SEARCH

SEARCH BY CITATION

Keywords:

  • Induced pluripotent stem cells;
  • Neural stem cells;
  • Reprogramming;
  • Transcriptional factors

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by ectopic expression of specific sets of transcription factors. Oct4, Sox2, and Klf4, factors that share many target genes in embryonic stem (ES) cells, are critical components in various reprogramming protocols. Nevertheless, it remains unclear whether these factors function together or separately in reprogramming. Here we show that Klf4 interacts directly with Oct4 and Sox2 when expressed at levels sufficient to induce iPS cells. Endogenous Klf4 also interacts with Oct4 and Sox2 in iPS cells and in mouse ES cells. The Klf4 C terminus, which contains three tandem zinc fingers, is critical for this interaction and is required for activation of the target gene Nanog. In addition, Klf4 and Oct4 co-occupy the Nanog promoter. A dominant negative mutant of Klf4 can compete with wild-type Klf4 to form defective Oct4/Sox2/Klf4 complexes and strongly inhibit reprogramming. In the absence of Klf4 overexpression, interaction of endogenous Klf4 with Oct4/Sox2 is also required for reprogramming. This study supports the idea that direct interactions between Klf4, Oct4, and Sox2 are critical for somatic cell reprogramming. STEM CELLS 2009;27:2969–2978


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

The recent development of methods to reprogram somatic cells to induced pluripotent stem (iPS) cells using retroviral or lentiviral transduction of four genes (Oct3/4, Sox2, c-Myc, and Klf4) represents a major breakthrough in stem cell research [1–3]. Further analysis shows that three of these genes, Oct4, Sox2, and Klf4, are critical to the process and that c-Myc functions to enhance reprogramming efficiency [4–6]. Additionally, inducible systems have been developed to better control transgene expression [7]. Novel methods requiring no viral integration have also been developed [8–12], as have strategies using small molecules to promote reprogramming efficiency [13–15].

As a key factor in reprogramming, Kruppel-like factor 4 (Klf4/GKLF/EZF) functions as both a transcriptional activator and repressor to regulate proliferation and differentiation of different cell types [16]. RNA interference experiments confirm that Klf4 is redundant with two other family members, Klf2 and Klf5, in regulating expression of pluripotency-related genes [17]. In embryonic stem (ES) cells, Klf4 has been shown to be important to activate Lefty1 together with Oct4 and Sox2 [18]. Genome-wide chromatin immunoprecipitation with microarray analysis (ChIP-Chip) demonstrates that the DNA binding profile of Klf4 overlaps with that of Oct4 and Sox2 on promoters of genes specifically underlying establishment of iPS cells, suggesting transcriptional synergy among these factors [19]. Furthermore, studies also suggest that Klf4 may function in establishing an “authentic” and “metastable” pluripotent state in various pluripotent cell types [20, 21].

The fact that other Klf family members can substitute for Klf4 in reprogramming [6] suggests that motifs common to this family are important for reprogramming activity. The only structural similarities common to Klf family proteins are C-terminal tandem zinc finger motifs [22]. Interactions between Klf1 (EKLF) and GATA-1 suggest that Klf family C2H2 zinc fingers can also bind other transcriptional partners [23], which may be functionally conserved among Klf family members [24]. However, little is known about potential interaction partners of Klf4 and the significance of these interactions in reprogramming.

Here we show that Klf4 interacts directly with Oct4 and Sox2 in iPS and ES cells. Three C-terminal Klf4 zinc fingers mediate both interactions and are required for transcriptional activation of the target gene Nanog. We also show that specific Klf4 mutants can compete with either overexpressed or endogenous wild-type (WT) Klf4 to form transcriptionally defective complexes with Oct4 and Sox2, inhibiting reprogramming efficiency. These results indicate that Oct4, Sox2, and Klf4 function via direct interaction to regulate downstream targets and facilitate reprogramming.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

Plasmid Construction

To generate the doxycycline-inducible viral expression vector, the digested rtTA2S-M2 fragment [25] was inserted into the vector FUIPW, containing an internal ribosomal entry site followed by the puromycin resistance gene. The ubiquitin promoter of FUW [26] was replaced with a tetracycline-responsive element (TRE) containing a cytomegalovirus minimal promoter to construct FTRE. cDNAs encoding Oct4, Sox2, Klf4, and Klf4 mutants were subsequently cloned into FTRE and FUIPW. Klf4 and its mutants were also cloned into pCS2 as described [16].

Cell Culture, Lentivirus Preparation, iPS Cell Generation, and In Vitro Differentiation

293T cells were maintained in Dulbecco's modified Eagle's medium (Cellgro; Mediatech, Inc., Manassas, VA, http://www.cellgro.com/shop/customer/home.php) containing 10% fetal bovine serum (FBS). FTRE-based lentiviruses were generated in 293T cells as described previously [27]. Virus-containing medium was collected at 48 hours after transfection and virus was concentrated by ultracentrifugation at 28,000 rpm for 2 hours. Concentrated viruses were reconstituted in phosphate-buffered saline (PBS).

Reprogramming of primary mouse embryonic fibroblasts (MEFs) was performed as described [28]. Briefly, primary MEFs were generated from embryonic day (E)-13.5 mouse embryos carrying the green fluorescent protein (GFP) transgene under control of the Oct4 promoter [29]. MEFs (6 × 105) were seeded in 100-mm dishes and transduced twice with a cocktail of five lentiviruses, including those expressing the four reprogramming factors plus rtTA. Mouse ES medium (Glasgow minimum essential medium with 15% FBS, 2 mM glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, 1% sodium pyruvate, leukemia-inhibitory factor [LIF] at 10 ng/ml) plus 0.1 μg/ml of doxycycline was added after 2 days and changed every day afterward. Three weeks later, mature iPS colonies were isolated by manual cutting, and individual lines were maintained and characterized.

For in vitro differentiation, iPS cells were maintained on feeder layers of irradiated MEFs in mouse ES (mES) growth medium. To obtain neural stem cells (NSCs), iPS cells were detached from the MEF layer with 1 mg/ml collagenase for 15 minutes at 37°C and clumps were transferred to 9-cm bacterial dishes as suspension cultures in mouse ES medium lacking LIF. After 4 days, differentiating clusters resembling embryoid bodies were transferred to tissue culture dishes in 2% B27 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) defined medium with 20 ng/ml basic fibroblast growth factor for 14 days of differentiation with a change of medium every 2 days to form neurospheres (NSs). NSs were collected and treated with 500 μl of 0.05% trypsin at 37°C for 10 minutes, and then triturated and neutralized with 1 mg/ml trypsin inhibitor (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). NSCs were subsequently cultured as a monolayer on poly-L-lysine- and fibronectin-coated dishes.

Reverse-Transcription Polymerase Chain Reaction Analysis

Total RNA was isolated from iPS cells using an RNAeasy mini kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). Two μg of RNA was subjected to the reverse-transcription (RT) reaction using Superscript II (Invitrogen). Semiquantitative polymerase chain reaction (PCR) was performed to evaluate total gene expression, using primers previously described [3].

Immunostaining and Alkaline Phosphatase Staining

iPS cells grown on feeders were fixed in 2% paraformaldehyde in PBS for 10 minutes. Cells were incubated with stage-specific embryonic antigen 1 (SSEA1) primary antibody (1:400; Developmental Studies Hybridoma Bank, Iowa City, IA, http://dshb.biology.uiowa.edu) for 1 hour, washed with PBS, and incubated with secondary antibody (goat anti-mouse IgM, 1:1000; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) for 30 minutes. Alkaline phosphatase staining was done using the manufacturer's protocol (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com).

Teratoma Formation and Histological Analysis

iPS cells were suspended at 1 × 107 cells per milliliter in PBS. Cell suspension (100 μl) was injected subcutaneously into the dorsal flank of severe combined immunodeficient (SCID) mice (Charles River Laboratories, Wilmington, MA, http://www.criver.com). Six weeks later, samples were fixed in Bouin's fixation buffer. Sections were stained with hematoxylin and eosin and evaluated for differentiation.

Coimmunoprecipitation and Western Blotting

Coimmunoprecipitation and Western blotting were performed as previously described [27]. Antibodies used were anti-Flag (Sigma-Aldrich), anti-HA (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), anti-Klf4 (a gift from Dr. Ng), anti-Sox2 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), anti-Oct4 (Santa Cruz Biotechnology Inc.), and anti-Myc (Santa Cruz Biotechnology Inc.). Relative quantities of Western blotting bands were quantified using ImageJ (National Institutes of Health, Bethesda, MD).

Purification of Recombinant Proteins and In Vitro Binding

Glutathione S-transferase (GST)-Klf4 (300-483) was constructed in pGEX41T, expressed in BL-21 Escherichia coli, and purified by affinity chromatography using glutathione-Sepharose (GE Healthcare, Little Chalfont, U.K., http://www.gehealthcare.com) as described [30]. HA-Oct4 and HA-Sox2 were cloned in to pGEX41T and further purified by the same method. GST-tagged Oct4 and Sox2 were further cleaved by thrombin (GE Healthcare) at room temperature for 2 hours. After further preclearing with glutathione-Sepharose, the supernatant was checked by Western blotting using anti-HA antibody to confirm that Oct4 and Sox2 in the supernatant were completely cleaved. In vitro binding was assayed by mixing glutathione-Sepharose bound GST-Klf4 (300-483) or GST with Oct4 (HA) and Sox2 (HA) at 4°C overnight. Sepharose beads were washed, boiled, and ready for Western blotting.

Luciferase Assays

Luciferase reporter assays were performed as described [31].

Electrophoretic Mobility Shift Assays

293T cells were transfected with vectors expressing wild-type or mutant Klf4. Cells transfected with GFP vector served as a control. Nuclear extracts were prepared as described [27], with modifications.

For electrophoretic mobility shift assay (EMSA), Klf-binding [17] oligonucleotides were synthesized, labeled with IRD800 dye (Integrated DNA Technologies, Coralville, IA, http://www.idtdna.com), and annealed at 5 mM. For DNA binding reactions, 1 μl of DNA was added to a 10-μl reaction containing 1 μl of nuclear extract, 2 μg of salmon sperm DNA (Gibco, Grand Island, NY, http://www.invitrogen.com), and 1 μl of 10× binding buffer (100 mM Tris, 10 mM EDTA, 1 M KCl, 1 mM dithiothreitol, 50% glycerol). After a 30-minute incubation, the mixture was resolved on prerun 10% native polyacrylamide gel electrophoresis gels in 1× TBE(Tris/Borate/EDTA) buffer. Gels were imaged directly on glass plates using (Licor, Lincoln, NE, www.licor.com).

ChIP Assay

ChIP assays with mouse ES cells (or iPS cells) were carried out as described [32]. For all ChIP experiments, 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 (primer 1), which was defined as 1.0.

Reprogramming Efficiency Assay

To perform a secondary generation iPS cell efficiency assay, 3 × 105 NSCs derived in vitro from the differentiation of iPS cells or second-generation MEFs derived from chimeric mice were plated on 10-cm plates. Equal amounts of viruses encoding mutant forms of Klf4 or control virus were added and the medium was changed to fresh medium after 24 hours. Forty-eight hours later, cells were trypsinized and replated into 6-well plates at 5 × 104 cells per well. NSCs were then seeded on irradiated MEF-coated plates, whereas MEFs were seeded on 0.1% gelatin. Duplicate or triplicate wells were usually prepared for each sample. The medium was again changed to mouse ES cell medium and doxycycline added to 1 μg/ml. Medium was changed every day and supplemented with doxycycline throughout the induction period. Fluorescence-activated cell sorting (FACS) analysis was performed after 1-2 weeks. GFP-positive colonies were counted at various time points.

To evaluate the number of alkaline phosphatase (AP)-positive colonies in primary MEFs infected with Oct4, Sox2, and Klf4 mutants without exogenous wild-type Klf4 expression, 2 × 105 or 1 × 105 primary MEFs isolated from Oct4-GFP mice were seeded in 60-mm dishes or in 1 well of a 6-well plate, respectively, and transduced with the combinations of lentiviruses indicated in the text. Alkaline phosphatase staining was performed 2 weeks after transduction.

Flow Cytometry

Cells were trypsinized, washed once in PBS, resuspended in PBS, and stained with anti-SSEA1 antibody (Developmental Studies Hybridoma Bank) diluted 1:250 for 15 minutes. After one washing with PBS, a secondary antibody (cyanin 3-conjugated goat anti-mouse IgM; Jackson Immunoresearch Laboratories) diluted 1:800 was added for 15 minutes. Cells were then washed twice in PBS and resuspended in PBS for analysis.

Lentivirus Transduction of mES Cells

FUIPW-based Klf4 mutants or control vectors were used for transduction in the presence of 8 μg/ml polybrene to enhance transduction. Forty-eight hours later, 0.5 μg/ml of puromycin was added, and the medium was changed every day afterward. Six days later, cells were stained and SSEA1-positive cells were counted.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

Generation of Secondary iPS Cells Using Genetically Homogeneous Cells

Previous studies showed that the efficiency of direct reprogramming is generally low and variable [5]. To investigate the function of Klf4 and other factors in reprogramming events in detail, it was necessary to establish a high efficiency system with consistent readout. To do so, we set up a secondary iPS cell induction system using inducible expression of reprogramming factors (Fig. 1A). MEFs were transduced by lentivirus expressing Klf4, Oct4, and Sox2 whose expression was controlled by doxycycline induction, along with lentivirus expressing the doxycycline coactivator rtTA. After 2 weeks of induction in ES cell medium in the presence of doxycycline, doxycycline was removed and many colonies emerged. Stable first-generation iPS cell lines were isolated, characterized, and then differentiated in vitro to generate NSCs. After several passages under differentiation conditions, this NSC population became homogeneous, and second-generation iPS cells were induced by addition of doxycycline. In addition, first-generation iPS cells were injected into mouse blastocysts to generate chimeric mice in order to generate MEFs. When isolated, these MEFs can also be reprogrammed to generate second-generation iPS cells by addition of doxycycline.

thumbnail image

Figure 1. Establishment of an inducible reprogramming system using defined factors. (A): Schematic representation of the experimental design showing first-generation iPS cell derivation and secondary iPS cells generated from NSCs differentiated in vitro from iPS cells and MEFs from chimeric mice. iPS cells are generated by doxycycline-induced expression of Oct4, Sox2, c-Myc, and Klf4 in MEF cells using lentiviral transduction. MEFs are derived from embryonic day (E)-13.5 mouse embryos containing an Oct4-driven GFP transgene. The construct encoding rtTA for doxycycline induction expresses the puromycin resistance gene. Stable iPS cell lines are isolated, characterized, and then differentiated in vitro to generate NSCs. After several passages under differentiation conditions, this NSC population becomes homogeneous and second-generation iPS cells can be induced by addition of doxycycline. First-generation iPS cells are also injected into mouse blastocysts to generate chimeric mice. iPS cell-derived MEFs are isolated from E13.5 chimeric embryos using puromycin selection. These cells can form second-generation iPS cells after addition of doxycycline. (B): Pluripotency markers are expressed in (Oct4-GFP) iPS cell lines. iPS cell lines were positive for endogenous Oct4 as indicated by GFP expression, and also positive for AP and SSEA1 (Bars = 5 μm). (C): Hematoxylin and eosin staining of histological sections of a teratoma derived from iPS cells shows differentiation of iPS cells into cartilage (mesoderm), muscle (mesoderm), gut-like structures (endoderm), and neural epithelium (ectoderm). (D): Homogeneous NSCs derived from iPS cells can be reprogrammed to second-generation iPS cells at high efficiency following Dox treatment with or without VPA (n = 3; error bars indicate sd). NSCs derived from in vitro differentiation were seeded 5 × 104 cells/well. Doxycycline was added at 0.5 μg/ml or 1 μg/ml. Where indicated, VPA was added at a final concentration of 2 μM for 14 days. GFP expression is driven by the Oct4 promoter. The number of GFP-positive colonies was determined after 3 weeks of induction. Abbreviations: AP, alkaline phosphatase; Dox, doxycycline; GFP, green fluorescent protein; iPS, induced pluripotent stem; MEF, mouse embryonic fibroblast; SSEA1, stage-specific embryonic antigen 1; VPA, valproic acid.

Download figure to PowerPoint

To establish first-generation iPS cell lines, lentiviral vectors expressing Oct4, Sox2, Klf4, and c-Myc under doxycycline control (TRE promoter) (supporting information Fig. 1A), together with a constitutively active lentivirus expressing rtTA with a puromycin selection cassette (supporting information Fig. 1A), were transduced into MEFs isolated from E13.5 transgenic mouse embryos expressing GFP driven by the pluripotent cell-specific Oct4 promoter (Oct4-GFP) [29]. After 3 weeks of induction (supporting information Fig. 1B), ES cell-like, GFP-positive colonies emerged (supporting information Fig. 1C). Further characterization suggested that, in addition to Oct4 (as indicated by GFP expression), these iPS cell lines were positive for the pluripotency markers AP and SSEA1 (Fig. 1B). RT-PCR analysis confirmed expression of pluripotency-related genes in these lines (supporting information Fig. 1D). All lines were SSEA1 and AP positive, although some did not express endogenous Oct4 or Nanog, indicating that they may be partially reprogrammed. We chose lines most similar to ES cells in pluripotency gene expression for further experiments. To further examine pluripotency, we injected different iPS cell lines into SCID mice and evaluated teratoma formation. After 7-9 weeks, multiple lines developed into teratomas, which contained derivatives of all three germ layers (Fig. 1C). These lines, which contained transgenes encoding reprogramming factors as shown by PCR analysis (supporting information Fig. 1E), were injected into mouse blastocysts to generate chimeric mice, confirmed by PCR analysis (supporting information Fig. 1F).

To derive genetically homogeneous NSCs directly from first-generation iPS cells, embryoid bodies were generated from iPS cells (supporting information Fig. 2A), and cells were then further differentiated into neural progenitors. Oct4-dependent GFP expression disappeared at later stages of embryoid bodies, and no Oct4-driven GFP-positive cells were detectable after passages in monolayer NSC culture conditions (supporting information Fig. 2B), indicating that no pluripotent cells remained.

To derive second-generation iPS cells, NSCs derived from in vitro differentiation were plated at the same density in the presence of doxycycline in multiple wells with mES medium (supporting information Fig. 2C). Colonies emerged within a week, and mature Oct4-GFP-positive colonies appeared in 2 weeks (supporting information Fig. 2D). We calculated reprogramming efficiency by counting the number of GFP-positive colonies after 3 weeks (Fig. 1D). To optimize conditions favoring iPS cell induction, NSCs were treated with 0.5 μg/ml or 1 μg/ml doxycycline and with or without the histone deacetylase inhibitor valproic acid (VPA) [13]. iPS cell induction efficiency increased with both increased doxycycline concentration and VPA treatment (Fig. 1D). Quantification of GFP-positive colonies after 3 weeks indicated that the estimated reprogramming efficiency of NSCs was approximately 0.04% (without VPA) to 0.2% (with VPA), a frequency higher than that obtained by directly generating iPS cells from transduction of neural progenitors [33]. These iPS colonies also exhibited SSEA1 and AP expression (supporting information Fig. 2E).

To obtain MEFs specifically derived from first-generation iPS cells, we used chimeric mice generated from blastocysts injected with iPS cells. MEFs from E13.5 chimeric embryos were isolated and wild-type cells were eliminated by puromycin selection. After doxycycline treatment to induce iPS cells, Oct4-GFP-positive colonies started to appear within 2 weeks (data not shown). These iPS cells were also SSEA1 and AP positive (data not shown). These secondary iPS cells induced from NSCs or MEFs were used for the reprogramming assays reported below.

Klf4 Interacts Directly with Oct4 and Sox2

Klf4, Sox2, and Oct4 are critical for reprogramming, suggesting that they may function in a complex. To determine whether they physically interact, we first examined potential interactions in 293T cells. In 293T cells overexpressing Flag-tagged Klf4 and untagged Oct4, Oct4 was coimmunoprecipitated by an anti-Flag antibody (Fig. 2A). Alternative experiments in which antibodies were reversed showed that Klf4 was coimmunoprecipitated by an anti-Oct4 antibody (Fig. 2A), indicating that Klf4 binds to Oct4. Similar results were obtained analyzing Klf4 interaction with Sox2 (Fig. 2B). To confirm interaction of endogenous Oct4, Sox2, and Klf4 in iPS cells, we performed coimmunoprecipitations from lysates of iPS cells not treated with doxycycline. When Klf4 was immunoprecipitated, Oct4 was detected by Western blotting (Fig. 2C). Oct4 and Sox2 interaction was also demonstrated by immunoprecipitating Sox2 and blotting for Oct4 (Fig. 2D). Interaction between endogenous Klf4 and Sox2 in iPS cells was undetectable (data not shown), possibly due to either weak interactions or low sensitivity of the Sox2 antibody. To determine whether these interactions were unique to iPS cells, we performed the same experiments in mouse ES cells and detected similar interactions (Fig. 2E, 2F). These data indicate that Klf4 interacts with Oct4 and Sox2 in iPS and ES cells.

thumbnail image

Figure 2. Klf4 interacts with Oct4 and Sox2. (A, B): Klf4 interacts with Oct4 (A) and Sox2 (B) when overexpressed in 293T cells. Constructs encoding Flag-tagged Klf4 or untagged Oct4 were transfected into 293T cells alone or together. Cell lysates were immunoprecipitated using anti-Flag antibody followed by Western analysis with anti-Oct4 antibody. Klf4 and Oct4 expression in whole-cell lysates was determined by Western blot. (C–F): Endogenous Klf4 interacts with endogenous Oct4 in iPS (C) and embryonic stem (ES) (E) cells, and endogenous Oct4 and Sox2 interact with each other in iPS (D) and ES (F) cells. iPS cells were maintained and passaged without doxycycline. Abbreviations: FL, full-length; IB, immunoblot; IP, immunoprecipitation; iPS, induced pluripotent stem; mES, mouse embryonic stem.

Download figure to PowerPoint

The Klf4 C terminus contains three consecutive, highly conserved C2H2 zinc finger motifs, which are generally thought to mediate DNA binding [34, 35]. To investigate which domains of Klf4 are required for Oct4 and Sox2 interaction, we generated Klf4 deletion mutants, including those deleted in one or more zinc fingers (Fig. 3A). Interactions of these mutants with Oct4 and Sox2 were determined by coimmunoprecipitation. Comparison of mutants having truncations of the last one (Klf4ΔZF3), last two (Klf4ΔZF2-3), or all three (Klf4ΔZF1-3) zinc fingers indicated that deletion of all three zinc finger motifs reduced interaction of Klf4 with Oct4 (Fig. 3B), whereas deleting all three motifs completely abolished Klf4's interaction with Sox2 (Fig. 3C). Deletion of the two C-terminal zinc fingers (Klf4ΔZF2-3) resulted in an intermediate reduction in Sox2 interaction (Fig. 3C). Other mutants, such as Klf4ΔM, which lacks the middle of the protein but retains all three zinc fingers, did not alter interaction of Klf4 with Oct4 or Sox2 (Fig. 3B, 3C). These results indicate that the Klf4 C terminus is important for binding to Oct4/Sox2, and also that Oct4 and Sox2 may bind to different regions of Klf4. We also found that neither the Klf4 middle region nor the N terminus is sufficient for binding Oct4 and Sox2 in 293T cells (Fig. 3D, 3E), whereas Klf4's C terminus is sufficient for interaction with both Oct4 and Sox2. In addition, a recombinant GST fusion of the Klf4 C terminus directly bound to bacterially purified recombinant HA-tagged Oct4 and Sox2 in in vitro pull-down assays (Fig. 3F, 3G). Competitive binding assays performed to determine whether Oct4 and Sox2 bind to the same domain in Klf4 showed that increasing amounts of Sox2 protein did not alter Klf4 and Oct4 interaction, supporting the idea that Oct4 and Sox2 do not compete for the same Klf4 binding site (Fig. 3H).

thumbnail image

Figure 3. The Klf4 C terminus interacts directly with Oct4 and Sox2. (A): Design of Klf4 deletion mutants used in the experiments. (B): Interaction between Klf4 and Oct4 requires zinc finger motifs at the Klf4 C terminus. Deletion of all three zinc finger motifs (Klf4ΔZF1-3) significantly decreased Klf4/Oct4 interaction. Deletion of the C terminal or two last C-terminal zinc fingers (Klf4ΔZF3 and Klf4ΔZF2-3), or deletion of the middle region (Klf4ΔM), did not affect the interaction. The relative quantity of each band was measured and listed below each blot. (C): Klf4 and Sox2 interaction requires zinc finger motifs at the Klf4 C terminus. Deletion of all three Klf4 zinc fingers abolishes Klf4/Sox2 interaction, whereas deleting the last two zinc fingers significantly reduces the interaction. The relative quantity of each band was measured and reported below each blot. (D, E): The Klf4 C terminus interacts with Sox2 (D) and Oct4 (E) when overexpressed in 293T cells, whereas the Klf4 N terminus or middle region alone does not. (F, G): Recombinant GST-Klf4 (300-483) interacts with bacterially purified recombinant Oct4 (F) and Sox2 (G) in vitro. Asterisks indicate intact GST-Klf4 (300-483). (H): Oct4 and Sox2 do not compete for interaction with Klf4. Klf4/Oct4 interaction was not disrupted by increasing Sox2 expression. (I): Klf4 and Oct4 co-occupy the Nanog promoter, shown schematically above, in induced pluripotent stem cells. Cross-linked chromatin was first immunoprecipitated with Oct4 antibody and then with a control IgG or anti-Klf4 antibody. The precipitated DNA was amplified by polymerase chain reaction, normalized by control IgG, and then normalized by the first pair of primers. Results indicate that Oct4 and Klf4 co-occupy the Nanog proximal promoter. Abbreviations: C, C terminus; FL, full-length; GST, glutathione S-transferase; IB, immunoblot; IP, immunoprecipitation; M, middle region; N, N terminus; WB, Western blot; WT, wild type; ZF, zinc finger.

Download figure to PowerPoint

We next asked whether a Klf4, Oct4, and Sox2 complex co-occupies a candidate promoter. Sequential ChIP using Oct4 antibody followed by Klf4 antibody plus PCR analysis using six primer pairs spanning approximately 1.5 kb of the Nanog proximal promoter [36] was performed in iPS cells (Fig. 3I). The results suggested that Klf4 and Oct4 co-occupy the same region of the Nanog promoter.

Klf4 Mutants Compete with Wild-Type Klf4 To Interact with Oct4 and Sox2 and Significantly Reduce Reprogramming Efficiency

We hypothesize that Klf4 recruits Oct4 and Sox2 through direct interaction and activates downstream targets required for reprogramming. Since different zinc finger deletions of the Klf4 C terminus alter its interaction affinities for Oct4 and Sox2, mutants interacting with Oct4 and Sox2 but lacking transcriptional activation capacity should be able to compete with wild-type Klf4 and serve as dominant negative constructs when introduced into an inducible reprogramming system. To evaluate transactivation potential of Klf4 mutants, we used the Nanog proximal promoter in a reporter assay and found that Klf4 activates the Nanog promoter (Fig. 4A). None of the C-terminal zinc finger deletion mutants, including Klf4ΔZF3, Klf4ΔZF2-3, or Klf4ΔZF1-3, activated the promoter, indicating that zinc fingers are critical for target gene activation (Fig. 4B). Furthermore, EMSA showed that all zinc finger deletion mutants lacked DNA binding ability (Fig. 4C), confirming that the zinc fingers are critical for DNA binding.

thumbnail image

Figure 4. Dominant negative Klf4 mutants compete with wild-type Klf4 for binding with Oct4 and Sox2, resulting in disruption of reprogramming. (A): Klf4 can activate the Nanog luciferase reporter in a dose-dependent manner. (B): Deletion of C-terminal zinc finger motifs abolishes Klf4's transcriptional activation capacity. Compared with wild-type Klf4, deleting the last (Klf4ΔZF3), the last two (Klf4ΔZF2-3), or all three zinc finger motifs (Klf4ΔZF1-3) significantly decreased Nanog-luciferase activity. (C): Electrophoretic mobility shift assay shows that only wild-type Klf4 binds DNA, whereas deletion of the last, the last two, or all three zinc fingers abolishes DNA binding capability. (D, E): The dominant negative mutant (Klf4ΔZF3) inhibits interaction of wild-type Klf4 with Oct4 (D) or Sox2 (E) by competing with wild-type Klf4. However, Klf4ΔZF1-3, which exhibits low binding affinity for Oct4 or Sox2, does not compete with wild-type Klf4 to bind Oct4 or Sox2. Cell lysates were immunoprecipitated using anti-HA antibody followed by immunoblotting with anti-HA, Myc, or Flag antibodies. The relative quantity of each band was measured and reported below each blot. (F): Dominant negative Klf4 mutants show significantly reduced reprogramming capacity (n = 3; error bars indicate sd; **, p < .01) in genetically homogeneous secondary NSCs. In vitro differentiated NSCs were transduced with lentivirus expressing Klf4 mutants and induced with doxycycline. The number of GFP-positive colonies was determined after 3 weeks of doxycycline induction. Klf4ΔZF3 and Klf4ΔZF2-3 strongly inhibited reprogramming, whereas Klf4ΔZF1-3 did not. (G): Fluorescence-activated cell sorting (FACS) analysis of the stage-specific embryonic antigen 1 (SSEA1)-positive (cyanin-3 labeled) cells in experiments described in (F). Samples were collected at day 21. Numbers indicate percentages of SSEA1-positive cells. (H): Klf4 mutants inhibit reprogramming in secondary mouse embryonic fibroblast (MEFs) in a manner similar to NSCs. Secondary MEFs (5 × 104) from chimeric mice were plated per well. Puromycin was used at 0.5 μg/ml for 3 days before transduction with new virus. The total number of GFP-positive colonies in triplicate wells was determined after 12 days of doxycycline induction (n = 3; **, p < .01). Similarly, Klf4ΔZF3 and Klf4ΔZF2-3 strongly inhibited reprogramming, whereas Klf4ΔZF1-3 did not. (I): FACS analysis of the SSEA1-positive population in sample from (H). Numbers indicate percentages of SSEA1-positive cells. Abbreviations: GFP, green fluorescent protein; IB, immunoblot; IP, immunoprecipitation; WT, wild type; ZF, zinc finger.

Download figure to PowerPoint

To further investigate competition between wild-type and mutant forms of Klf4 in interacting with Oct4 and Sox2, we performed a competition assay between wild-type Klf4 and mutants lacking only the last zinc finger (Klf4ΔZF3), or all three zinc fingers (Klf4ΔZF1-3), respectively. Constructs encoding either of these Flag-tagged Klf4 mutants were cotransfected with constructs encoding Myc-tagged wild-type Klf4 and HA-tagged Oct4, and the amount of Flag-tagged mutant Klf4 and Myc-tagged wild-type Klf4 associated with HA-tagged Oct4 was determined by immunoprecipitation of Oct4, followed by immunoblotting with anti-Flag and anti-Myc antibodies. In cells expressing Klf4ΔZF3, Flag-tagged Klf4ΔZF3 directly bound to Oct4, whereas the interaction between Myc-tagged wild-type Klf4 and Oct4 was significantly reduced (Fig. 4D), suggesting that Klf4ΔZF3 significantly competes with wild-type Klf4. As a control, Klf4ΔZF1-3 did not compete with wild-type Klf4 in binding to Oct4 (Fig. 4D). Similar results were obtained in analyses of Klf4 and Sox2 interactions (Fig. 4E). These results suggest that KlfΔZF3 functions as a dominant negative mutant in competing with wild-type Klf4 to form complexes with Oct4 and Sox2.

With a dominant negative form of Klf4 (Klf4ΔZF3) in hand, we determined whether interaction of Klf4, Sox2, and Oct4 was critical for iPS cell induction. The same inducible lentiviral system was used to introduce Flag-tagged mutant or wild-type forms of Klf4 into iPS cell-derived second-generation homogeneous NSCs or MEFs described above. High-titer viruses were generated to ensure transduction efficiency of all NSCs or MEFs, and equivalent transduction efficiency in different samples was confirmed by Flag immunostaining (supporting information Fig. 3). Thus, differences in reprogramming efficiency observed among various samples were assumed to be due primarily to different activities of overexpressed products. Cells from the same passage NSCs or MEFs were split and plated at the same density. Klf4ΔZF3- and Klf4ΔZF2-3-expressing cells showed significantly inhibited reprogramming efficiency: the numbers of Oct4-GFP-positive colonies and percentage of SSEA1-positive cells in these samples were significantly reduced compared with cells overexpressing wild-type Klf4 (Fig. 4F, 4G). To exclude the possibility that this effect was limited to NSCs, MEFs derived from chimeric mice were similarly analyzed. Although reprogramming efficiency in MEFs was significantly lower than in NSCs, we observed a similar inhibition pattern in MEFs as in NSCs (Fig. 4H, 4I). As Klf4ΔZF3 or Klf4ΔZF2-3 interact with Oct4 and Sox2 in a manner similar to wild-type Klf4, we conclude that these mutants compete with wild-type Klf4 to form complexes that cannot activate transcription, possibly due to the mutant Klf4's inability to bind DNA. Klf4ΔZF1-3, however, did not inhibit reprogramming, as the number of GFP-positive colonies and the proportion of SSEA1-positive cells were similar to those observed in controls (Fig. 4F–4I). This finding is consistent with the observation that Klf4ΔZF1-3 cannot interact with Sox2 and only poorly interacts with Oct4. Thus, there was no competition between Klf4ΔZF1-3 and wild-type Klf4 in forming an Oct4/Sox2/Klf4 complex.

Endogenous Klf4 in MEF Is Required for Reprogramming

Although Klf4 has been shown to be important for reprogramming, protocols have been developed to induce iPS cells from mouse or human fibroblasts in the absence of exogenous Klf4 by expressing Oct4 and Sox2 combined with chemicals such as VPA [13, 37–39]. However, it has been shown that endogenous Klf4 is expressed in MEFs [39, 40]. Thus it is critical to determine whether endogenous Klf4 is present in an Oct4/Sox2/Klf4 complex during reprogramming in these protocols. To address this question, we used short hairpin RNA (shRNA)-mediated knockdown to determine whether endogenous Klf4 is required for iPS cell induction from wild-type MEFs using only overexpressed Oct4 and Sox2, as described before [13]. Treatment of MEFs with Klf4 shRNA prior to initiation of reprogramming with only Oct4 and Sox2 significantly decreased the number of AP-positive colonies compared with controls (Fig. 5A, 5B), indicating that endogenous Klf4 is required for reprogramming.

thumbnail image

Figure 5. Endogenous Klf4 is critical for reprogramming and embryonic stem (ES) cell self-renewal. (A): Knockdown of endogenous Klf4 results in decreased numbers of AP-positive colonies compared with controls. Primary mouse embryonic fibroblasts (MEFs; 1 × 105) were seeded in 1 well of 6-well plates and transduced with lentivirus expressing Oct4 and Sox2, plus either Klf4shRNA (right) or a control scramble shRNA vector (left). Valproic acid (VPA) was added to both at 0.5 μM. AP staining was performed 2 weeks after induction of transgenes expression. (B): Quantification of AP-positive colonies in (A) (n = 3 independent experiments; **, p < .01). (C): Primary MEFs overexpressing Klf4ΔZF3 fail to produce AP-positive cells, whereas overexpression of Klf4ΔZF1-3 has no significant effect compared with controls. MEFs (2 × 105) were seeded in 60-mm dishes. MEFs were transduced by lentivirus expressing Oct4 and Sox2, along with indicated Klf4 mutants. Control samples were transduced with empty vector. VPA was added to all samples at 0.5 μM. AP staining was performed 2 weeks after induction of transgene expression. (D): Quantification of AP-positive colonies in (C) (n = 3 independent experiments; **, p < .01). (E): Overexpression of Klf4ΔZF3 inhibits normal self-renewal of wild-type mouse ES cells, whereas a substantial proportion of cells overexpressing Klf4ΔZF1-3 remain SSEA1 positive. Wild-type mouse ES cells were transduced with lentiviral vectors containing indicated Klf4 mutant constructs followed by an internal ribosome entry site and a puromycin resistance gene. Empty vector served as a control. Cells were selected in puromycin for 6 days and then stained for SSEA1 (red) and 4′,6-diamidino-2-phenylindole (blue). Bars = 10 μm. (F): Quantification of the percentage of SSEA1-positive cells in (E) revealed a statistically significant decrease (n = 3 independent experiments; *, p < .05; **, p < .01) in Klf4ΔZF3-overexpressing cells compared with both control cells and cells overexpressing Klf4ΔZF1-3. Abbreviations: AP, alkaline phosphatase; shRNA, short hairpin RNA; SSEA1, stage-specific embryonic antigen 1; WT, wild type, ZF, zinc finger.

Download figure to PowerPoint

To further determine whether endogenous Klf4 facilitates reprogramming by forming a complex with Oct4 and Sox2, we expressed the dominant negative forms of Klf4 described above together with Oct4 and Sox2 in primary MEFs using a similar protocol as described before [13]. When dominant negative mutant Klf4ΔZF3 was overexpressed, the number of AP-positive colonies was significantly reduced compared with controls treated with either empty vector or with the Klf4ΔZF1-3 construct, which does not function as a dominant negative mutant (Fig. 5C, 5D). These results strongly suggest that endogenous levels of Klf4 are sufficient to form a complex with exogenous Oct4 and Sox2 during reprogramming and that inclusion of Klf4 in this complex is required for efficient iPS cell induction.

An Oct4/Sox2/Klf4 Complex Is Required for Self-Renewal in Wild-Type Mouse ES Cells

A requirement for Klf4 in maintaining ES cell self-renewal has been investigated [17]. Since our findings indicate that Klf4/Oct4/Sox2 complex formation is required for somatic cell reprogramming, we asked whether this complex is also required for ES cell self-renewal. To address this question, we transduced lentiviral constructs expressing the Klf4ΔZF3 dominant negative mutant or control constructs plus a puromycin resistance gene into mouse ES cells. After 6 days of selection, we immunostained surviving cells with SSEA1 antibody and counted SSEA1-positive cells (Fig. 5E). Overexpression of Klf4ΔZF3 resulted in differentiation and almost complete loss of SSEA1-positive cells (Fig. 5E, 5F). By contrast, only a small proportion of cells overexpressing Klf4ΔZF1-3, which does not function as a dominant negative mutant, differentiated (Fig. 5E, 5F). Collectively, these data indicate that disruption of the wild-type Oct4/Sox2/Klf4 complex by inclusion of a dominant negative form of Klf4 interferes with normal self-renewal of ES cells.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

Klf4 has been suggested to play an important role in ES cell self-renewal [17]. Its target genes overlap with those of Oct4, Sox2, and Nanog. Previous studies indicate that Oct4 and Sox2 colocalize on target gene promoters [36, 41]. Here, we found that Klf4 interacts directly with Oct4 and Sox2 through its C terminus, which contains three zinc finger motifs. Oct4 and Sox2 do not compete for binding to Klf4, indicating that each likely binds to a different site. This result was confirmed by the fact that loss of two zinc finger motifs significantly decreased Klf4 binding affinity for Sox2, whereas the same mutant did not differ from wild-type Klf4 in terms of Oct4 interaction. We hypothesize that a complex containing Klf4, Oct4, and Sox2 activates downstream targets required for reprogramming. Interestingly, Klf4 alone can activate the Nanog promoter in 293T cells in a transfection assay, whereas Oct4 or Sox2 alone cannot (data not shown), suggesting that Klf4 may function as the transactivator in the complex.

To analyze Klf4's function in reprogramming, we analyzed the ability of mutant forms of Klf4 to induce reprogramming. We found that Klf4ΔZF3 and KlfΔZF2-3, which interact with Oct4 and Sox2 but lack DNA binding activity, significantly inhibit normal reprogramming. However, Klf4ΔZF1-3, which lacks all three zinc fingers, fails to suppress reprogramming. The striking differences between Klf4ΔZF1-3 and Klf4ΔZF3/Klf4ΔZF2-3 activity indicate that Klf4's binding with Oct4 and Sox2, which is disrupted by deletion of the three zinc finger motifs, is critical for reprogramming.

Only recently has the mechanism underlying direct reprogramming begun to be understood [19, 42]. Profiling of Oct4, Sox2, and Klf4's DNA binding in the whole genome in both ES cells and iPS cells suggests that these three factors colocalize on promoters of essential pluripotency genes [17, 19]. However, whether this colocalization reflects active recruitment and assembly of a specific functional protein complex remains elusive. In our model, a complex of Klf4, Oct4, and Sox2, which is assembled via direct interaction, is likely required to activate transcription of critical pluripotency genes, such as Nanog. The requirement for complex formation may also play a regulatory role: not only the presence but also the stoichiometry of these factors may determine whether a sufficient level of complexes is available to reprogram a single cell. The recent finding that reprogramming is more efficient when factors are expressed on a polycistronic vector [10, 43] supports this idea. Such design may guarantee more efficient colocalization of these factors immediately after translation or ensure that equivalent amounts of each factor are produced. These conditions could facilitate complex formation and optimize reprogramming efficiency.

ChIP-on-Chip studies indicate that common targets of Klf4, Oct4, and Sox2 are most differentially bound between fully reprogrammed and partially reprogrammed iPS cells, compared with other promoters bound by only one or two of them [19]. This interesting phenomenon suggests that binding of target promoters by Oct4, Sox2, and Klf4 may not be through individual binding but may require assembly of a functional complex containing all three factors. The observation that these three proteins are abundant (when expressed ectopically) even in partially reprogrammed cells suggests that a fully functional complex may require interacting partners, particularly factors that can antagonize repressive chromatin structures in key target genes. Identifying those partners is critical for further investigation.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

Altogether, our results demonstrate that direct interactions among the reprogramming factors Klf4, Oct4, and Sox2 are required for success of reprogramming. Klf4 interacts directly with Oct4 and Sox2 and they co-occupy the Nanog promoter. When formation of the complex is disrupted by introducing dominant negative forms of Klf4 or by shRNA-mediated knockdown, reprogramming efficiency is significantly reduced. Further studies of this interaction should facilitate our understanding of reprogramming mechanisms.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

We thank Dr. D. Johnson for critical reading of the manuscript, H. Ng and X. Chen for generous sharing of the Klf4 antibody, P. Robson for Nanog-luciferase constructs, C. Cunningham for FACS analysis, the University of Southern California (USC) Stem Cell Core Facility for providing feeder cells, and the USC Transgenic Core Facility for generating chimeric embryos. Z.W. and Y.Y. contributed equally to this work.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information
  • 1
    Jaenisch R, Young R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 2008; 132: 567582.
  • 2
    Hochedlinger K, Plath K. Epigenetic reprogramming and induced pluripotency. Development 2009; 136: 509523.
  • 3
    Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126: 663676.
  • 4
    Wernig M, Lengner CJ, Hanna J et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat Biotechnol 2008; 26: 916924.
  • 5
    Feng B, Ng JH, Heng JC et al. Molecules that promote or enhance reprogramming of somatic cells to induced pluripotent stem cells. Cell Stem Cell 2009; 4: 301312.
  • 6
    Nakagawa M, Koyanagi M, Tanabe K et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 2008; 26: 101106.
  • 7
    Brambrink T, Foreman R, Welstead GG et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2008; 2: 151159.
  • 8
    Kaji K, Norrby K, Paca A et al. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 2009; 458: 771775.
  • 9
    Stadtfeld M, Nagaya M, Utikal J et al. Induced pluripotent stem cells generated without viral integration. Science 2008; 322: 945949.
  • 10
    Okita K, Nakagawa M, Hyenjong H et al. Generation of mouse induced pluripotent stem cells without viral vectors. Science 2008; 322: 949953.
  • 11
    Woltjen K, Michael IP, Mohseni P et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 2009; 458: 766770.
  • 12
    Zhou H, Wu S, Joo JY et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 2009; 4: 381384.
  • 13
    Huangfu D, Osafune K, Maehr R et al. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol 2008; 26: 12691275.
  • 14
    Shi Y, Desponts C, Do JT et al. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 2008; 3: 568574.
  • 15
    Marson A, Foreman R, Chevalier B et al. Wnt signaling promotes reprogramming of somatic cells to pluripotency. Cell Stem Cell 2008; 3: 132135.
  • 16
    Evans PM, Zhang W, Chen X et al. Kruppel-like factor 4 is acetylated by p300 and regulates gene transcription via modulation of histone acetylation. J Biol Chem 2007; 282: 3399434002.
  • 17
    Jiang J, Chan YS, Loh YH et al. A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat Cell Biol 2008; 10: 353360.
  • 18
    Nakatake Y, Fukui N, Iwamatsu Y et al. Klf4 cooperates with Oct3/4 and Sox2 to activate the Lefty1 core promoter in embryonic stem cells. Mol Cell Biol 2006; 26: 77727782.
  • 19
    Sridharan R, Tchieu J, Mason MJ et al. Role of the murine reprogramming factors in the induction of pluripotency. Cell 2009; 136: 364377.
  • 20
    Guo G, Yang J, Nichols J et al. Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 2009; 136: 10631069.
  • 21
    Hanna J, Markoulaki S, Mitalipov SM et al. Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 2009; 4: 513524.
  • 22
    Philipsen S, Suske G. A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res 1999; 27: 29913000.
  • 23
    Merika M, Orkin SH. Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the Kruppel family proteins Sp1 and EKLF. Mol Cell Biol 1995; 15: 24372447.
  • 24
    Wolfe SA, Nekludova L, Pabo CO. DNA recognition by Cys2His2 zinc finger proteins. Ann Rev Biophys Biomol Struct 2000; 29: 183212.
  • 25
    Urlinger S, Baron U, Thellmann M et al. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci U S A 2000; 97: 79637968.
  • 26
    Lois C, Hong EJ, Pease S et al. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 2002; 295: 868872.
  • 27
    Lyu J, Yamamoto V, Lu W. Cleavage of the Wnt receptor Ryk regulates neuronal differentiation during cortical neurogenesis. Dev Cell 2008; 15: 773780.
  • 28
    Takahashi K, Okita K, Nakagawa M et al. Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc 2007; 2: 30813089.
  • 29
    Szab´o PE, Hubner K, Scholer H et al. Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev 2002; 115: 157160.
  • 30
    Harper S and Speicher DW. Expression and purification of GST fusion proteins. Curr Protoc Protein Sci 2008; Chapter 6:Unit 6 6.
  • 31
    Lu W, Yamamoto V, Ortega B et al. Mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth. Cell 2004; 119: 97108.
  • 32
    Wells J, Farnham PJ. Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation. Methods 2002; 26: 4856.
  • 33
    Eminli S, Utikal JS, Arnold K et al. Reprogramming of neural progenitor cells into iPS cells in the absence of exogenous Sox2 expression. Stem Cells 2008; 26: 24672474.
  • 34
    Mahatan CS, Kaestner KH, Geiman DE et al. Characterization of the structure and regulation of the murine gene encoding gut-enriched Kruppel-like factor (Kruppel-like factor 4). Nucleic Acids Res 1999; 27: 45624569.
  • 35
    Xie D, Cai J, Chia NY et al. Cross-species de novo identification of cis-regulatory modules with GibbsModule: application to gene regulation in embryonic stem cells. Genome Res 2008; 18: 13251335.
  • 36
    Rodda DJ, Chew JL, Lim LH et al. Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem 2005; 280: 2473124737.
  • 37
    Feng B, Jiang J, Kraus P et al. Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb. Nat Cell Biol 2009; 11: 197203.
  • 38
    Kim JB, Sebastiano V, Wu G et al. Oct4-induced pluripotency in adult neural stem cells. Cell 2009; 136: 411419.
  • 39
    Lyssiotis CA, Foreman RK, Staerk J et al. Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4. Proc Natl Acac Sci U S A 2009; 106: 89128917.
  • 40
    Rowland BD, Bernards R, Peeper DS. The KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene. Nat Cell Biol 2005; 7: 10741082.
  • 41
    Wang J, Rao S, Chu J et al. A protein interaction network for pluripotency of embryonic stem cells. Nature 2006; 444: 364368.
  • 42
    Maherali N, Sridharan R, Xie W et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 2007; 1: 5570.
  • 43
    Yu J, Hu K, Smuga-Otto K et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science 2009; 324: 797801.

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. References
  11. Supporting Information

Additional supporting information available online.

FilenameFormatSizeDescription
STEM_231_sm_suppinfo.pdf648KSupporting Information

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.