Short Report 2629 Klf5 is involved in self-renewal of mouse embryonic stem cells

Silvia Parisi1,2,*, Fabiana Passaro1,3,*, Luigi Aloia1,2, Ichiro Manabe4, Ryozo Nagai5, Lucio Pastore1,3 and Tommaso Russo1,3,‡ 1CEINGE Biotecnologie Avanzate, 80145 Napoli, Italy 2European School of Molecular Medicine, SEMM, 80145 Napoli, Italy 3Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy 4Nano-Bioengineering Education Program and 5Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 15 May 2008 Journal of Cell Science 121, 2629-2634 Published by The Company of Biologists 2008 doi:10.1242/jcs.027599

Summary Self-renewal of embryonic stem cells (ESCs) is maintained by undifferentiated state by Klf5 is, at least in part, due to the a complex regulatory mechanism involving transcription factors control of Nanog and Oct3/4 transcription, because Klf5 directly Oct3/4 (Pou5f1), Nanog and . Here, we report that Klf5, a binds to the promoters of these and regulates their Zn-finger of the Kruppel-like family, is transcription. involved in ESC self-renewal. Klf5 is expressed in mouse ESCs, blastocysts and primordial germ cells, and its knockdown by RNA interference alters the molecular phenotype of ESCs, Supplementary material available online at thereby preventing their correct differentiation. The ability of http://jcs.biologists.org/cgi/content/full/121/16/2629/DC1 Klf5 to maintain ESCs in the undifferentiated state is supported by the finding that differentiation of ESCs is prevented Key words: Differentiation, Kruppel-like factors, Nanog, Oct3/4, when Klf5 is constitutively expressed. Maintenance of the Self-renewal

Introduction Results and Discussion ESCs derived from the inner cell mass of the blastocyst can Klf5 is expressed in mouse ESCs and its knockdown

Journal of Cell Science differentiate into primitive ectoderm, primitive endoderm and suppresses normal ESC differentiation trophoectoderm cells, and in turn into all cell types present in the The screening of a collection of short hairpin RNAs (shRNAs) embryo. ESCs are maintained in the undifferentiated state during designed to target mouse mRNAs allowed us to observe that the self-renewal by a complex regulatory network involving three shRNA targeting Klf5 mRNA was able to interfere with ESC transcription factors, namely Oct3/4 (Pou5f1) (Nichols et al., 1998), differentiation (see supplementary material Fig. S1). Klf5, also Nanog (Chambers et al., 2003; Mitsui et al., 2003) and Sox2 (Avilion known as intestinal-enriched factor and basic transcription et al., 2003). These factors regulate their own expression and that element binding 2, is a Zn-finger transcription factor of many other genes (Boyer et al., 2005; Loh et al., 2006). Other belonging to the Sp/Kruppel-like family. In adults, it is expressed transcription factors play important roles in ESC pluripotency and in the proliferating crypt cells of the intestinal epithelium and at self-renewal (Niwa, 2007). These data illustrate the complexity of low levels in the testis, uterus, placenta, lung and in the proliferating basal layer of the epidermis (Ohnishi et al., 2000). transcription regulation in ESCs, which is still not completely Klf5 knockout causes early embryonic lethality (Shindo et al., understood. 2002), which suggests that this factor plays a key role during Recently, several results indicate that transcription factors early development. belonging to the Kruppel-like family could have an important role Klf5 mRNA and protein are present in undifferentiated ESCs in the regulation of ESCs. Ectopic expression of , together and levels rapidly decrease after induction of ESC differentiation with Oct3/4, Sox2 and , results in the conversion of by two different approaches (Fig. 1A,B). Immunostaining differentiated cells into pluripotent ES-like cells (Takahashi et al., demonstrated that Klf5 is expressed at various levels in almost all 2006; Okita et al., 2007; Werning et al., 2007; Maherali et al., undifferentiated ESCs (Fig. 1C) and that it substantially colocalises 2007). Recent findings indicate that Klf2 or Klf5 can replace Klf4 with Oct3/4 and Nanog [964 out of 1100 cells (87.6%) were positive in the combination inducing cell reprogramming (Nakagawa for both Oct3/4 and Klf5; 512 out of 678 cells (75.5%) were Nanog et al., 2008) and that triple knockdown (KD) of Klf2, Klf4 and and Klf5 positive; 625 out of 720 cells (86.8%) expressed both Klf5 abolishes the undifferentiated phenotype of ESCs (Jiang et Oct3/4 and Nanog]. After 3 days in differentiation conditions, the al., 2008). Klf5 signal disappeared from most cells, as observed for Oct3/4 In this study, we demonstrate that KD of even only Klf5 and Nanog (supplementary material Fig. S2). The expression of abolishes the ESC undifferentiated phenotype, whereas its Klf5 mRNA in vivo is in agreement with that observed during in constitutive expression prevents ESC differentiation. vitro differentiation of ESCs. In fact, we found the Klf5 transcript 2630 Journal of Cell Science 121 (16)

in blastocysts at embryonic day (E)3.5 but not in the epiblast of E6.5 embryos (Fig. 1D). Immunostaining of pre-implantation embryos revealed nuclear Klf5 in the morula (Fig. 1E) and in many cells of the blastocyst at Theiler stage 4 (Fig. 1E,F), when the blastocoelic cavity is formed. At these stages, the cells also expressed Oct3/4 and Nanog. At a later stage, the expression of Klf5 persisted (supplementary material Fig. S2). Immunostaining of sections from E11.5 embryos revealed that Klf5 is also expressed in small groups of cells in the genital ridge, coincident with primordial germ cells expressing Oct3/4 (Fig. 1H).

ESCs lose their undifferentiated phenotype as a consequence of Klf5 KD The observation that Klf5 expression is restricted to undifferentiated ESCs both in vitro and in vivo and is tightly regulated when differentiation occurs suggests that it might be required to maintain ESCs in an undifferentiated state. To address this hypothesis, we explored the effects of Klf5 KD in undifferentiated ESCs. To this aim, ESCs were transfected with the previously used Klf5 shRNA or with a mixture of four short interfering RNAs (siRNAs), all targeting different regions of the Klf5 mRNA. Whereas non-silencing (CRL)- shRNA-transfected cells were indistinguishable from untransfected cells, Klf5 KD resulted in the appearance of clusters of enlarged flattened cells (supplementary material Fig. S3). To verify that these morphological changes result from loss of the undifferentiated phenotype, we counted alkaline-phosphatase-positive colonies (APcs), as a marker of undifferentiated ESCs, in cells plated at clonal density (~80 cells/cm2). Although grown in the presence

Journal of Cell Science of leukaemia inhibitory factor (LIF) and serum, Klf5- KD cells lost their undifferentiated phenotype, as witnessed by the drastic reduction of APcs (Fig. 2A). Accordingly, Oct3/4 and Nanog expression was significantly decreased, as demonstrated by immunostaining (Fig. 2B) and reverse-transcriptase (RT)-PCR (Fig. 2C). Fig. 1. Klf5 expression in ESCs. (A,B) Klf5 mRNA (A) and protein (B) levels decrease soon after the induction of ESC differentiation. ESCs were differentiated as embryoid Furthermore, Oct3/4 KD decreased levels of the bodies (EBs) or in monolayer. (C) Klf5 is highly expressed in undifferentiated ESCs and cognate mRNA and protein by >50% and was colocalised with Oct3/4 and Nanog. Scale bars: 50 μm. (D) RT-PCR of Klf5 in ESCs, accompanied by a significant reduction of Sox2 and and in E3.5 and E6.5 embryos. (E) Klf5 is expressed in morulae together with Oct3/4 Nanog mRNA levels. In these conditions, Klf5 mRNA and Nanog. (F,G) E3.5 embryos express Klf5, Oct3/4 and Nanog in the inner cell mass. μ was significantly decreased (Fig. 2D), which reinforces Scale bars: 20 m. (H) Two consecutive sections of an E11.5 embryo were stained with anti-Klf5 and -Oct3/4 antibodies. The arrowheads indicate the groups of cells in the the concept that the expression of Klf5 is restricted to the genital ridge (primordial germ cells) expressing both Klf5 and Oct3/4. The arrow ESC undifferentiated state. indicates the stomach epithelium, where Klf5 is also expressed (Ohnishi et al., 2000). To explore phenotypic changes induced by Klf5 KD, we analysed several markers of cell fate. As shown in Fig. 2E, markers of endoderm (Gata4, Hnf4 and Sox17), ectoderm Klf5 constitutive expression prevents ESC differentiation (Sox1) and visceral endoderm (Afp) were undetectable in both We then examined ESC clones stably expressing Klf5 under the Klf5- and CRL-shRNA-transfected cells; mRNAs for the control of a constitutive β-actin promoter (see supplementary mesoderm markers and Meox1, and for the trophoblast material Fig. S5). Mock-transfected ESC clones plated at ~80 markers Cdx2, Eomes and PL-1, appeared only in Klf5 KD cells. cells/cm2 showed only a few APcs (60±3.8 APcs/100-mm plate, Immunostaining of Klf5 KD cells showed that 12.3±0.2% of cells n=4) 6 days after LIF withdrawal, whereas, despite LIF withdrawal, (n=920) expressed brachyury and 11.2±0.6% (n=950) expressed ESCs stably expressing exogenous Klf5 showed 912.5±33 Cdx2 (supplementary material Fig. S4). The remaining cells did APcs/100-mm plate (n=4, Fig. 3A). As shown in Fig. 3B-D, after not express any examined differentiation markers, and some of 5 days in differentiation conditions, exogenous Klf5 expression these cells still expressed Nanog (28.5±3%) and/or Oct3/4 sustains Oct3/4 and Nanog expression whereas, in mock-transfected (37.8±3.5%). cells, Oct3/4 and Nanog expression is strongly decreased. Klf5 in self-renewal of ESCs 2631

immunoprecipitation (ChIP) experiments, which demonstrated the direct interaction of Klf5 with the Nanog promoter (Fig. 4B). Also, in the case of the Oct3/4 promoter, a significant enrichment of chromatin that co- immunoprecipitated with Klf5 was observed. To confirm the presence of a Klf5 cis-element in the Nanog promoter, we performed electrophoretic mobility shift assay (EMSA) experiments. The probe on which the Oct4- Sox2 cis-element is present was not supershifted by the anti-Klf5 antibody and no changes in the band pattern were seen after challenging the probe with extracts from Klf5- overexpressing cells (supplementary material Fig. S6). On the contrary, by using a second probe covering a region downstream of the previous one, an additional shifted band appeared when it was challenged with extracts from 3ϫFLAG-Klf5-expressing cells (Fig. 4C). This band was erased by pre-treatment with anti-Klf5 antibody. Furthermore, one of the bands obtained by challenging the probe with wild-type ESC extracts was significantly decreased when the extract was pre-treated with anti-Klf5 antibody. The sequence of this region contains at least one element that is compatible with the known consensus of Klf factors. An oligonucleotide bearing mutations that disrupt this cis-element does not compete for either exogenous or endogenous Klf5 candidate bands (Fig. 4C). Systematic analyses of Oct3/4 and Nanog target genes revealed numerous candidate

Journal of Cell Science Fig. 2. Klf5 is required to maintain ESCs in an undifferentiated state. (A) Klf5 KD causes a decrease genes, including Klf5 [see supplementary in the number of APcs. CRL- or Klf5-shRNA transfected ESCs were plated at 80 cells/cm2 and grown information from Boyer et al. and Loh et al. for 6 days. The number of APcs was 2040±55 per 100-mm plate (n=5) for CRL shRNA (upper panel) (Boyer et al., 2005; Loh et al., 2006)]. Thus, = and 158±14 per 100-mm plate (n 5) for Klf5 shRNA (lower panel). Scale bar: 1 mm. (B) ESCs shown we examined the mouse Klf5 genomic region in A were stained with anti-Oct3/4 or -Nanog antibodies. In Klf5-KD cells, the expression of these two is drastically decreased. Scale bars: 100 μm. (C) Klf5 KD is accompanied by a significant and found that it contains a Nanog candidate (P<0.01) decrease of Oct3/4, Sox2 and Nanog mRNAs. β-actin mRNA was used as a control. cis-element. Alignment of this region with all Standard errors of three independent experiments are reported. (D) Silencing of Oct3/4 downregulates available genomic sequences revealed a 100% Klf5 expression. (E) RT-PCR analysis of mRNAs shows that Klf5 KD causes illegitimate expression conservation of these cis-elements in Klf5 of brachyury and Meox (mesoderm), and of Cdx2, PL-1 and Eomes (trophoblast). orthologues in all mammalian species and also in the chicken orthologue (supplementary material Fig. S7). ChIP experiments designed Klf5 directly regulates the transcription of Oct3/4 and Nanog to determine whether Nanog interacts directly with the Klf5 The transcription-factor network implicated in ESC self-renewal promoter showed a significant enrichment for Nanog binding to consists of positive- and negative-feedback loops involving Oct3/4 Klf5 chromatin, comparable to that observed for Nanog binding to and Nanog. The effects of Klf5 overexpression or silencing on the the Oct3/4 and Nanog promoters (Fig. 4D). expression of these genes and on the ESC undifferentiated phenotype suggest that this transcription factor could play a direct Klf4 is not involved in the regulation of Klf5 in ESCs role in this regulatory network. To address this point, Klf5 was co- Experimental evidence indicates a regulatory crosstalk between Klf5 transfected with reporter vectors that drive the expression of and the Sp/Kruppel-like factor Klf4. At least in adult cells, this luciferase under the control of Nanog or Oct3/4 promoters. Under crosstalk seems to be involved in cell proliferation (McConnell et these conditions, Klf5 overexpression was accompanied by a al., 2007), but Klf5 KD or overexpression does not appear to significant induction of the Nanog and Oct3/4 promoters (Fig. 4A). significantly alter ESC proliferation, as demonstrated by the BrdU- In addition, in Klf5 KD cells, transcription from both the Oct3/4 incorporation assay (supplementary material Fig. S8). To address and Nanog promoters was about 50% lower, which confirms that whether Klf4 is involved in Klf5 regulation, we analysed the effects Klf5 drives the activity of these two genes (Fig. 4A). of Klf4 KD or overexpression in ESCs, and found that changing To evaluate whether Klf5 regulates Oct3/4 and Nanog by directly Klf4 expression levels does not significantly modify the levels of interacting with their promoters, we performed chromatin Klf5, and also of Oct3/4 and Sox2, whereas Klf4 KD induces a 2632 Journal of Cell Science 121 (16)

Conclusions Our results demonstrate that Klf5 is an essential factor of the core regulatory network that maintains ESCs in the undifferentiated state. Klf5 KD does not modify levels of Klf2 and Klf4, thus suggesting that the presence of these two factors cannot completely substitute for the reduction or absence of Klf5. The relevance of Klf5 is also supported by the observation that Klf5–/– embryos die before E8.5 (Shindo et al., 2002), whereas Klf4–/– mice die at birth (Segre et al., 1999) and Klf2–/– embryos die between E12.5 and E14.5 (Kuo et al., 1997). Nevertheless, it is worth noting that Klf2 and Klf4 are more efficient than Klf5 in adult-cell reprogramming (Nakagawa et al., 2008), and their expression is modified by Oct3/4 or Nanog suppression, respectively (Fig. 4), thus indicating that these three Klf proteins are all important for ESC functions. The regulation of Klf5 is closely related to that of Oct3/4 and Nanog. In particular, Klf5 directly regulates both Nanog and Oct3/4 promoters. Furthermore, Klf5 KD impairs differentiation induced by removal of LIF and serum from the ES culture medium as observed for the KD of Nanog (Chambers et al., 2003), and both Klf5 and Nanog overexpressions are able to sustain the ESC undifferentiated state in the absence of LIF (Chambers et al., 2003; Mitsui et al., 2003).

Materials and Methods Plasmid construction The α1-tubulin–EGFP vector was derived from pEGFP (Clontech), containing the G418 resistance gene, by replacing the CMV promoter with the rat α1-tubulin promoter from nucleotide –1050 to +5 (Schmandt et al., 2005). Klf5 cDNA (NIH Mammalian Gene Collection, Invitrogen) was sub-cloned into p3ϫFLAG-CMV7.1 vector (Sigma-Aldrich). The 3ϫFLAG-tagged Klf5 cDNA was then subcloned into the pCBA-GFP vector (kind gift of Francesca Tuorto, CNR, Naples, Italy), by replacing GFP with 3ϫFLAG-Klf5. The CBA promoter, containing the CMV immediate early enhancer fused to the chicken β-actin promoter, is active in both undifferentiated and differentiated ESCs (Chung et al., 2002). A 379-bp fragment encompassing the promoter region of Nanog (Kuroda et al., 2005), and a 2.2-kb fragment encompassing the promoter region of Oct3/4 (Chew et al., 2005) were cloned into the promoterless luciferase vector pGL3-Basic (Promega).

Journal of Cell Science Cell culture, transfections and luciferase assay E14Tg2a (BayGenomics) mouse ESCs were maintained as described elsewhere (Niwa et al., 2000). Plasmids and siRNA smart pools (Dharmacon) were transfected using either ArrestIn (Open Biosystems) or Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The sequences of shRNA used are listed in supplementary material Table S1. To generate the reporter cell lines, we electroporated ESCs with α1-tubulin–EGFP plasmid. Recombinant clones were selected with 380 μg/ml G418 for 7 days (Invitrogen). One randomly selected clone (C7) was Fig. 3. Klf5 constitutive expression prevents ESC differentiation. (A) ESCs characterised for the co-expression of EGFP and β3-tubulin under differentiation that were mock-transfected (right panel) or transfected with Klf5 expression conditions, and for self-renewal and in vitro pluripotency (see supplementary material vector (left panel) were plated at low density (80 cells/cm2) and grown for 6 Fig. S1). For luciferase assay, reporter plasmids were co-transfected with an internal days without LIF. Scale bars: 1 mm. (B,C) Mock- or Klf5-transfected ESCs control plasmid and, 24 hours later, Firefly and Renilla luciferase activities were were grown for 5 days in differentiation conditions and stained with anti- measured using a dual-luciferase reporter system (Promega). Nanog and -Oct3/4 (B) or -Klf5 and -Nanog (C) antibodies. Scale bars: 100 μm. (D) Western blot analyses of Oct3/4 and Nanog in undifferentiated and ESC differentiation differentiated ESCs. Protein extracts are from four clones stably expressing To induce neural differentiation, ESCs were plated onto gelatine-coated dishes at low ϫ 3 ϫ 3 2 Klf5-overexpressed (Klf5 ov.) and one from mock-transfected cells. density (1 10 -5 10 cells/cm ) in the following differentiation medium: knockout DMEM supplemented with 10% KSR, 2 mM glutamine, 100 U/ml penicillin/ streptomycin (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma). Embryoid-body differentiation was obtained as described elsewhere (Maltsev et al., 1993). significant decrease of Nanog mRNA (Fig. 4F). Furthermore, Klf4 and Klf2 mRNAs remained unchanged in Klf5-KD or Screening of the shRNA collection shRNA-expressing plasmids (Open Biosystems) were transfected in α1-tubulin–EGFP -overexpressing cells (Fig. 4G). On the contrary, the KD of Oct3/4 cells plated in 96-microwell plates. Transfected cells were selected with puromycin significantly decreased Klf2 mRNA levels, whereas Nanog (2 μg/ml, Sigma) for 3 days, then trypsinised and plated in differentiation medium. suppression led to a strong decrease of Klf4 mRNA. Taken together, EGFP-positive cells appeared within 4-5 days. The presence/absence of EGFP was evaluated at different time points (4, 7 and 10 days of differentiation) by using a these results suggest that Klf4 is not involved in the transcriptional fluorescence-inverted microscope (Leica Microsystems). regulation of Klf5 in ESCs. However, it is worth noting that Klf5, Klf4 and Klf2 mRNAs are all regulated as a consequence of Oct3/4 RT-PCR and western blotting and/or Nanog suppression, thus further supporting the results Total RNAs were extracted using Trizol (Invitrogen). RNA (2 μg/reaction) was reverse-transcribed using M-MuLV reverse transcriptase (Biolabs). Details on indicating that all these proteins have a role in the regulation of semiquantitative and real-time PCR are reported in supplementary material Tables ESCs. S2 and S3. Total cell lysates were obtained by using Laemmli lysis buffer and analysed Klf5 in self-renewal of ESCs 2633

Fig. 4. Klf5 is a component of the Oct3/4 and Nanog transcription-factor network. (A) Nanog-Luc or Pou5f1- Luc were co-transfected with Klf5 (Klf5 ov.), Klf5 shRNA or control (mock) vectors. (B) 3ϫFLAG-Klf5 transfected ESCs were processed for ChIP with antibody for the FLAG epitope or IgG as control. The amount of precipitated DNA was calculated relative to the total input chromatin. (C) The indicated oligonucleotide, designed on the basis of the Nanog promoter (Wu and Yao, 2005), was incubated with nuclear extracts of mock-transfected (lanes 2-4) or 3ϫFLAG-Klf5-transfected (lanes 6-9, 11,12) cells. Nuclear extracts were preincubated with 50-fold molar excess of unlabelled oligo probe (lanes 3 and 7) or unrelated oligo (lanes 4 and 8), or mutant oligo in which the sequence –78 to –72 (GGGTGGG, underlined) was changed into AGATAGA to disrupt the candidate cis-element of Klf5. In lane 12, nuclear extracts were preincubated with anti-Klf5 antibody. Relevant bands are indicated (arrows) as follows: (a) candidate band for endogenous Klf5; (b) specific shifted band not related to Klf5; (c) additional band due to exogenous 3ϫFLAG-Klf5. (D) ChIP experiments with anti-Nanog antibody on the Klf5 promoter. K1 and K2 indicate two oligonucleotide pairs that amplify the regions of the Klf5 gene (supplementary material Fig. S7). The differences with control antibody and control DNA were

Journal of Cell Science significant (P<0.001). (E) Real-time PCR of Klf5, Nanog, Oct3/4 and Sox2 mRNAs in Klf4-KD or -overexpressing cells. *P<0.001, **P<0.01. (F) mRNA levels of Klf2 and Klf4 in ESCs transfected with Oct3/4, Nanog or Klf5 shRNAs or with Klf5 overexpressed (Klf5 ov). *P<0.001.

by western blot using the following antibodies: Klf5 (KM1784), Oct3/4, GAPDH Oligonucleotide pairs are reported in supplementary material Table S4. EMSAs were (Santa Cruz), anti-Flag (Sigma), Nanog (Calbiochem), secondary HRP-conjugated performed as described by Bevilacqua et al. (Bevilacqua et al., 2005). antibodies (Santa Cruz and Amersham Biosciences). KM1784 was raised in rabbits against human BTEB2 (orthologue of mouse Klf5). The specificity of this antibody This work is supported by grants to T.R. from Ministry of Research, was checked by immunostaining and western blot (supplementary material Fig. S9). Regione Campania, Italy. We are grateful to Antonio Simeone, Dario Immunostaining of cells and embryos Acampora, Caterina Missero and Riccardo Cortese for precious ESCs and embryos were fixed in 4% paraformaldehyde and stained with antibodies suggestions, and to Jean Ann Gilder for reading the manuscript. recognizing βIII-tubulin (1:400; Sigma), Oct3/4 (1:100; Santa Cruz), Nanog (1:100, R&D Systems), brachyury (1:100, Santa Cruz), Cdx2 (Biogenex), BrdU (1:10, Roche) and Klf5 (1:300, KM1784), and with appropriate secondary antibody (Molecular References Avilion, A. A.,Nicolis, S. K., Pevny, L. H., Perez, L., Vivian, N. and Lovell-Badge, R. Probes). Images were captured with an inverted microscope (DMI4000, Leica (2003). Multipotent cell lineages in early mouse development depend on SOX2 function. Microsystems) or with a confocal microscope (LSM 510 META, Zeiss). Genes Dev. 17, 126-140. Immunohistochemistry of E11.5 embryos was performed as described by Puelles et Bevilacqua, M. A., Iovine, B., Zambrano, N., D’Ambrosio, C., Scaloni, A., Russo, T. al. (Puelles et al., 2006). and Cimino, F. (2005). Fibromodulin gene transcription is induced by ultraviolet irradiation, and its regulation is impaired in senescent human fibroblasts. J. Biol. Chem. ChIP assay and EMSA 280, 31809-31817. Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine, S. S., Zucker, J. P., and formaldehyde was then inactivated by the addition of 125 mM glycine. The Guenther, M. G., Kumar, R. M., Murray, H. L., Jenner, R. G. et al. (2005). Core chromatin was then sonicated to an average DNA-fragment length of 200-1000 bp. transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947-956. Soluble chromatin extracts were immunoprecipitated using anti-FLAG (Sigma, cat. Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S. and Smith, F3165) or -Nanog (R&D Systems, cat. AF2729) antibodies. Supernatant obtained A. (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor in without antibody was used as input control. The amount of precipitated DNA was embryonic stem cells. Cell 113, 643-655. calculated by real-time PCR relative to the total input chromatin, and expressed as Chew, J. L., Loh, Y. H., Zhang, W., Chen, X., Tam, W. L., Yeap, L. S., Li, P., Ang, Y. Δ percent of total chromatin according to the following formula: 2 Ct ϫ 10, where Ct S., Lim, B., Robson, P. et al. (2005). Reciprocal transcriptional regulation of Pou5f1 represents the cycle threshold and ΔCt = Ct(input) – Ct(immunoprecipitation) (Frank and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol. Cell. Biol. 25, 6031- et al., 2002). The Student’s t-test was used to measure statistical significance. 6046. 2634 Journal of Cell Science 121 (16)

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