Klf4 interacts directly with Oct4 and Sox2 to promote reprogramming.
ABSTRACT 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.
- SourceAvailable from: Yi-Ching Wang[Show abstract] [Hide abstract]
ABSTRACT: Overexpression of Oct4, a stemness gene encoding a transcription factor, has been reported in several cancers. However, the mechanism by which Oct4 directs transcriptional program that leads to somatic cancer progression remains unclear. In this study, we provide mechanistic insight into Oct4-driven transcriptional network promoting drug-resistance and metastasis in lung cancer cell, animal and clinical studies. Through an integrative approach combining our Oct4 chromatin-immunoprecipitation sequencing and ENCODE datasets, we identified the genome-wide binding regions of Oct4 in lung cancer at promoter and enhancer of numerous genes involved in critical pathways which promote tumorigenesis. Notably, PTEN and TNC were previously undefined targets of Oct4. In addition, novel Oct4-binding motifs were found to overlap with DNA elements for Sp1 transcription factor. We provided evidence that Oct4 suppressed PTEN in an Sp1-dependent manner by recruitment of HDAC1/2, leading to activation of AKT signaling and drug-resistance. In contrast, Oct4 transactivated TNC independent of Sp1 and resulted in cancer metastasis. Clinically, lung cancer patients with Oct4 high, PTEN low and TNC high expression profile significantly correlated with poor disease-free survival. Our study reveals a critical Oct4-driven transcriptional program that promotes lung cancer progression, illustrating the therapeutic potential of targeting Oc4 transcriptionally regulated genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.Nucleic Acids Research 01/2015; · 8.81 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Induced pluripotent stem (iPS) cells can be generated from somatic cells by coexpression of four transcription factors: Sox2, Oct4, Klf4, and c-Myc. However, the low efficiency in generating iPS cells and the tendency of tumorigenesis hinder the therapeutic applications for iPS cells in treatment of human diseases. To this end, it remains largely unknown how the iPS process is subjected to regulation by upstream signaling pathway(s). Here, we report that Akt regulates the iPS process by modulating posttranslational modifications of these iPS factors in both direct and indirect manners. Specifically, Akt directly phosphorylates Oct4 to modulate the Oct4/Sox2 heterodimer formation. Furthermore, Akt either facilitates the p300-mediated acetylation of Oct4, Sox2, and Klf4, or stabilizes Klf4 by inactivating GSK3, thus indirectly modulating stemness. As tumorigenesis shares possible common features and mechanisms with iPS, our study suggests that Akt inhibition might serve as a cancer therapeutic approach to target cancer stem cells.Cancer Medicine 07/2014; 3(5).
- [Show abstract] [Hide abstract]
ABSTRACT: Interactions among transcriptional factors (TFs), cofactors and other proteins or enzymes can affect transcriptional regulatory capabilities of eukaryotic organisms. Post-translational modifications (PTMs) cooperate with TFs and epigenetic alterations to constitute a hierarchical complexity in transcriptional gene regulation. While clearly implicated in biological processes, our understanding of these complex regulatory mechanisms is still limited and incomplete. Various online software have been proposed for uncovering transcriptional and epigenetic regulatory networks, however, there is a lack of effective web-based software capable of constructing underlying interactive organizations between post-translational and transcriptional regulatory components. Here, we present an open web server, post-translational hierarchical gene regulatory network (PTHGRN) to unravel relationships among PTMs, TFs, epigenetic modifications and gene expression. PTHGRN utilizes a graphical Gaussian model with partial least squares regression-based methodology, and is able to integrate protein-protein interactions, ChIP-seq and gene expression data and to capture essential regulation features behind high-throughput data. The server provides an integrative platform for users to analyze ready-to-use public high-throughput Omics resources or upload their own data for systems biology study. Users can choose various parameters in the method, build network topologies of interests and dissect their associations with biological functions. Application of the software to stem cell and breast cancer demonstrates that it is an effective tool for understanding regulatory mechanisms in biological complex systems. PTHGRN web server is publically available at web site http://www.byanbioinfo.org/pthgrn.Nucleic Acids Research 05/2014; · 8.81 Impact Factor
EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS
Klf4 Interacts Directly with Oct4 and Sox2 to Promote
ZONG WEI,a,b,cYANG YANG,a,b,ePEILIN ZHANG,a,bROSEMARY ANDRIANAKOS,a,bKOUICHI HASEGAWA,a,d
JUNGMOOK LYU,a,bXI CHEN,fGANG BAI,eCHUNMING LIU,fMARTIN PERA,a,dWANGE LUa,b,c
aEli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research,bDepartment of Biochemistry
and Molecular Biology,cGraduate Program in Genetics, Molecular and Cellular Biology, anddDepartment of Cell
and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA;
eCollege of Life Sciences, Nankai University, Tianjin, People’s Republic of China;fMarkey Cancer Center,
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky, USA
Key Words. Induced pluripotent stem cells•Neural stem cells•Reprogramming•Transcriptional factors
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. En-
dogenous 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 endoge-
nous Klf4 with Oct4/Sox2 is also required for reprogram-
ming. 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
Disclosure of potential conflicts of interest is found at the end of this article.
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 . Novel methods requir-
ing no viral integration have also been developed [8–12], as
have strategies using small molecules to promote reprogram-
ming 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 . RNA interference experiments con-
firm that Klf4 is redundant with two other family members,
Klf2 and Klf5, in regulating expression of pluripotency-
related genes . In embryonic stem (ES) cells, Klf4 has
been shown to be important to activate Lefty1 together with
Oct4 and Sox2 . Genome-wide chromatin immunoprecipi-
tation 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 estab-
lishment of iPS cells, suggesting transcriptional synergy
among these factors . Furthermore, studies also suggest
that Klf4 may function in establishing an ‘‘authentic’’ and
‘‘metastable’’ pluripotent state in various pluripotent cell types
The fact that other Klf family members can substitute for
Klf4 in reprogramming  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 . Interactions
between Klf1 (EKLF) and GATA-1 suggest that Klf family
Author contributions: Z.W.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing;
Y.Y.: collection and assembly of data, data analysis and interpretation; P.Z., R.A., and K.H.: collection and assembly of data; J.L., X.C.,
G.B., and C.L.: provision of study material; M.P.: data analysis and interpretation; W.L.: conception and design, financial support, data
analysis and interpretation, manuscript writing, final approval of manuscript.
Correspondence: Wange Lu, PhD, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Department of
Biochemistry and Molecular Biology, Graduate Program in Genetics, Molecular and Cellular Biology, Department of Cell and Neurobiology,
Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA. Telephone: (þ1) 323-442-1618; Fax:
(þ1) 323-442-4040; e-mail: firstname.lastname@example.org
online in STEM CELLS EXPRESS October 8, 2009. V
STEM CELLS 2009;27:2969–2978 www.StemCells.com
Received June 9, 2009; accepted for publication September 21, 2009; first published
C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.231
C2H2 zinc fingers can also bind other transcriptional partners
, which may be functionally conserved among Klf family
members . However, little is known about potential inter-
action partners of Klf4 and the significance of these interac-
tions 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 spe-
cific Klf4 mutants can compete with either overexpressed or
endogenous wild-type (WT) Klf4 to form transcriptionally de-
fective complexes with Oct4 and Sox2, inhibiting reprogram-
ming efficiency. These results indicate that Oct4, Sox2, and
Klf4 function via direct interaction to regulate downstream
targets and facilitate reprogramming.
MATERIALS AND METHODS
To generate the doxycycline-inducible viral expression vector, the
digested rtTA2S-M2 fragment  was inserted into the vector
FUIPW, containing an internal ribosomal entry site followed by
the puromycin resistance gene. The ubiquitin promoter of FUW
 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 .
Cell Culture, Lentivirus Preparation, iPS Cell
Generation, and In Vitro Differentiation
293T cells were maintained in Dulbecco’s modified Eagle’s me-
dium (Cellgro; Mediatech, Inc., Manassas, VA, http://www.cell-
gro.com/shop/customer/home.php) containing 10% fetal bovine
serum (FBS). FTRE-based lentiviruses were generated in 293T
cells as described previously . 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 . Briefly, primary MEFs
were generated from embryonic day (E)-13.5 mouse embryos car-
rying the green fluorescent protein (GFP) transgene under control
of the Oct4 promoter . MEFs (6 ? 105) were seeded in 100-
mm dishes and transduced twice with a cocktail of five lentivi-
ruses, including those expressing the four reprogramming factors
plus rtTA. Mouse ES medium (Glasgow minimum essential me-
dium with 15% FBS, 2 mM glutamine, 0.1 mM b-mercaptoetha-
nol, 1% nonessential amino acids, 1% sodium pyruvate, leuke-
mia-inhibitory factor [LIF] at 10 ng/ml) plus 0.1 lg/ml of
doxycycline was added after 2 days and changed every day after-
ward. Three weeks later, mature iPS colonies were isolated by
manual cutting, and individual lines were maintained and
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 differentia-
tion with a change of medium every 2 days to form neurospheres
(NSs). NSs were collected and treated with 500 ll of 0.05% tryp-
sin 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
Total RNA was isolated from iPS cells using an RNAeasy mini
kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). Two lg
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 .
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 em-
bryonic antigen 1 (SSEA1) primary antibody (1:400; Develop-
mental Studies Hybridoma Bank, Iowa City, IA, http://dshb.biolo-
gy.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.jack-
sonimmuno.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 ? 107cells per milliliter in PBS.
Cell suspension (100 ll) 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 . Antibodies used
(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.rndsys-
tems.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 . 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
Luciferase reporter assays were performed as described .
Interaction Between Reprogramming Factors
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 , with
For electrophoretic mobility shift assay (EMSA), Klf-binding
 oligonucleotides were synthesized, labeled with IRD800 dye
(Integrated DNA Technologies, Coralville, IA, http://www.idtd-
na.com), and annealed at 5 mM. For DNA binding reactions, 1 ll
of DNA was added to a 10-ll reaction containing 1 ll of nuclear
extract, 2 lg of salmon sperm DNA (Gibco, Grand Island, NY,
http://www.invitrogen.com), and 1 ll 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, http://www.licor.com).
ChIP assays with mouse ES cells (or iPS cells) were carried out
as described . For all ChIP experiments, relative occupancy
values were calculated by determining the apparent immunopreci-
pitate 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
Reprogramming Efficiency Assay
To perform a secondary generation iPS cell efficiency assay, 3 ?
105NSCs 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 me-
dium was again changed to mouse ES cell medium and doxycy-
cline added to 1 lg/ml. Medium was changed every day and sup-
plemented with doxycycline throughout the induction period.
Fluorescence-activated cell sorting (FACS) analysis was per-
formed 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 ? 105or
1 ? 105primary MEFs isolated from Oct4-GFP mice were
seeded in 60-mm dishes or in 1 well of a 6-well plate, respec-
tively, and transduced with the combinations of lentiviruses indi-
cated in the text. Alkaline phosphatase staining was performed 2
weeks after transduction.
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 wash-
ing 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 lg/ml polybrene to enhance
transduction. Forty-eight hours later, 0.5 lg/ml of puromycin was
added, and the medium was changed every day afterward. Six
days later, cells were stained and SSEA1-positive cells were
Generation of Secondary iPS Cells Using Genetically
Previous studies showed that the efficiency of direct reprog-
ramming is generally low and variable . 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 reprog-
ramming factors (Fig. 1A). MEFs were transduced by lentivi-
rus 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-genera-
tion 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.
To establish first-generation iPS cell lines, lentiviral vec-
tors expressing Oct4, Sox2, Klf4, and c-Myc under doxycy-
cline control (TRE promoter) (supporting information Fig.
1A), together with a constitutively active lentivirus expressing
rtTA with a puromycin selection cassette (supporting informa-
tion Fig. 1A), were transduced into MEFs isolated from E13.5
transgenic mouse embryos expressing GFP driven by the plu-
ripotent cell-specific Oct4 promoter (Oct4-GFP) . 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 pluripo-
tency-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 tera-
toma 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 pluripo-
tent 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
Wei, Yang, Zhang et al.
(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 condi-
tions favoring iPS cell induction, NSCs were treated with 0.5
lg/ml or 1 lg/ml doxycycline and with or without the histone
deacetylase inhibitor valproic acid (VPA) . iPS cell induc-
tion efficiency increased with both increased doxycycline con-
centration and VPA treatment (Fig. 1D). Quantification of
GFP-positive colonies after 3 weeks indicated that the esti-
mated 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 . These iPS colo-
nies also exhibited SSEA1 and AP expression (supporting in-
formation 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 puromy-
cin 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
Klf4 Interacts Directly with Oct4 and Sox2
Klf4, Sox2, and Oct4 are critical for reprogramming, suggest-
ing 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 coimmunoprecipi-
tated by an anti-Flag antibody (Fig. 2A). Alternative experi-
ments 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
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 transduc-
tion. 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 af-
ter 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 ¼ 5 lm). (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 ¼ 3; error bars indicate sd). NSCs derived from in vitro differentiation
were seeded 5 ? 104cells/well. Doxycycline was added at 0.5 lg/ml or 1 lg/ml. Where indicated, VPA was added at a final concentration of 2
lM for 14 days. GFP expression is driven by the Oct4 promoter. The number of GFP-positive colonies was determined after 3 weeks of induc-
tion. 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.
Establishment of an inducible reprogramming system using defined factors. (A): Schematic representation of the experimental design
Interaction Between Reprogramming Factors
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.
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 inter-
action, 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 coim-
munoprecipitation. Comparison of mutants having truncations
of the last one (Klf4DZF3), last two (Klf4DZF2-3), or all
three (Klf4DZF1-3) 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 abol-
ished Klf4’s interaction with Sox2 (Fig. 3C). Deletion of the
two C-terminal zinc fingers (Klf4DZF2-3) resulted in an
intermediate reduction in Sox2 interaction (Fig. 3C). Other
mutants, such as Klf4DM, which lacks the middle of the pro-
tein but retains all three zinc fingers, did not alter interaction
of Klf4 with Oct4 or Sox2 (Fig. 3B, 3C). These results indi-
cate 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).
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  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
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.
Klf4 interacts with Oct4 and Sox2. (A, B): Klf4 interacts with Oct4 (A) and Sox2 (B) when overexpressed in 293T cells. Constructs
Wei, Yang, Zhang et al.
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 Klf4DZF3,
Klf4DZF2-3, or Klf4DZF1-3, activated the promoter, indicat-
ing that zinc fingers are critical for target gene activation
(Fig. 4B). Furthermore, EMSA showed that all zinc finger de-
letion mutants lacked DNA binding ability (Fig. 4C), confirm-
ing that the zinc fingers are critical for DNA binding.
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 (Klf4DZF3), or all
three zinc fingers (Klf4DZF1-3), 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-
action between Klf4 and Oct4 requires zinc finger motifs at the Klf4 C terminus. Deletion of all three zinc finger motifs (Klf4DZF1-3) signifi-
cantly decreased Klf4/Oct4 interaction. Deletion of the C terminal or two last C-terminal zinc fingers (Klf4DZF3 and Klf4DZF2-3), or deletion of
the middle region (Klf4DM), 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 interac-
tion, 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 termi-
nus 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 plu-
ripotent 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.
The Klf4 C terminus interacts directly with Oct4 and Sox2. (A): Design of Klf4 deletion mutants used in the experiments. (B): Inter-
Interaction Between Reprogramming Factors
Myc antibodies. In cells expressing Klf4DZF3, Flag-tagged
Klf4DZF3 directly bound to Oct4, whereas the interaction
between Myc-tagged wild-type Klf4 and Oct4 was signifi-
cantly reduced (Fig. 4D), suggesting that Klf4DZF3 signifi-
Klf4DZF1-3 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 KlfDZF3 functions as a dominant negative mutant in
competing with wild-type Klf4 to form complexes with Oct4
With a dominant negative form of Klf4 (Klf4DZF3) 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-genera-
tion 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 dif-
ferent samples was confirmed by Flag immunostaining (sup-
porting information Fig. 3). Thus, differences in reprogram-
ming efficiency observed among various samples were
assumed to be due primarily to different activities of overex-
pressed products. Cells from the same passage NSCs or MEFs
were split and plated at the same density. Klf4DZF3- and
Klf4DZF2-3-expressing cells showed significantly inhibited
reprogramming efficiency: the numbers of Oct4-GFP-positive
colonies and percentage of SSEA1-positive cells in these sam-
ples were significantly reduced compared with cells overex-
pressing wild-type Klf4 (Fig. 4F, 4G). To exclude the possi-
bility that this effect was limited to NSCs, MEFs derived
from chimeric mice were similarly analyzed. Although
ming. (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 (Klf4DZF3), the last two (Klf4DZF2-3), or all three
zinc finger motifs (Klf4DZF1-3) 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 (Klf4DZF3) inhibits interaction of wild-type Klf4 with Oct4 (D) or Sox2 (E) by competing with wild-type Klf4. However,
Klf4DZF1-3, 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 ¼ 3; error bars indicate sd; **, p < .01) in genetically homogeneous secondary NSCs. In vitro differentiated NSCs were transduced with lenti-
virus expressing Klf4 mutants and induced with doxycycline. The number of GFP-positive colonies was determined after 3 weeks of doxycycline
induction. Klf4DZF3 and Klf4DZF2-3 strongly inhibited reprogramming, whereas Klf4DZF1-3 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 lg/ml for 3 days before transduction with new virus. The total number of GFP-positive colonies in triplicate wells was determined af-
ter 12 days of doxycycline induction (n ¼ 3; **, p < .01). Similarly, Klf4DZF3 and Klf4DZF2-3 strongly inhibited reprogramming, whereas
Klf4DZF1-3 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.
Dominant negative Klf4 mutants compete with wild-type Klf4 for binding with Oct4 and Sox2, resulting in disruption of reprogram-
Wei, Yang, Zhang et al.
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 Klf4DZF3 or Klf4DZF2-3
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.
Klf4DZF1-3, 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 con-
trols (Fig. 4F–4I). This finding is consistent with the observa-
tion that Klf4DZF1-3 cannot interact with Sox2 and only
poorly interacts with Oct4. Thus, there was no competition
between Klf4DZF1-3 and wild-type Klf4 in forming an Oct4/
Endogenous Klf4 in MEF Is Required for
Although Klf4 has been shown to be important for reprogram-
ming, 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 endog-
enous 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 endoge-
nous Klf4 is required for iPS cell induction from wild-type
MEFs using only overexpressed Oct4 and Sox2, as described
before . Treatment of MEFs with Klf4 shRNA prior to
initiation of reprogramming with only Oct4 and Sox2 signifi-
cantly decreased the number of AP-positive colonies com-
pared with controls (Fig. 5A, 5B), indicating that endogenous
Klf4 is required for reprogramming.
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 . When dominant
negative mutant Klf4DZF3 was overexpressed, the number of
AP-positive colonies was significantly reduced compared with
controls treated with either empty vector or with the
Klf4DZF1-3 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 com-
plex with exogenous Oct4 and Sox2 during reprogramming
and that inclusion of Klf4 in this complex is required for effi-
cient 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 . 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 Klf4DZF3
dominant negative mutant or control constructs plus a puro-
mycin resistance gene into mouse ES cells. After 6 days of
selection, we immunostained surviving cells with SSEA1 anti-
body and counted SSEA1-positive cells (Fig. 5E). Overex-
pression of Klf4DZF3 resulted in differentiation and almost
complete loss of SSEA1-positive cells (Fig. 5E, 5F). By
contrast, only a small proportion of cells overexpressing
Klf4DZF1-3, 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 com-
plex by inclusion of a dominant negative form of Klf4 inter-
feres with normal self-renewal of ES cells.
Klf4 has been suggested to play an important role in ES cell
self-renewal . 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 Klf4DZF3 and KlfDZF2-3, which interact with
Oct4 and Sox2 but lack DNA binding activity, significantly in-
hibit normal reprogramming. However, Klf4DZF1-3, which
lacks all three zinc fingers, fails to suppress reprogramming.
The striking differences between Klf4DZF1-3 and Klf4DZF3/
Klf4DZF2-3 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 reprog-
ramming 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 colocal-
ize on promoters of essential pluripotency genes [17, 19]. How-
ever, whether this colocalization reflects active recruitment and
assembly of a specific functional protein complex remains elu-
sive. 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 . 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 observa-
tion that these three proteins are abundant (when expressed
ectopically) even in partially reprogrammed cells suggests
Interaction Between Reprogramming Factors
that a fully functional complex may require interacting part-
ners, particularly factors that can antagonize repressive chro-
matin structures in key target genes. Identifying those partners
is critical for further investigation.
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
reduced. Further studies of this interaction should facilitate
our understanding of reprogramming mechanisms.
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.contributedequally tothis work.
DISCLOSURE OF POTENTIAL CONFLICTS
The authors indicate no potential conflicts of interest.
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 lM. AP staining was performed 2 weeks after induction of transgenes expression.
(B): Quantification of AP-positive colonies in (A) (n ¼ 3 independent experiments; **, p < .01). (C): Primary MEFs overexpressing Klf4DZF3
fail to produce AP-positive cells, whereas overexpression of Klf4DZF1-3 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 lM. AP staining was performed 2 weeks after induction of transgene
expression. (D): Quantification of AP-positive colonies in (C) (n ¼ 3 independent experiments; **, p < .01). (E): Overexpression of Klf4DZF3
inhibits normal self-renewal of wild-type mouse ES cells, whereas a substantial proportion of cells overexpressing Klf4DZF1-3 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 40,6-diamidino-2-phenylindole (blue). Bars ¼ 10 lm. (F): Quantification of the percentage of SSEA1-positive cells
in (E) revealed a statistically significant decrease (n ¼ 3 independent experiments; *, p < .05; **, p < .01) in Klf4DZF3-overexpressing cells
compared with both control cells and cells overexpressing Klf4DZF1-3. Abbreviations: AP, alkaline phosphatase; shRNA, short hairpin RNA;
SSEA1, stage-specific embryonic antigen 1; WT, wild type, ZF, zinc finger.
Endogenous Klf4 is critical for reprogramming and embryonic stem (ES) cell self-renewal. (A): Knockdown of endogenous Klf4
Wei, Yang, Zhang et al.
1Jaenisch R, Young R. Stem cells, the molecular circuitry of pluripo-
tency and nuclear reprogramming. Cell 2008;132:567–582.
Hochedlinger K, Plath K. Epigenetic reprogramming and induced plu-
ripotency. Development 2009;136:509–523.
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors. Cell
Wernig M, Lengner CJ, Hanna J et al. A drug-inducible transgenic
system for direct reprogramming of multiple somatic cell types. Nat
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;4:301–312.
Nakagawa M, Koyanagi M, Tanabe K et al. Generation of induced
pluripotent stem cells without Myc from mouse and human fibroblasts.
Nat Biotechnol 2008;26:101–106.
Brambrink T, Foreman R, Welstead GG et al. Sequential expression
of pluripotency markers during direct reprogramming of mouse so-
matic cells. Cell Stem Cell 2008;2:151–159.
Kaji K, Norrby K, Paca A et al. Virus-free induction of pluripotency
and subsequent excision of reprogramming factors. Nature 2009;458:
Stadtfeld M, Nagaya M, Utikal J et al. Induced pluripotent stem cells
generated without viral integration. Science 2008;322:945–949.
10 Okita K, Nakagawa M, Hyenjong H et al. Generation of mouse
induced pluripotent stem cells without viral vectors. Science 2008;
11 Woltjen K,MichaelIP,MohseniPetal.piggyBactranspositionreprograms
12 Zhou H, Wu S, Joo JY et al. Generation of induced pluripotent stem
cells using recombinant proteins. Cell Stem Cell 2009;4:381–384.
13 Huangfu D, Osafune K, Maehr R et al. Induction of pluripotent stem
cells from primary human fibroblasts with only Oct4 and Sox2. Nat
14 Shi Y, Desponts C, Do JT et al. Induction of pluripotent stem cells
from mouse embryonic fibroblasts by Oct4 and Klf4 with small-mole-
cule compounds. Cell Stem Cell 2008;3:568–574.
15 Marson A, Foreman R, Chevalier B et al. Wnt signaling promotes
reprogramming of somatic cells to pluripotency. Cell Stem Cell 2008;
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;282:33994–34002.
17 Jiang J, Chan YS, Loh YH et al. A core Klf circuitry regulates self-
renewal of embryonic stem cells. Nat Cell Biol 2008;10:353–360.
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;26:7772–7782.
19 Sridharan R, Tchieu J, Mason MJ et al. Role of the murine reprogram-
ming factors in the induction of pluripotency. Cell 2009;136:364–377.
20 Guo G, Yang J, Nichols J et al. Klf4 reverts developmentally pro-
grammed restriction of ground state pluripotency. Development 2009;
21 Hanna J, Markoulaki S, Mitalipov SM et al. Metastable pluripotent
states in NOD-mouse-derived ESCs. Cell Stem Cell 2009;4:513–524.
22 Philipsen S, Suske G. A tale of three fingers: the family of mammalian
Sp/XKLF transcription factors. Nucleic Acids Res 1999;27:2991–3000.
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;15:2437–2447.
24 Wolfe SA, Nekludova L, Pabo CO. DNA recognition by Cys2His2
zinc finger proteins. Ann Rev Biophys Biomol Struct 2000;29:
25 Urlinger S, Baron U, Thellmann M et al. Exploring the sequence
space for tetracycline-dependent transcriptional activators: novel muta-
tions yield expanded range and sensitivity. Proc Natl Acad Sci U S A
26 Lois C, Hong EJ, Pease S et al. Germline transmission and tissue-spe-
cific expression of transgenes delivered by lentiviral vectors. Science
27 Lyu J, Yamamoto V, Lu W. Cleavage of the Wnt receptor Ryk regu-
lates neuronal differentiation during cortical neurogenesis. Dev Cell
28 Takahashi K, Okita K, Nakagawa M et al. Induction of pluripotent
stem cells from fibroblast cultures. Nat Protoc 2007;2:3081–3089.
29 Szab´o PE, Hubner K, Scholer H et al. Allele-specific expression of
imprinted genes in mouse migratory primordial germ cells. Mech Dev
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 core-
ceptor required for stimulation of neurite outgrowth. Cell 2004;119:
32 Wells J, Farnham PJ. Characterizing transcription factor binding sites
using formaldehyde crosslinking and immunoprecipitation. Methods
33 Eminli S, Utikal JS, Arnold K et al. Reprogramming of neural progen-
itor cells into iPS cells in the absence of exogenous Sox2 expression.
Stem Cells 2008;26:2467–2474.
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;
35 Xie D, Cai J, Chia NY et al. Cross-species de novo identification of
cis-regulatory modules with GibbsModule: application to gene regula-
tion in embryonic stem cells. Genome Res 2008;18:1325–1335.
36 Rodda DJ, Chew JL, Lim LH et al. Transcriptional regulation of
nanog by OCT4 and SOX2. J Biol Chem 2005;280:24731–24737.
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;11:197–203.
38 Kim JB, Sebastiano V, Wu G et al. Oct4-induced pluripotency in
adult neural stem cells. Cell 2009;136:411–419.
39 Lyssiotis CA, Foreman RK, Staerk J et al. Reprogramming of murine
fibroblasts to induced pluripotent stem cells with chemical com-
plementation of Klf4. Proc Natl Acac Sci U S A 2009;106:
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;7:1074–1082.
41 Wang J, Rao S, Chu J et al. A protein interaction network for pluripo-
tency of embryonic stem cells. Nature 2006;444:364–368.
42 Maherali N, Sridharan R, Xie W et al. Directly reprogrammed fibro-
blasts show global epigenetic remodeling and widespread tissue con-
tribution. Cell Stem Cell 2007;1:55–70.
43 Yu J, Hu K, Smuga-Otto K et al. Human induced pluripotent stem
cells free of vector and transgene sequences. Science 2009;324:
Interaction Between Reprogramming Factors