MOLECULAR AND CELLULAR BIOLOGY, Sept. 2011, p. 3723–3733
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 18
Cooperative Transcriptional Activation by Klf4, Meis2, and Pbx1?
Glen A. Bjerke, Cathy Hyman-Walsh, and David Wotton*
Department of Biochemistry and Molecular Genetics and Center for Cell Signaling, University of Virginia, Charlottesville, Virginia
Received 21 December 2010/Returned for modification 18 January 2011/Accepted 30 June 2011
The Kruppel-like factor Klf4 is implicated in tumorigenesis and maintaining stem cell pluripotency, and
Klf4 can both activate and repress gene expression. We show that the Pbx1 and Meis2 homeodomain proteins
interact with Klf4 and can be recruited to DNA elements comprising a Klf4 site or GC box, with adjacent Meis
and Pbx sites. Meis2d and Pbx1a activate expression of p15Ink4aand E-cadherin, dependent on the Meis2d
transcriptional activation domain. In HepG2 cells, reducing expression of endogenous Meis2 or Pbx1 decreases
p15 gene expression and increases the number of cells entering S phase. Although DNA binding by all three
proteins contributes to full cooperative activation, the sequence requirements for binding by Meis2 and Pbx1
are variable. In the E-cadherin promoter, a Pbx-like site is required for full activation, whereas in the p15
promoter, the Klf4 site appears to play the major role. Through a bioinformatics search we identified
additional genes with conserved binding sites for Klf4, Meis2, and Pbx1 and show that at least some of these
genes can be activated cooperatively by Klf4 and Meis2/Pbx1. We suggest a model in which genes with Klf4 sites
can be cooperatively activated by Meis2/Pbx1 and Klf4, dependent primarily on recruitment by Klf4. This
provides a mechanism to modulate transcriptional regulation by the multifunctional Klf4 transcription factor.
Homeodomain proteins comprise a large evolutionarily con-
served family of DNA binding proteins, with diverse functions
in organisms from yeast to mammals (17, 39, 40). The homeo-
domain is an approximately 60 amino-acid domain consisting
of three alpha helices. The third helix is primarily responsible
for DNA binding, whereas helices 1 and 2 play a structural role
and are responsible for protein-protein interactions (10, 18, 49,
52). Many homeodomain proteins bind to DNA in complex
with other proteins (9, 29, 30, 37). Meis2 and Pbx1 are mem-
bers of the TALE superfamily of homeodomains, in which
alpha helices 1 and 2 are separated by an extra three-amino-
acid loop extension (hence TALE) (5, 7, 44). The TALE su-
perfamily includes proteins which are transcriptional activa-
tors, such as Meis1 and Meis2, and repressors, such as Tgif1
and Tgif2 (5, 20, 41, 64, 65). In mice and humans, there are
three Meis paralogs (Meis1, 2, and 3), as well as the related
Prep1 and Prep2 (4, 15). Meis/Prep proteins share a highly
conserved TALE homeodomain and a second conserved do-
main, termed the homothorax homology domain (Hth), which
is present in the fly homothorax protein (42, 53). The Hth
domain of Meis family proteins mediates interaction with Pbx
proteins via the conserved PBC-A and PBC-B domains (22, 54,
57). Interaction of Meis and Pbx partners with each other
facilitates their cooperative binding to a composite DNA ele-
ment. Additionally, Meis proteins can be recruited to DNA by
interaction with a Pbx partner and other homeodomain pro-
teins, with out the need for Meis to bind DNA (34, 57). Meis
and Pbx family proteins can contribute to the formation of
DNA-bound transcription factor complexes with non-home-
odomain proteins. For example, Pbx1 can recruit MyoD to
promoter elements at which Pbx1 is prebound, prior to MyoD
expression (2, 28). Some members of the Meis family, includ-
ing Meis1 and Meis2, have transcriptional activation domains
(20, 21), whereas Prep1 and 2 appear to be much weaker
activators and may contribute primarily to the formation of a
DNA-bound complex (3). Pbx proteins are unable to activate
transcription alone but contribute to the binding of Meis pro-
teins to DNA and may facilitate transcriptional activation by
Meis partner proteins (35). Multiple splice variants of Meis2
have been identified (65). Most of the alternate splicing of
Meis2 affects the transcriptional activation domain. However,
one major splice variant (Meis2e) inserts a translational stop
close to the amino terminus of the homeodomain, generating
a variant lacking the activation domain and most of the homeo-
Mammalian Meis1 (myeloid ecotropic insertion site 1) was
identified as a common site of viral integration in mouse my-
eloid leukemia cells (43), and the related Meis2 and Meis3
genes were identified based on sequence similarity (46, 48).
Meis1 plays a key role in the progression of acute myeloid
leukemia (AML) and mixed lineage leukemia (MLL), and
fusion proteins generated by chromosomal rearrangements in
MLL can induce increased expression of Meis1 (55, 61, 63).
The Meis relative Prep1 plays a role in hematopoietic stem cell
function and in early T cell development (12, 50, 51). Pbx
proteins, which are common partners of Meis family members,
have also been implicated in tumorigenesis. The Pbx1 gene is
fused to the transcription factor E2A as a result of the t(1;19)
translocation in pre-B cell leukemia (25, 26). This fusion pre-
vents interaction with Meis proteins and converts Pbx1 to a
transcriptional activator. Thus, there is significant evidence for
deregulation of Meis and Pbx family proteins promoting tu-
morigenesis in lymphoid and hematopoietic cells.
Sp1 is a zinc finger-containing transcription factor which
regulates a large number of genes via binding to a GC-rich
consensus site (6, 13, 24). The related Sp3 protein binds to the
same sequence and is highly homologous over the carboxyl-
terminal zinc finger domain but is less well conserved outside
* Corresponding author. Mailing address: Center for Cell Signaling,
University of Virginia, Box 800577, HSC, Charlottesville, VA 22908.
Phone: (434) 243-6752. Fax: (434) 924-1236. E-mail: dw2p@virginia
?Published ahead of print on 11 July 2011.
this region (27). Sp1 has been shown to regulate the p15 pro-
moter and to activate p15 cooperatively with transforming
growth factor ? (TGF-?)-regulated Smads (32, 33). Sp1 and
Sp3 belong to a larger family of Kruppel-related zinc finger
transcription factors that includes more than 15 KLF (for
Kruppel-like factor) proteins (23, 60). The KLF family includes
GKLF (gut-enriched KLF), also known as Klf4, which al-
though highly expressed in gut epithelium is also expressed in
a wide variety of other tissues (14) and plays a role in the
maintenance of pluripotent stem cells (36, 47, 62). Klf4 has the
characteristic three zinc fingers of the KLF family located at its
carboxyl terminus and contains both a transcriptional repres-
sion domain and an activation domain that can interact with
p300/CBP (19, 66). Thus, Klf4 can activate or repress transcrip-
tion, possibly depending on the context. Klf4 also plays appar-
ently contradictory roles in tumorigenesis, as there is evidence
for Klf4 being both a tumor suppressor and an oncogene (14,
38). For example, Klf4 levels are decreased in colorectal cancer
and in medulloblastoma, both by hypermethylation and by
mutation (45, 68). In contrast, Klf4 expression in keratinocytes
can induce squamous epithelial dysplasia (16). Consistent with
a role as an oncogene, Klf4 expression is increased in a number
of cancers, including mammary carcinomas and some squa-
mous cell cancers (38).
Here we show that Klf4 can recruit Meis2 and Pbx1 to a
suboptimal Meis/Pbx site adjacent to a GC box in the p15
promoter. The GC box is essential for Meis2 and Pbx1 recruit-
ment and for the transcriptional activation by Meis/Pbx, which
requires the Meis2 transcriptional activation domain. Using a
combination of bioinformatics and functional analysis we iden-
tified a number of other genes that are candidates for cooper-
ative regulation by Klf4-Meis/Pbx complexes. This work sug-
gests that Meis/Pbx dimers may promote transcriptional
activation over repression by DNA-bound Klf4, dependent on
adjacent Meis or Pbx binding sites.
MATERIALS AND METHODS
Plasmids and oligonucleotides. TGIF expression and short hairpin RNA
(shRNA) plasmids have been described previously (1). p15 reporter constructs
were created in pGL2 or pGL3 (Promega) or in pGL2 basic into which a minimal
TATA element from the adenovirus major late promoter (MLP) had been
inserted. The E-cad-luc reporter contains sequences from 178 bp upstream of the
transcriptional start to ?92 from the mouse gene. The four-copy SBR2 and
two-copy Meis/Pbx reporters are as described previously (21, 56). Meis2, Pbx1,
KLF, and Sp1 expression constructs were created in a modified pCMV5 with
either a Flag or T7 epitope tag. KLF4 was also expressed from within pCDNA3.
Meis2 and Pbx1 mutants and deletion constructs are as described previously (21).
Pax3 and Etv1 luciferase reporters are as described previously (8, 11).
Cell culture and siRNA knockdown. HepG2 (ATCC HB-8065), Mcf7, NMuli,
and HeLa cells were maintained in Dulbecco’s modified Eagle medium
(DMEM) with 10% fetal bovine serum (FBS), and COS1 cells were grown in
DMEM with 10% bovine growth serum (BGS). For knockdown, cells were
plated in 12-well plates and transfected with Dharmacon SMARTpool oligonu-
cleotides by using DharmaFECT reagent 1, according to the manufacturer’s
instructions. Small interfering RNA (siRNA) sequences are available on request.
RNA was isolated 60 h after transfection. The control pool (mouse siGENOME
nontargeting siRNA pool 3) was used for the nontargeting control. For EdU
labeling, cells were labeled with 10 ?M EdU for 1 h at 37°C, and after fixation
in 4% paraformaldehyde, were permeabilized with Triton X-100 for 30 min at
room temperature. EdU was detected with an Alexa Fluor 488 EdU detection kit
(Click-iT EdU [Molecular Probes]), according to the manufacturer’s protocol,
and DNA was stained with Hoechst 33342. Images were captured on a Zeiss
AxioObserver with Volocity.
RNA analysis. RNA was isolated and purified using an Absolutely RNA kit
(Stratagene). For quantitative real-time PCR (qRT-PCR), cDNA was generated
using Superscript III (Invitrogen) and analyzed in triplicate by real-time PCR
using a Bio-Rad MyIQ cycler and Sensimix Plus SYBR green plus fluorescein
isothiocyanate (FITC) mix (Quantace). Intron-spanning primer pairs were se-
lected using Primer3 (http://frodo.wi.mit.edu/). Primer sequences for qRT-PCR
are available on request. Expression was normalized to cyclophilin using the
cycle threshold (??CT) method and is shown as the mean plus standard devia-
tion (SD) from triplicate experiments.
Immunoprecipitation and Western blotting. COS1 cells were transfected using
Lipofectamine (Invitrogen). Forty hours after transfection, cells were lysed by
sonication in phosphate-buffered saline (PBS) with 1% NP-40, 1 mM dithiothre-
itol (DTT), and protease inhibitors. Immunocomplexes were precipitated with
Flag M2-agarose (Sigma). Following SDS-polyacrylamide gel electrophoresis,
proteins were electroblotted to Immobilon-P (Millipore) and incubated with
antisera specific for Flag (Sigma) or T7 (Novagen). Proteins were visualized with
horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit Ig (Pierce)
and ECL (Amersham Pharmacia Biotech). For testing dependence on DNA
binding, ethidium bromide was added to a concentration of 1 ?M to cell lysates
prior to precipitation.
DNA affinity precipitation. For isolation of protein complexes on double-
stranded DNA oligonucleotides, lysates were prepared from 75% of a confluent
15-cm dish of COS1 cells for each condition, in MSLD (100 mM NaCl, 20 mM
HEPES, pH 7.8, 10% glycerol, 0.1% Tween 20, with 1 mM DTT) and protease
inhibitors. Lysates were precleared with protein A-agarose (Pierce) and incu-
bated in 1 ml with 100 ng of biotinylated double-stranded oligonucleotide and 1
?g poly(dI-dC) ? poly(dI-dC). Complexes were isolated on streptavidin-agarose,
washed 4 times in binding buffer, and then subjected to analysis by SDS-PAGE
and Western blotting. Sp1 antibody is from Upstate (07-645), Meis2 is from
Abnova (H00004212-M01), and Pbx1 is from Abnova (H00005087-M01).
Luciferase assays. HepG2 cells were transfected using Exgen 500 (MBI Fer-
mentas) according to the manufacturer’s instructions. Cells were transfected with
firefly luciferase reporters, a Renilla transfection control (phCMVRLuc [Pro-
mega]), and the indicated expression constructs. After 40 h, firefly luciferase
activity was assayed using firefly substrate (Biotium) and Renilla luciferase was
assayed with 0.09 ?M coelenterazine (Biosynth), by using a Berthold LB953
luminometer. Mithramycin was added to a final concentration of 200 nM, 24 h
prior to analysis, where indicated.
ChIP. For transfected chromatin immunoprecipitation (ChIP) HeLa cells
were transfected with Exgen 500 (Fermentas) in 60-mm dishes and 5 ?g of DNA.
Two days after transfection, cells were washed with PBS, fixed in PBS with 1%
formaldehyde for 15 min, and then quenched with 0.125 M glycine for 5 min.
Plates were washed twice with cold PBS, scraped into 1 ml of cold radioimmu-
noprecipitation assay (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.5% deoxy-
cholate, 0.1% SDS, 50 mM Tris pH 8, 5 mM EDTA) with protease inhibitors,
and sonicated 15 times for 10 s. Lysates were centrifuged at 21,000 relative
centrifugal force (RCF) for 15 min to remove cell debris and then precleared
with protein G-agarose (Pierce) for 2 h. Immunoprecipitation was carried out
overnight with 15 ?l Flag-agarose (in PBS, 1 mg/ml bovine serum albumin [BSA],
0.3 mg/ml salmon sperm DNA). Precipitates were washed twice with RIPA, 4
times with Szak’s IP wash buffer (100 mM Tris-HCl, pH 8.5, 500 mM LiCl, 1%
NP-40, 1% deoxycholate), twice more with RIPA, and twice with 1? Tris-EDTA
(TE). One hundred microliters of 1.5? Talianidis elution buffer (70 mM Tris-
HCl, pH 8, 1 mM EDTA, 1.5% SDS, 300 mM NaCl) was added to precipitates
(and inputs) in 50 ?l TE and samples were incubated at 65°C for 5 h. Samples
were treated with 10 ?g of proteinase K for 30 min at 45°C, and DNA was
isolated using QIAquick columns (Qiagen) in 100 ?l of water. Amounts of
immunoprecipitated DNA were analyzed by qPCR on a Bio-Rad MyIQ cycler
with Sensimix Plus SYBR green plus FITC mix (Quantace). Primer sequences
for ChIP are available on request. Signal was expressed as bound versus input by
the ??CT method. For endogenous ChIP, Mcf7 cells were fixed and harvested as
described above. One 15-cm dish was used per sample. Immunoprecipitation was
carried out overnight using 3 ?g of antibody and 15 ?l of protein G-agarose
(Pierce) with BSA and salmon sperm DNA, as described above. Antibody for
Klf4 is from Santa Cruz (sc-20691) and Pbx1 was from Abnova (H00005087-
M01). Immunoprecipitated fractions were washed and analyzed as described
In silico site search. Mouse and human genomic databases were searched using
the Site Search program (59): http://www.sitesearch.mshri.on.ca/Genome/index
.html. We searched 2 kb upstream of the predicted start site of each gene, for the
combination of a Klf4 site (RRGGYSY ) with both Meis and Pbx consensus
sites (TGACA and CAATC) within 40 bp either side of the Klf4 site. This
combination had to be present in both mouse and human, and we then accepted
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