC cells by chromatin immunoprecipitation assays with anti-OCT4 and anti-SOX2 antibodies (Fig. 5E). Exogenously expressed OCT4 and SOX2 bound to the Octamer and Sox elements, respectively, in both R1 ES and F9 EC cells, as seen for the Fgf4 Octamer and Sox elements.
A novel factor binds to Octamer and Sox elements in ES cell extracts. R1 ES cells, TMA-58G EG cells, and F9 EC cells share important properties, which include a robust capacity for self-renewal and expression of NANOG, OCT4, and SOX2 (Fig. 5A). However, full pluripotency is retained only by ES cells. Notably, R1 ES cells were isolated from normal inner cell mass cells of blastocyst-stage E3.5 embryos, while F9 EC cells were derived from E6.0 embryos through carcinogenesis and TMA-58G EG cells were derived from homing primordial germ cells in genital ridges of E12.5 embryos. Thus, some of the factors involved in maintaining pluripotency in ES cells may not be present in EC or EG cells. We therefore repeated the EMSA, binding competition, and supershift assays with nuclear extracts from R1 ES cells and TMA-58G EG cells. Strikingly, the major complex that formed when ES cell extracts were used was clearly distinct from the OCT4/SOX2 complex detected in EC cell extracts or in EG cell extracts (Fig. 6A). The same complex was detected in ES cell extracts with both the Nanog and the Fgf4 probes. A decrease in the band intensity of the ES cell-specific major complex in binding competition assays with the Octmut or Soxmut competitor probe demonstrated that the ES cell major complex required both the Octamer and the Sox elements for stable binding (Fig. 6B). This finding was confirmed by a lack of competition when the Octmut/Soxmut competitor probe was used. Supershift assays revealed that ES cell major complex mobility was reduced following incubation with anti-OCT4 antibody whereas, in contrast to the findings for F9 cell nuclear extracts (Fig. 5D), anti-SOX2 antibody had little effect on complex mobility (Fig. 6C). This finding was confirmed with a second anti-SOX2 antibody. These data showed that OCT4 is an essential component of the ES cell major complex but that SOX2 is not. Thus, another as-yet-undefined component (PSBP) preferentially associates with OCT4 to form an ES cell-specific complex on the Octamer and Sox elements in the Nanog promoter region.
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DISCUSSION |
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It is evident that the Octamer and Sox elements are required for the up-regulation of mouse and human Nanog transcription and that factors that bind to these elements act to promote Nanog transcription through synergistic molecular interactions. Adjacent Octamer and Sox elements have been identified as cis regulatory elements in the Fgf4, Sox2, Utf1, and Fbx15 genes, which are expressed in EC and ES cells and during embryogenesis (17, 33, 34, 42). Sox2 is a member of the Sox (SRY-related HMG box) family that bears DNA-binding HMG domains and that is implicated in transcriptional regulation. The gene is expressed in pluripotential embryonic cells and neuronal cells (1). In pluripotential embryonic cells, expression is governed by at least two regulatory regions, the 5'-flanking region containing the CCAAT box and the 3'-flanking region containing the Octamer and Sox elements (34, 37). Fgf4 is a member of the fibroblast growth factor family that is expressed in blastocyst inner cell mass cells and in developing embryos, as well as in ES and EC cells (19). Both Octamer and Sox elements are located in the intragenic 3' untranslated region, while a GT-box motif is located in the 3'-flanking region. Both regions are required for mediating optimal transcriptional Fgf4 activation (13, 14). Thus, the expression of some pluripotential embryonic cell-specific genes appears to require the action of Octamer and Sox elements in combination with other gene-specific cis regulatory elements. For Nanog, luciferase assays with ES cell extracts demonstrated that activity controlled by the –2342 5'-end genomic fragment was about 15% higher than that controlled by the –332 5'-end genomic fragment (Fig. 2B); this finding implied that an unidentified cis regulatory element(s) lying in the region from position –332 to position –2342 may function, in combination with the Octamer and Sox elements, in enhancing and determining the specificity of Nanog expression.
There is significant evidence that Nanog plays a key role in maintaining the pluripotency of ES cells and embryonic cells. Nanog-deficient ES and embryonic cells show a complete loss of pluripotency (9, 15), whereas Nanog overexpression results in the clonal expansion of ES cells via the bypassing of regulation by LIF-STAT3 signaling and maintenance of OCT4 levels (3). Therefore, it is important to understand how the expression of Nanog acts in harmony with the expression of other embryonic factors through molecular communications in the stem cell-specific regulatory network. Our data clearly show that the ternary protein-DNA complexes of OCT4/PBSP and Octamer and Sox elements efficiently formed even in the presence of SOX2. The data also suggest that this complex formation is essential for the activation of Nanog transcription in ES cells. During embryonic development, mouse Nanog RNA and protein expression can be detected from the morula stage to the epiblast stage of E7.5 embryos (8, 9, 15). The pattern of expression of Oct4 and Sox2 is temporally similar. Interestingly, in E7.5 embryos, Nanog expression is spatially enhanced in the caudal region (primitive streak region) of the epiblast (8, 9), whereas Sox2 expression is restricted to the presumptive neuroectoderm in the anterior and is excluded from the posterior (primitive streak region) (1). These data suggest that the OCT4/PSBP complex may activate Nanog expression in vivo. In primordial germ cells of E7.5 embryos, Oct4 and PGC7/Stella are expressed, whereas Nanog is repressed (9, 23, 40). However, Nanog is expressed in primordial germ cells at E11.5 and in EG cells derived from E12.5 embryos (3, 9). Our EMSA data (Fig. 6A) suggest that OCT4 and SOX2 may function in this up-regulation of Nanog transcription, although the profile of expression of SOX2 in germ cells is not fully understood. Thus, even though the control of Nanog expression is closely linked to the Oct4 regulatory network, Oct4 expression alone is insufficient for inducing Nanog expression. We suggest that the expression of Nanog is tightly regulated by competing coactivators (SOX2 and PSBP) in different cell types, which have different affinities for the Nanog Sox element sequence.
To understand the relationship between Nanog and Oct4, Nanog transcription in Oct4-deficient embryos was analyzed (3). mRNA in situ hybridization analysis demonstrated that Nanog expression was maintained in the blastocyst inner cell mass cells, suggesting that other pluripotential cell-specific factors may contribute to alternative transcriptional regulatory mechanisms. It has been shown that OCT1 and OCT6 are expressed in pluripotential embryonic cells (24, 29) and have the capacity to bind to Octamer elements in the Fgf4, Sox2, Utf1, and Rex-1 (Zfp-42) promoter regions (2, 5, 17, 34). Thus, instead of OCT4, OCT1 or OCT6 may participate to form the DNA-protein complex with SOX2 or PSBP on the Nanog Octamer and Sox elements. In fact, it has been reported that OCT6 but not OCT1 can form a complex with SOX2 on the Octamer and Sox elements in Sox2 (34). However, the affinity of OCT1 and OCT6 for binding to the Nanog Octamer element (TTTTGCAT) is low relative to that for binding to the consensus Octamer element (ATTAGCAT) (18). It remains to be explored whether other pluripotential genes contribute to the up-regulation of Nanog activity through interactions with the Octamer and Sox elements and whether Nanog expression in Oct4-null mutants is quantitatively equivalent to that in normal embryos.
ES cells promise to serve as an unlimited cell source of therapeutic materials for use in regenerative medicine. In clinical applications, it would be crucial to monitor the undifferentiated state of ES cells through numerous cell divisions and to selectively eliminate populations of spontaneously differentiated cells in cultures. Furthermore, following tissue-specific cell differentiation induction, a tool for the selective elimination of pluripotential ES cells is desirable to avoid contamination with a potential source for generating malignant tumors in vivo. For such purposes, the Nanog minimum promoter encompassing the Octamer and Sox elements may be a suitable tool for positive and negative selection of undifferentiated stem cells. Furthermore, genetically engineered human stem cells containing the herpes simplex virus thymidine kinase gene have been generated for selective elimination of undifferentiated ES cells with ganciclovir after in vitro and in vivo differentiation (25). In this context, the human Nanog promoter is an ideal candidate element for regulating the expression of a pluripotential cell-specific suicide gene. Further understanding of the mechanisms that regulate Nanog gene expression will also contribute to the field of stem cell engineering and its application to regenerative medicine.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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