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Oct4 and Sox2 directly regulate expression of another pluripotency

transcription factor Zfp206 in embryonic stem cells*

Zheng-Xu Wang§¶, Christina Hui-Leng Teh§, Jacqueline LL. Kueh§, Thomas Lufkin§‡, Paul Robson§‡, and Lawrence W. Stanton§‡

From the §Stem Cell & Developmental Biology, Genome Institute of Singapore, Singapore 138672 and the ¶Department of General Surgery, Jin Cheng Hospital of Lan Zhou, Gansu Province 730050, P.R. China and the ‡Department of Biological Sciences, National University of Singapore, Singapore 117543

Running Title: Zfp206 is regulated by Oct4 and Sox2

Address correspondence to: Lawrence W Stanton, Genome Institute of Singapore, 60 Biopolis Street Singapore 138672; Tel: (65) 6478 8000; Fax: (65) 6478 9051; E-mail: stantonl@gis.a-star.edu.sg

It is well known that Oct4 and Sox2 play an important role in the maintenance of embryonic stem cell pluripotency. These transcription factors bind to regulatory regions within hundreds of target genes to control their expression. Zfp206 is a recently characterized transcription factor that has a role in maintaining stem cell pluripotency. We now demonstrate that Zfp206 is a direct downstream target of Oct4 and Sox2. Two composite sox-oct binding sites have been identified within the first intron of Zfp206. We demonstrate binding of Oct4 and Sox2 to this region. In addition, we show that Oct4 or Sox2 alone can activate transcription via one of these sox-oct element, although the presence of both Oct4 and Sox2 give rise to a synergistic effect. These studies extend our understanding of the transcriptional network that operates to regulate the differentiation potential of embryonic stem cells.

Embryonic stem cells (ESCs)1 are derived from the inner cell mass of the blastocyst and exhibit both pluripotency and self-renewing capabilities. For proper developmental outcome ESCs must tightly regulate their differentiation status and the molecular basis of that regulation process is beginning to emerge. Systematic, genome-wide interrogations have identified hundreds of genes, including several transcription factors, which have expression patterns tightly correlated with ES cell differentiation (1-6). Two key transcription factors, Oct4 and Sox2, have

been identified that are crucial for maintenance of the pluripotent state of ESCs (7,8). ESCs lose the capacity to maintain pluripotency upon knockdown of expression of these transcription factors by RNA interference (9,10). Gene knockout studies confirm the importance of Oct4 and Sox2 for early embryonic development. It has been demonstrated by chromatin immunoprecipitation studies that Oct4 and Sox2 bind to a few thousand regulatory sites in the ES cell genome (11,12). It is likely that many of these target genes play a role in modulating ES cell differentiation. Indeed, the transcription factor Nanog, an established regulator of pluripotency, is transcriptionally regulated directly by Oct4 and Sox2 (13).

Zfp206 is a transcription factor that is highly expressed in mouse and human ESCs and down-regulated upon differentiation (3,14). Zfp206 contains a SCAN domain and 14 zinc-finger domains, which suggests that it may be a transcription factor that binds DNA directly. Zfp206 is expressed in the ICM, but not in trophectoderm suggesting that it may play a role in establishing cell fate decisions regarding embryonic versus extraembryonic tissue (15). There is wide temporal and spatial distribution of RNA and protein in the early embryos indicating that Zfp206 may regulate multiple cell fate decisions (14). Recent data has demonstrated that over-expression of Zfp206 promotes the formation of undifferentiated mouse ESC colonies in vitro (14). We have obtained similar results, and further found that over-expression of

http://www.jbc.org/cgi/doi/10.1074/jbc.M611814200The latest version is at JBC Papers in Press. Published on March 7, 2007 as Manuscript M611814200

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Zfp206 renders ESCs resistant to retinoic acid induced differentiation, whereas knocked down expression of Zfp206 made ESCs more easily differentiated by retinoic acid (data to be published elsewhere). The pattern of expression and functional impact of Zfp206 on ESC differentiation establishes that this transcription factor plays an important role in maintaining pluripotency.

Given that Zfp206 plays a key role in controlling ESC differentiation, it is important to understand how this transcription factor fits into the network of other transcription factors involved in maintaining pluripotency of ESCs, such as Oct4 and Sox2. Oct4 (also known as Oct3 and encoded by Pou5f1) is a POU domain containing transcription factor that binds an octamer sequence, ATGCAAAT (12,16). Sox2 is an HMG domain containing transcription factor that binds to the consensus motif, CATTGTT (17,18). Oct4 and Sox2 were shown to reciprocally regulate Pou5f1 and Sox2 transcription via the Oct4/Sox2 complex in ESCs (16). In addition, Oct4 and Sox2 positively regulate Nanog revealing that a tight transcriptional network is at work to maintain the undifferentiated state of ESCs (13,19). To identify target genes regulated by these pluripotent transcription factors, studies have been carried out to map the binding sites for Nanog, Oct4 and Sox2 on a genome-wide scale (11,12). Nanog, Oct4 and Sox2 binding sites have been identified within a large number of genes with diverse functions including other transcription factors. It has been observed that Oct4 binding within the enhancer of a number of genes are in close proximity to Sox2-binding sox elements. Such close proximity of the two binding sites suggests that these two transcription factors may interact with each other. Indeed, two structures for a POU/HMG ternary complex bound to composite sox-oct motifs have been solved, revealing that that the POU and HMG domains are capable of mediating specific protein-protein and DNA-protein interactions (20,21). This finding reinforces the importance of Oct4 and Sox2 at the top of the hierarchy in the maintenance of pluripotency in ESCs. Based on previous chromatin immunoprecipitation experiments, two Oct4 and two Sox2 binding sites were found on Zfp206 genomic locus (12). Each of the Oct4 binding sites lies in close proximity to the Sox2 binding site, suggesting

that Oct4 and Sox2 may be partners in regulating the expression of Zfp206. The oct-sox elements are located at the beginning and end of the first intron of Zfp206. In this study, we established that Oct4 and Sox2 are direct regulators of Zfp206 expression. These results position Zfp206 within the transcriptional regulatory network that maintains pluripotency and controls the differentiation of ESCs.

MATERIALS AND METHODS ES Cell Culture—E14 mouse ESCs (ATCC), either cultured on mouse primary embryonic fibroblast feeders (MEF) or under feeder-free conditions, were maintained in DMEM, supplemented with 15% FBS (GIBCO, ES qualified), 0.055 mM β -mercaptoethanol (GIBCO), 2 mM L-glutamine, 0.1 mM non-essential amino acid, 5,000 units/ml penicillin/streptomycin and 1,000 units/ml of LIF

(Chemicon), and maintained at 37°C with 5% CO2. The V6.4 (129S4/SvJae × C57BL/6J)F1 ES cell cultured on MEF, were maintained in DMEM containing 15% FBS, 0.055 mM β -mercaptoethanol, 4 mM L-glutamine, 40µg/ml Gentamicin Sulphate (GIBCO), 1mM Sodium Pyruvate (GIBCO), and 1,000 units/ml of LIF. Site-specific lacZ knock in stable cell line—RED/ET recombination method (Gene Bridges GmbH, Germany) was employed to make the site-specific knock-in construct which contain 5.43 kb of left homologous arm, and 10.374 kb of right homologous arm for the homologous recombination (20). Briefly, we inserted prokaryotic Pgb2 promoter into the plasmid pTCT5.2 (20) to generate a plasmid pTCT5.2-Pgb2 that contains both eukaryotic PGK and prokaryotic Pgb2 promoters. Genomic DNA of Zfp206 which is 16,147 bp in length was subcloned into a minimer vector by RED/ET method using the BAC subcloning kit (Gene Bridges GmbH, Germany) from the BAC clone RP24-440G8 (contains 172 kb of genomic DNA). The cassette of PCR product from pTCT5.2-Pgb2 plasmid was inserted into this subcloning plasmid to generate a novel plasmid in which part of the second exon (328 bp in length) of Zfp206 containing the translation initiation site has been replaced. For homologous recombination, V6.4 ESCs were stably transfected with the construct that contains the lacZ (β-galactosidase, β-Gal) reporter gene using a standard protocol of

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electroporation (20). Individual colonies were picked after selection with 300 µg/ml G418 for 10 days with cells grown on neomycin-resistant

mouse embryonic fibroblasts (DR4, ATCC). Southern blot was used to verify the site-specific integration into the genomic DNA. 20µg genomic DNA from different neomycin resistant cell clones was digested by Spe I (NEB), loaded on agarose gel and transferred onto nylon membrane. Southern blot was conducted using PCR product localized on the upstream of left homologous arm was labeled by PCR DIG Probe Synthesis Kit (Roche) following the manufacturer’s instruction. Promoter reporter constructs—We cloned Zfp206 promoter (1468bp in length) from the BAC clone (RP24-440G8) and Nanog (417 bp) promoter regions from the genomic DNA into pGL4-basic vector (Promega). The beginning of the 1st intron (1,062 bp) or the end of the 1st intron (938 bp) were cloned upstream of the Zfp206 promoter-luciferase or Nanog promoter-luciferase construct in the forward or reverse orientation. Sox2, Oct4, and both Oct4 and Sox2 binding site were modified using the QuikChange II site-directed mutagenesis kits (Stratagene) to incorporate 3-bp mutations, which were subsequently verified by sequencing. RNA interference—RNAi experiments were performed with Dharmacon siGENOME SMARTpool reagents against mouse Oct4, Sox2 or Nanog. The Dharmacon siCONTROL nontargeting siRNA pool was used as a negative control. All duplexed RNA oligonucleotides were synthesized at Dharmacon as 21-mers with 3′-UU overhangs forming a 19 bp duplex core. Based on the manufacturer's instruction, four duplex individual siRNA have been combined into a signal pool for one target gene. Mouse ESCs were transfected according to manufacturer’s instructions. Briefly, E14 cells have been transfected with gene specific SMARTpool or siCONTROL non-targeting siRNA pool for three times at day 0, day 2 and day 4, using DharmFECT 2 transfection reagent (Dharmacon). The RNA was extracted at day 1, day 3 and day 5 for RT-PCR assay. Total RNA was isolated using Trizol reagent (Invitrogen) and purified with the RNeasy kit (Qiagen). The first strand cDNA was synthesized using the High-Capacity cDNA Archive Kit (Applied Biosystems) with 1,500 ng of total RNA and quantitative PCR analyses were performed in real time using an ABI PRISM 7900 Sequence Detection System (Applied

Biosystems). Expression was normalized to β-actin and each sample was analyzed in duplicate or triplicate. A threshold cycle (Ct) of >33 indicates undetected transcripts. A 19 bp gene-specific RNAi sequence for Oct4 and Sox2 (13) was synthesized and cloned into pSUPER.puro vector (Oligoengine), which can express short hairpin RNAs (shRNAs) as described previously (22). Luciferase and lacZ Reporter Assays—200 ng of luciferase reporter plasmid, 1µg of Oct4 or Sox2 shRNA in plasmid pSUPER.puro, and 20 ng of plasmid pRL-SV40 were co-transfected into E14 cells in a 24-well plate by Lipofectamine 2000 (Invitrogen) following the supplied protocol. Puromycin (1µg/ml) was applied 24 hrs post-transfection. The pRL-SV40 plasmid served as an internal control for normalizing the transfection efficiency. After 48 hours of puromycin selection, the E14 cells were lysed and luciferase activity determined with the Dual Luciferase System (Promega) using a Centro LB960 96-well luminometer (Berthold Technologies). Transfections were done in duplicate and on two independent occasions. To quantitate β-galactosidase activity (lacZ expression) in V6.4 ESCs upon Sox2 and Oct4 knock down, 1µg of Sox2 or Oct4 shRNA in plasmid pSUPER.puro, and 3 ng of plasmid pRL-SV40 were co-transfected into V6.4 heterozygous ESC in a 96-well plate. Puromycin (1µg/ml) was applied 24 hrs post-transfection. The 96-well plates were equilibrated to room temperature before adding Beta-Glo Reagent (Promega) after 48 hours of puromycin selection. 100µl of reagent is added into the culture medium. The sample is mixed and incubated for 1 hour at room temperature, and is measured using a luminometer. The V6.4 cells were lysed and Renilla activity was determined with the Dual Luciferase System (Promega) at the same time. Electrophoretic mobility shift assays (EMSAs) — Nuclear extracts were prepared from E14 mouse ESCs grown under feeder-free conditions using the method of Rodda et al (13). Equal molar of oligonucleotides labeled with cy5 at the 5' termini of both sense and antisense strands were annealed in 95°C water until cooling to room temperature. The double-stranded DNA has been diluted to 1µM for EMSA assay. EMSA was performed in 10 µl mixtures containing 20 mM HEPES, pH 7.9, 100 mM KCl, 1.66 mM DTT,

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0.83 mM EDTA, 20% glycerol, 1µl of cy5-labelled double-stranded DNA (1µM), 12-16 µg nuclear extracts and 1 µl of poly dGdC (2µg/µl , Amersham), 1% protease inhibitor cocktail (Roche). Antibodies (Santa Cruz) were added after the initial incubation (25 min) for additional 25 min as follows: 2 µl of anti-Oct4 (sc-9081), 8 µl of anti-Sox2 (sc-17320), or 2 µl of anti-Oct1 (sc-232), 8 µl of anti-Oct6 (sc-11660), 8 µl of

anti-Sox4 (sc-17326), 2µl anti-JunB (sc-46x). For competitive studies, a specified 1µl (25µM) of unlabeled double-stranded competitor was added with Cy5 labeled DNA together. Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays with E14 mouse ESC were carried out as described (12, 13). Briefly, cells were cross-linked with 1% formaldehyde for 10 min at room temperature, and formaldehyde was inactivated by the addition of 125 mM glycine. Sonicated chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using Oct4 (N19), Sox2 (Y17) or GST control polyclonal antibodies (Santa Cruz Biotechnology). For all ChIP

experiments, quantitative PCR analyses were performed in real time using the ABI PRISM 7900 sequence detection system and 2xSYBR Green master mix (Applied Biosystems) as

described (12, 13). Relative occupancy values were calculated by determining the apparent immunoprecipitate efficiency (ratios of the amount of immunoprecipitated DNA over that of the input sample) and normalized to the level observed at a control region, which was defined as 1.0. Primer pairs were as follows: for Oct4/Sox2 binding site 1 (BS1): region 1, 5’-TGGGGGCACATGTGGAAGTCAG-3’ and 5’-TGGAGGCAGGCCTCTTTCAGCT-3’; region 2, 5’-CTGTTCCTTGGAATGCAACCCC-3’ and 5’-CGTGCTTGCTGTGTGTAGGCCT-3’; region 3, 5’-CCGGCCAGATTTCACT AAAGAGC-3’ and 5’-CCTACCCCATG AAATTTTGCCAG-3’; region 4, 5’-TGGGG TAGGACGCAAATAGTGGG-3’ and 5’-TGCCTGCTGCTCACTTCATCCC-3’; region 5, 5’-TTGTATGAATGTGCATCTGCGCA-3’ and 5’-GCATTTCGCTGTCTGAAGAAGCA-3’; region 6, 5’-GCTTCCCACATGTTTG GTAG-3’ and 5’-CAAAGTACGGTTCAG GGAAA-3’. For Oct4/Sox2 binding site 2 (BS2): region 7, 5’-GCTGATCGCAAAG CTAGGCAGTG-3’ and 5’-CAGGGATCCCAG

GTAAGCACACA-3’; region 8, 5’-GAATGT GTGTGGTGCATATGGCC-3’ and 5’-GAAAG ACCAAGGCCCAGGAAGAT-3’; region 9, 5’-ATCCCTGGCTGGAATTCACTT TG-3’ and 5’-AAAATTGAAGCCAGGTGTGGTGG-3’; region 10, 5’-ACTGTGTGGAGGTGTT TCGCCTG-3’ and 5’-TGCGGGGAAGCC AACCTCTA-3’; region 11, 5’-GTGGCCC ACCAGCTTTTCAGA-3’ and 5’-TTCTTG GTGTGCAGAGCCGGT-3’; region 12, 5’-AGAAACCTGCAAATTCCACTGGC-3’ and 5’-GCGCCACTGAAAGGATCAACT CT-3’; region 13, 5’-CCTGGCTGTCCAAT CTGTCTGTG-3’ and 5’-TGGGTGTGATG GTGAACGACCT-3’.

RESULTS

Expression of Zfp206 is regulated by Oct4 and Sox2— A recent report (14) and our own data (to be published elsewhere) have identified Zfp206 as a transcription factor that plays a key role in maintaining the pluripotency of ESCs. The expression pattern of Zfp206 is quite similar to the patterns of expression for Nanog, Oct4, and Sox2 in ESCs; they are all expressed in undifferentiated cells and that expression is quickly repressed upon differentiation. Since Nanog, Oct4 and Sox2 are key transcription factors in the maintenance of pluripotency in ESCs, we set out to determine if they can regulate the expression of Zfp206. Therefore, we examined the expression of Zfp206 in response to the knockdown of Nanog, Oct4 and Sox2 expression by RNA interference (RNAi). We were able to achieve efficient and substantial knockdown of the target genes in ESCs by a repeated transfection on alternative days with small inhibitory RNA (siRNA). When mouse ESCs were transfected with gene-specific siRNAs against Nanog, Oct4 and Sox2, a 50% knockdown of expression was observed one day after transfection and a 70-90% reduction of the targeted gene was observed at day 5 (Fig. 1). These effects were gene-specific as there was no knockdown of beta-actin mRNA expression by these three siRNAs, and a non-targeting siRNA had no impact on the expression of Nanog, Oct4 and Sox2. The expression level of Zfp206 was examined in these transfected cells. Upon Oct4 or Sox2 knockdown (Fig. 1A, 1C), a down-regulation of Zfp206 transcript level to less than

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30% of control levels was observed. In the Nanog RNAi cells, the transcript level of Zfp206 was reduced to only 70%. These results suggest that Zfp206 is regulated by Oct4 and Sox2, perhaps directly or indirectly, as part of the regulatory network that control ESC pluripotency.

Oct4 and Sox2 regulate the activity of Zfp206 in vivo — To further examine the regulation of Zfp206 expression in ESCs, we have knocked a lacZ reporter into the Zfp206 locus by homologous recombination. We have constructed a targeting vector that contains a 5.5 kb genomic fragment upstream of the translational start site of the Zfp206 and a 10.4 kb genomic fragment downstream of 3' end of the second exon. The construct includes the entire first exon and intron. The first 134 aa of Zfp206 protein, which includes the SCAN domain in the second exon, was replaced with a construct containing the IRES-lacZ-Neomycin-3’ UTR reporter and selection cassette (Supplemental Fig. 1A). The targeting construct was electroporated into V6.4 ESCs and 196 neomycin resistant colonies were assessed for homologous recombination by Southern blotting. We identified 4 independent clones that had the correct restriction digest pattern (Supplemental Fig. 1B). All four of the undifferentiated, heterozygous colonies were positive for lacZ indicating appropriate expression of this reporter from the Zfp206 locus (Fig. 2A upper). To determine whether down-regulation of lacZ expression was linked to endogenous Zfp206 expression, ESC clones were differentiated by retinoic acid (RA). After 5 days of continuous RA treatment, the cell morphology changed significantly and lacZ became undetectable (Fig. 2A lower). There was also reduced lacZ expression in embryoid bodies generated by aggregation of ESCs in the absence of LIF (Fig. 2B). lacZ expression was not detectable in the endoderm-like cells on the EB surface. It was noted that lacZ expression appeared in the centre of the embryoid bodies (which are undifferentiated) but not at the rim (differentiated cells), indicating that the regulatory region of Zfp206 contained elements that regulate Zfp206 expression in undifferentiated ESC.

To determine if Oct4 and Sox2 had an effect on the regulatory region of Zfp206, we knocked down the expression of Oct4 and Sox2 by RNAi. Immunoblotting showed that effective knockdowns of Oct4 and Sox2 were achieved

(Figure 2C). When these cells were assayed for lacZ reporter activity, it was found that Zfp206 expression was reduced by more than 40% with Oct4, Sox2, or both Oct4 and Sox2 RNAi, as compared to either vector control or Gfp RNAi (Fig. 2D). These results indicate that both Oct4 and Sox2 have a positive regulatory effect on the genomic region of Zfp206, since the knock down of these two proteins is able to decrease the expression of an exogenous gene (lacZ) under the control of the regulatory element of Zfp206. This confirms that the regulation of Oct4 and Sox2 on Zfp206 is at the transcriptional level in vivo.

Identification of two composite oct-sox elements within the first intron on Zfp206 gene — We sought to determine if Zfp206 is a direct target of regulation by Oct4 and Sox2. Previous work from our institute had comprehensively mapped Oct4 (12) and Sox2 (unpublished) binding sites in mouse ESCs by a new chromatin immunoprecipitation (ChIP) strategy called ChIP-PET (23). A search of our ChIP-PET database revealed two regions within the first intron of Zfp206 where both Oct4 and Sox2 occupancy had been mapped. We call these binding site 1 (BS1) and binding site 2 (BS2), which map at the 5’ and 3’ end, respectively, of intron 1 (Supplemental Fig. 2). There were multiple, independent paired-end tags (PETs) for both sites, which provides confidence that they are real. Co-localization of Oct4 and Sox2 binding at BS1 and BS2 further confirms their validity since these two transcription factors bind DNA as a heterodimer (20). Closer inspection of the sequence within BS1 and BS2 revealed the presence of consensus binding motifs for Oct4 and Sox2.

To further confirm that Sox2 and Oct4 do indeed directly bind to BS1 and BS2 within the first intron of Zfp206 in ESCs, ChIP-PCR was carried out with Sox2 and Oct4 antibodies. The ChIP material was amplified by PCR with 13 pairs of primers for regions within the promoter, exon 1, intron 1, exon 2 and intron 2 of Zfp206 (Fig. 3A). One pair of primers was specific for BS1 and another for BS2, regions 3 and 10, respectively (Fig 3A). DNA fragments from region 3 containing the composite oct-sox element of BS1 were enriched up to 12- and 6-fold with the Sox2 and Oct4 antibodies respectively (Fig. 3B). Likewise, for region 10 (BS2), there were 7- and 4-fold enrichments with Sox2 and Oct4 antibodies, respectively (Fig. 3C).

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The neighboring regions on both sides of the oct-sox composite elements were not significantly enriched, showing the specificity of the binding. An irrelevant antibody against GST provided no enrichments. The ChIP-PCR data confirm the ChIP-PET results and indicate in vivo occupancy of Oct4 and Sox2 at two sites in intron 1 of Zfp206 in undifferentiated mouse ESCs.

Sox2 and Oct4 bind directly to BS1 and BS2—Compared to previously identified composite sox-oct elements from different mouse target genes (Supplemental Fig. 3) that are known to bind Sox2 and Oct4, the sox and oct elements within BS1 (Fig. 4A) are in reverse orientation with respect to each other whereas the orientation of the sox and oct elements within BS2 (Figure 4B) is similar to those of the other target genes. However, in both BS1 and BS2, the oct and sox elements are separated by 11bp; this is in contrast to the enhancer regions of Fgf4, Utf1, Pou5f1, Nanog, Fbx15 and Sox2 (Supplemental Fig. 3), in which the sox and oct elements are less than 3 bp apart (24-30).

To confirm our results and more precisely map the Oct4 and Sox2 binding sites within BS1 and BS2 we performed electrophoretic mobility shift assays (EMSA). Duplexed oligonucleotide probes (39 bp) encompassing the oct-sox element of either BS1 or BS2 were combined with nuclear extracts from mouse ESCs. Three major protein-DNA complexes were observed using the BS1 probe (Fig. 4C, lane 1), and four protein-DNA complexes were found with the BS2 probe (Fig. 4D, lane 1). The addition of antibodies specific for either Oct4 (Figure 4C and 4D, lane 5) or Sox2 (Figure 4C and 4D, lane 8) resulted in supershifts of Oct4 or Sox2 band, respectively. The extra band observed with BS2 (Figure 4D, lane 1) is likely due to binding of an Oct4-Sox2 heterodimer since this band was supershifted by both Oct4 and Sox2 antibody (Figure 4D, lanes 5 and 8). An additional band is likely due to binding of Oct1 as it was supershifted by an Oct1 antibody (Figure 4C and 4D, lane 6). No supershifts were observed with antibodies to Oct6 (Figure 4C and 4D, lane 7), JunB (Figure 4C and 4D, lane 9) or Sox4 (Figure 4C and 4D, lane 10) indicating specificity of our EMSA assay. The addition of a 25-fold excess of wild-type unlabeled probe successfully competed for binding to the Oct4/Sox2 complex (Figure 4C and 4D, lane 2). However, when probe that has

the oct binding site mutated was used for competition, the Oct4 complex remained while the Sox2 complex was competed away (Figure 4C and 4D, lane 3). Conversely, when probe that has the sox binding site mutated was used for competition, the Sox2 complex remained while the Oct4 complex was competed (Figure 4C and 4D, lane 4), indicating the DNA binding specificity of the Oct4/Sox2 complex. The above results confirm our ChIP results and further refine the binding sites for Oct4 and Sox2 within the first intron of Zfp206. In addition, an Oct4-Sox2 heterodimer is able to bind within BS2, despite the large spacer region between the oct and sox element, implying that a spacer size of 11 bp is still able to accommodate binding of an Oct4-Sox2 heterodimer.

The sox-oct composite element within BS1 of Zfp206 conferred activation on different promoters—We have shown that Oct4 and Sox2 are able to bind directly to BS1 and BS2 of Zfp206. We were interested to determine if these DNA-protein interactions have a functional role in regulating expression of Zfp206. To answer this question, we tested whether the BS1 and BS2 regions would have an effect on two separate promoters—the Zfp206 promoter and the Nanog promoter. Luciferase reporter constructs were therefore generated. A 1062 bp genomic sequence of Zfp206, which contain the BS1, and another 938 bp genomic fragment of Zfp206, which contain the BS2 were isolated. These two genomic fragments were individually cloned into either a Zfp206 or Nanog promoter-driven luciferase reporter plasmids (Figure 5A). BS1 was able to activate the expression of the reporter gene up to 8-fold when Zfp206 promoter was used and ~13 fold when driven by a Nanog promoter (Figure 5B). When the sox element within BS1 was mutated (BS1mS), this activation dropped to almost half of that for wild-type BS1 for both promoters. Upon mutation of the oct element within BS1 (BS1mO), the level of activation decreased to about one-quarter that of the wild-type BS1 for both promoters. When both sox and oct elements were mutated in BS1 (BS1mSmO), the activation was completely abolished, suggesting that the activation observed for BS1 is entirely due to the sox-oct element within BS1 and this activation effect is not promoter-specific.

Similar experiments were carried out with BS2 but no activation of the reporter gene

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was observed. Mutation of either sox or oct or both elements within BS2 did not result in any significant difference in reporter gene expression. This was somewhat surprising given that we have demonstrated that Oct4 and Sox2 are bound to these sites. It is possible that occupancy at the BS2 site requires co-regulatory factors and that Oct4 and Sox2 occupancy at this site is not sufficient to regulate Zpf206 expression.

We went on to determine if BS1 could function as an enhancer by reversing the orientation of BS1 in the reporter construct. It was observed that BS1 in reverse orientation was able to activate reporter gene expression and that mutation of either the sox, oct or both element decreased this activation (Figure 5C). This showed that both orientations of BS1 with respect to the promoter are functional and suggests that BS1 may functions as an enhancer.

Oct4 and Sox2 are required for the activation of Zfp206 —To determine in ESCs if the activation conferred by the sox-oct composite element within BS1 of Zfp206 is due to Oct4 and Sox2 proteins, we carried out luciferase assays using the constructs in Figure 5A, but this time with knocked down expression of either Oct4 or Sox2 by RNAi. Endogenous Oct4 and Sox2 mRNA levels in ESCs were measured by real-time PCR after transfection with the respective RNAi constructs containing a puromycin resistance gene. Transfection of ESCs with shRNAs targeting Sox2, Oct4 or both Oct4 and Sox2 resulted in a decrease of Oct4 transcript level by 30%, 40% and 60% respectively (data not shown), whereas Sox2 transcript level was reduced by 60%, 30% and 50% respectively (data not shown). The activation mediated by BS1 on the Zfp206 promoter was abolished upon knockdown of Oct4, Sox2, or both Oct4 and Sox2. Similar results were observed with BS1 in the reverse orientation (Figure 6A). The same effect was also observed when Zfp206 promoter was replaced with the Nanog promoter (Figure 6A). All these results suggest that in ESCs the activation conferred by the sox-oct composite element within BS1 of Zfp206 is dependent on the presence of Sox2 and Oct4 proteins.

To determine if there is an absolute requirement of both the sox and oct element within BS1 and Oct4 and Sox2 proteins for transcriptional activation effect, we transfected ES cells with different constructs in which BS1 had been mutated at either the sox or oct or both

elements, in combination with different RNAi plasmids. Although mutation of sox element (BS1mS) reduced the reporter gene level to about a quarter compared to that of the wild type BS1 (BS1), knocking down Oct4, Sox2 or both Oct4 and Sox2 were able to reduce the activation mediated by BS1mS further (Figure 6B). Similarly, mutation of the oct element (BS1mO) also decreased the activation of BS1 to the same level as BS1mS and knocking down Oct4, Sox2 or both Oct4 and Sox2 reduced the activation mediated by BS1mO even further. On the contrary, mutations of both oct and sox element (BS1mOmS) totally abolished the activation mediated by BS1 and knocking down Oct4, Sox2 or both Oct4 and Sox2 did not further reduce the reporter gene level. Similar results were obtained for BS1 in the reverse orientation. Collectively, these results demonstrate that Oct4 or Sox2 individually activate expression Zfp206 at the BS1 site, but in combination they act synergistically to enhance expression.

DISCUSSION Zfp206 has been identified as a gene that

is highly expressed in undifferentiated ESCs and down-regulated upon differentiation (3, 14). It was also shown that Zfp206 is expressed in the pluripotent cells of the inner cell mass of the blastocyst (15). This pattern of expression is similar to that observed for pluripotency regulating transcription factors such as Oct4, Sox2, and Nanog. The linkage between Zfp206 expression and differentiation status in ESCs and embryonic development suggested that this transcription factor may play a role in regulating pluripotency. It has now been established that Zfp206 does play a role in maintaining ES cell pluripotency as over-expression renders ESCs resistant to differentiation, while knock down of expression causes ESCs to spontaneously differentiate and be more sensitive to induced differentiation (14 and our data to be published elsewhere). Thus, Zfp206 joins Oct4 Sox2, and Nanog as ESC-specific transcription factors that regulate pluripotency. In the study presented here, we have shown that expression of Zfp206 is positively regulated by Oct4 and Sox2 in mouse ESCs. Upon knockdown of Oct4 or Sox2 by RNA interference, Zfp206 level decreased (Fig. 1). Chromatin immunoprecipitation experiments had identified binding sites for Oct4 and Sox2 in

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Zfp206 (11,12), which indicated that it may be a direct target of regulation by these established pluripotency transcription factors. This prompted us to further analyze the Zfp206 gene for functionally important Oct4 and Sox2 binding sites. The previous ChIP studies had identified two Oct4 and two Sox2 binding sites in the first intron of Zfp206. Both of these Oct4 binding sites are in close proximity to the Sox2 binding sites, thus forming 2 composite oct-sox elements, one located near the start of intron 1 on Zfp206 and the other one near the end of the same intron, BS1 and BS2, respectively. ChIP-PCR with Oct4 or Sox2 antibodies confirmed that Oct4 and Sox2 are indeed bound at both BS1 and BS2 in mouse ESCs (Fig. 3).

We tested BS1 and BS2 further by EMSA to precisely map the Oct4 and Sox2 binding sites. Our results show that both Oct4 and Sox2 bind at BS1 and BS2 (Fig. 4). Previous reports have shown that Oct4 and Sox2 can form a heterodimer that interacts with DNA at closely linked binding sites. We found that only BS2 bound Oct4 and Sox2 as a heterodimer. Composite oct-sox elements have been identified and found to be functional in the enhancer region of a number of different genes such as Fgf4, Utf1, Fbx15 and Sox2. In these instances the sox and oct elements are separated by 3 bp or less. In contrast, the sox and oct element in both BS1 and BS2 are separated by 11bp. Thus, 11bp separation between the Oct4 and Sox2 binding sites does not preclude heterodimer binding. It is not clear why a heterodimer was not detected at BS1; it may be related to the fact that the sox and oct elements are in a different orientation compared to those in the BS2 element and all the other known composite sox-oct elements.

We then went on to determine if there are any functional differences between BS1 and BS2. A series of luciferase assays confirmed that the sox-oct element within BS1 is able to confer transcriptional activation, regardless of the promoters used. Moreover, the transcriptional activation is dependent of Oct4 and Sox2

proteins – either Oct4 or Sox2 can confer activation via BS1, though a combination of these two proteins result in synergistic activation. This suggests that a tight Sox2-Oct4 heterodimer complex is not the only way that these two transcription factors transcriptionally activate genes in pluripotent cells. We also found that BS1 works in both orientations, suggesting that it operates as an enhancer to regulate Zfp206 expression in response to Oct4 and Sox2 binding (Fig. 5 and 6). This is further confirmed by an in vivo experiment which showed that a regulatory region of Zfp206 containing BS1 is able to control the expression of an exogenous gene (Fig. 2) in undifferentiated ESC and its differentiated derivatives. Surprisingly, we could not demonstrate any transcriptional effect mediated by BS2 even though this site binds Oct4 and Sox2 heterodimers. One possibility is that BS2 can not activate the Zfp206 promoter alone. Perhaps it is functional only in cooperation with other transcription factor binding sites. Another possibility is that Oct4 and Sox2 binding at BS2 could modulate the occupancy at nearby BS1, which in turn controls the Zfp206 promoter activity. Additional experiments are required to test this hypothesis.

The results presented here have expanded upon our understanding of the transcriptional network that is operative in ESCs to control differentiation. We have found that Zfp206 is directly regulated by Oct4 and Sox2. Oct4 and Sox2 are often described as master regulators of ES cell pluripotency, though the key downstream effectors of their action have not been established. It now appears that Zfp206 is one of downstream modulators of Oct4 and Sox2 regulation. It will be of interest to define direct targets of Zfp206, which is expressed primarily in early embryonic development. It is not clear if Zfp206 is a transcriptional activator or a repressor. In either case, identification of Zfp206 targets will improve upon our understanding of the molecular basis of early embryonic development and ESC differentiation.

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FOOTNOTES

*We thank Tan Siok Lay for the Chromatin-Immunoprecipitation work. We extend our gratitude to Linda Lim, Aina Hoi, Yvonne Yi-Ling Koh, Kee Yew Wong, Pin Li, Shi-Feng Xue for technical assistance; Hian-Cheong Ng, Evangeline Chew, Rubing Liu for making Zfp206 knock out ESCs. For their generosity of reagents, we thank David Rodda and Jonathan Loh. This work was supported by funding from the Singapore government through the Biomedical Research Council (BMRC) and Agency for Science, Technology and Research (A*STAR). 1The abbreviations used are: ESC, embryonic stem cell; EMSA, electrophoretic mobility shift assay; RNAi, RNA interference; LIF, leukemia inhibitory factor; MEF, mouse embryonic fibroblast; ChIP, chromatin immunoprecipitation; β-Gal, β-galactosidase.

FIGURE LEGENDS Fig.1. Oct4 and Sox2 can regulate Zfp206 expression. Changes in endogenous gene expression levels of Oct4, Nanog and Sox2 following gene-specific Oct4 (A), Nanog (B) and Sox2 (C) RNA interference, and corresponding changes in endogenous Zfp206 gene levels. cDNAs were prepared from the RNAi knockdown ESCs and analyzed using real-time PCR. The levels of the transcripts were normalized against values derived from E14 (no siRNA, has transfect reagent) at each time point. Fig.2. LacZ gene knocked in at Zfp206 locus is regulated by Oct4 and Sox2. A, Expression of lacZ in undifferentiated V6.4 ESC. lacZ expression was visualized by microscopy before (up), and after (down) ESC differentiation induced by retinoic acid (RA) for 5 days. B, Expression of lacZ restricted to undifferentiated ESCs located in the middle of 5-day-old EBs. C, Sox2 and Oct4 protein expression following Sox2 and Oct4 RNAi by Western blot. Tubulin was used as a loading control. D, lacZ expression following Sox2 and Oct4 knock down. V6.4 heterozygous ESC were transfected with Sox2 or Oct4 RNAi, after 48 hrs puromycin selection, lacZ activity was measured. An irrelevant shRNA to Gfp and an empty vector served as negative controls. The change of lacZ reporter activity resulting from each RNA interference is given relative to the empty vector. Fig. 3. Oct4 and Sox2 binds to BS1 and BS2 in vivo. A, Real-time PCR primer pairs along the 3kb region covering the Sox2 overlay region were used to quantify the ChIP-enriched DNA. B-C, ChIP was performed

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using anti-Sox2, anti-Oct4 and GST control antibodies. Fold enrichment measured by real time PCR, of six different amplicons, were compared for BS1; Amplicon 3 encompasses the composite oct-sox element, and the five others (Amplicons 1, 2, 4, 5 and 6) do not. Fold enrichment represents the abundance of enriched DNA fragments over a control region (amplicon 1, 1,522 bp upstream of the BS1). GST ChIP served as mock ChIP. For BS2, we designed 7 pairs of primers flanking this region for q-RT-PCR quantification. Immunoprecipitation showed enrichment of amplicon 10, which is quite close to this overlay region (upstream 149 bp of BS2), and the other six amplicons do not. Fold enrichment is compared with amplicon 7 which localize 1,542 bp upstream of the BS2. Fig. 4. Oct4 and Sox2 bind on the Zfp206 composite element. A-B, The alignment of the BS1 (A) and BS2 (B) sequences from mouse, rat, human, dog, and chimpanzee. The oct-sox composite element is indicated, as are the 3-bp replacement mutations for BS1 and BS2 with their corresponding names given. BS1 is highly conserved in five species and BS2 is not (only the mouse BS2 has the typical Sox2/Oct4 binding site). C-D, EMSA was performed for BS1 (C) and BS2 (D) using the labeled putative oct-sox element from the Zfp206 intron 1. We tested binding of factors in crude nuclear extracts of E14 embryonic stem cells. Protein-DNA complexes containing Oct4 and Sox2 were identified by the addition of antibodies (α). Binding specificity was tested using oligonucleotide competitors. Lane 1 to 10 for both BS1 and BS2 is represented: 1, only Cy5-labelled oligos; 2, contain unlabelled competitor (25 folds); 3, contain competitor of Oct4 element mutated (25 fold); 4, contain competitor of Sox2 element mutated (25 folds); 5-10, Oct4, Oct1, Oct6, Sox2, JunB and Sox4 antibody added. Fig. 5. Sox2 and Oct4 bind to the first intron of Zfp206 and regulate its activity. A, The Zfp206 enhancer constructs used to assay BS1 and BS2 activity in Zfp206 promoter or Nanog promoter driving luciferase reporter. The different reporter constructs used to assay for BS1 or BS2 activity are shown. The forward or reverse BS1 (1,062 bp) and forward BS2 (938 bp) region in the beginning and end of the 1st intron of Zfp206 were inserted either Zfp206 promoter driving luciferase gene (Zfp206-Luc) or Nanog promoter driving luciferase cDNA (Nanog-luc). The relative Sox2 or Oct4 binding site has been mutated in the different construct. The key is as follows: rBS1, the reverse orientation BS1 relative to the transcription start site; mO, mutated Oct4 binding site; mS, mutated Sox2 binding site; mOmS, mutated at both Oct4 and Sox2 binding sites. B, The effect of forward BS1 and BS2 in Zfp206 or Nanog promoter driving luciferase assay. Luciferase reporter assays of various Zfp206 or Nanog promoter constructs transiently transfected into E14 ESC. Sox2, Oct4 or both Sox2/Oct4 binding site were mutated in BS1 or BS2 and tested for activity in ESC. The luciferase activity of the Zfp206 or Nanog promoter (without BS1 or BS2) is arbitrarily set at 100%. C, The effect of reverse BS1 (rBS1) in Zfp206 or Nanog promoter driving luciferase assay. Fig. 6. Knockdown of Oct4 or Sox2 reduces activity of Zfp206 BS1 region. A, the luciferase reporter constructs of Zfp206 promoter plus BS1 region or control vector was co-transfected with Sox2, Oct4, or both Sox2/Oct4 RNAi, or control Gfp RNAi plasmid into murine ESCs and assayed for the luciferase activity 3 days after transfection. The effect of both forward BS1 and reverse BS1 (rBS1) in Zfp206 or Nanog promoter driving luciferase reporter following Sox2, Oct4, or both Sox2/Oct4 RNAi knocking down is measured by relative luciferase activity 3-day after transfection, with the empty vector set at 100%. B, The effect of different mutated forward BS1 or reverse orientation BS1 in Zfp206 promoter driving luciferase reporter following Sox2, Oct4 or both Sox2/Oct4 RNAi knocking down. The levels of the luciferase were normalized against values derived from Zfp206 promoter driving luciferase without BS1 or BS2 which is 100% set.

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Robson and Lawrence W. StantonZheng-Xu Wang, Christina Hui-Leng Teh, Jacqueline LL. Kueh, Thomas Lufkin, Paul

factor Zfp206 in embryonic stem cellsOct4 and Sox2 directly regulate expression of another pluripotency transcription

published online March 7, 2007J. Biol. Chem. 

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