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The Gut-enriched Krüppel-like Factor (Krüppel-like Factor 4)
Mediates the Transactivating Effect of p53 on the p21WAF1/Cip1
Promoter*
Weiqing Zhang‡, Deborah E. Geiman‡, Janiel M. Shields‡, Duyen T. Dang‡,§, Channing S.
Mahatan‡, Klaus H. Kaestner¶, Joseph R. Biggs||, Andrew S. Kraft||, and Vincent W.
Yang‡,**,‡‡
‡Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
**Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore,
Maryland 21205
¶Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
||Department of Medical Oncology, University of Colorado Health Science Center, Denver, Colorado 80262
Abstract
An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce
cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 gene. We
show that the gene encoding the gut-enriched Krüppel-like factor (GKLF, KLF4) is concurrently
induced with p21WAF1/Cip1 during serum deprivation and DNA damage elicited by methyl
methanesulfonate. The increases in expression of both Gklf and p21WAF1/Cip1 due to DNA damage
are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the
rise in Gklf mRNA level precedes that in p21WAF1/Cip1, suggesting that GKLF may be involved in
the induction of p21WAF1/Cip1. Indeed, GKLF activates p21WAF1/Cip1 through a specific Sp1-like
cis-element in the p21WAF1/Cip1 proximal promoter. The same element is also required by p53 to
activate the p21WAF1/Cip1 promoter, although p53 does not bind to it. Potential mechanisms by which
p53 activates the p21WAF1/Cip1 promoter include a physical interaction between p53 and GKLF and
the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic
induction of the p21WAF1/Cip1 promoter activity. The physiological relevance of GKLF in mediating
p53-dependent induction of p21WAF1/Cip1 is demonstrated by the ability of antisense Gklf
oligonucleotides to block the production of p21WAF1/Cip1 in response to p53 activation. These
findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of
p21WAF1/Cip1 and may be part of a novel pathway by which cellular responses to stress are modulated.
A principal function of the tumor suppressor p53 is to maintain genomic stability. It does so
by eliciting cellular changes in response to various forms of stress such as DNA damage,
hypoxia, and nucleotide deprivation (1–3). The amount of p53 protein increases in response
to these so-called genotoxic stresses. In addition, covalent modifications such as
phosphorylation are involved in its activation (4,5). Once activated, p53 exerts potent
regulatory effects on diverse aspects of cellular events that cumulate in cell cycle arrest or
apoptosis (3). Many of these “downstream” events are dependent upon the ability of p53 to
function as a transcription factor in activating the expression of “target” genes (2,6). Notably,
*This work was in part supported by grants from the National Institutes of Health (to V. W. Y., K. H. K., and A. S. K.).
‡‡ To whom correspondence should be addressed: Dept. of Medicine, Ross 918, The Johns Hopkins University School of Medicine, 720
Rutland Ave., Baltimore, MD 21205. Tel.: 410-955-9691; Fax: 410-955-9677; E-mail: vyang@welch.jhu.edu.
§Supported by a National Research Service Award from the National Institutes of Health.
NIH Public Access
Author Manuscript
J Biol Chem. Author manuscript; available in PMC 2008 February 6.
Published in final edited form as:
J Biol Chem. 2000 June 16; 275(24): 18391–18398.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
an important consequence of p53 activation is the transcriptional induction of the gene
encoding the cyclin-dependent kinase (Cdk)1 inhibitor p21 (also called WAF1 or Cip1) (7,8).
p21WAF1/Cip1 inhibits the activity of several cyclin-Cdk complexes such as cyclin D1-Cdk4,
cyclin E1-Cdk2, and cyclin A-Cdk2, which results in cell cycle arrest at the G1-S transition
checkpoint (9,10).
The gut-enriched Krüppel-like factor (GKLF, KLF4) (11) is a recently identified and
developmentally regulated transcription factor, the expression of which is enriched in the
epithelial cells of the gastrointestinal tract (12–14), skin (14,15), and thymus (16) and in
vascular endothelial cells (17). Both the in vivo (12–16) and in vitro (12) patterns of expression
of Gklf are indicative of a growth arrest-associated nature. Upon stimulation of quiescent
cultured cells by fresh serum, levels of Gklf mRNA are decreased significantly during the G1-
S transition phase of the cell cycle (12). Conversely, constitutive expression of GKLF inhibits
DNA synthesis (12). In vivo, Gklf transcripts are highly enriched in the population of terminally
differentiated, post-mitotic epithelial cells of the intestinal tract and skin (12–15). Moreover,
the intestinal expression of Gklf is down-regulated in two independent mouse models of
intestinal tumorigenesis or hyperproliferation (18,19). Taken together, these studies suggest
that GKLF is potentially a negative regulator of proliferation; however, the mechanism by
which it accomplishes this task is not well defined.
The established binding site for GKLF is rich in GC content (20) and overlaps with that for
the transcription factor Sp1 (21,22). By coincidence, the proximal promoter of the
p21WAF1/Cip1 gene contains a number of GC-rich elements (7), some of which have been shown
to bind Sp1 (23–29). These Sp1-binding sites have been shown to be important in controlling
expression of p21WAF1/Cip1 in several physiologically diverse processes, including the gene’s
responsiveness to phorbol ester (23), transforming growth factor-β (24–26), and sodium
butyrate (27), and in keratinocyte differentiation (28). As both Gklf and p21WAF1/Cip1 are
growth arrest-associated genes, we sought to determine whether GKLF is involved in
regulating p21WAF1/Cip1 expression. We demonstrate that GKLF not only transactivates the
p21WAF1/Cip1 proximal promoter, but also mediates the activating effects of p53 in response
to DNA damage on the same promoter. This study suggests that GKLF may be an important
component of the p53 tumor suppressor network of regulatory proteins.
EXPERIMENTAL PROCEDURES
Plasmid Constructs, Reagents, and Cell Lines
The eukaryotic expression vector PMT3 and its derivatives containing various forms of GKLF
were previously described (12,20,30,31). They include full-length GKLF (PMT3-GKLF-(1–
483)), truncated GKLF lacking the three zinc fingers (PMT3-GKLF-(1–401)), and truncated
GKLF containing the zinc fingers only (PMT3-GKLF-(350–483)). pC53-SN3 and pC53-
SX3, two cytomegalovirus-based expression constructs containing wild-type p53 and mutant
p53 with a missense mutation at codon 143 in the DNA-binding domain (DBD) of p53,
respectively, were kindly provided by B. Vogelstein and K. Kinzler (32). The reporter
constructs linking various regions of the p21WAF1/Cip1 promoter to chloramphenicol
acetyltransferase (CAT) have previously been described (23). They include the CAT reporter
linked to either a 2320-nt 5′-flanking sequence of the p21WAF1/Cip1 gene containing an
upstream p53-binding site at nt −2301 (33) or the same 2320-nt 5′-flanking sequence with a
1The abbreviations used are: Cdk, cyclin-dependent kinase; GKLF, gut-enriched Krüppel-like factor; KLF4, Krüppel-like factor 4; DBD,
DNA-binding domain; CAT, chloramphenicol acetyltransferase; nt, nucleotide(s); kb, kilobase(s); bp, base pair(s); MEFs, mouse embryo
fibroblasts; MMS, methyl methanesulfonate; PCR, polymerase chain reaction; HEK, human embryonic kidney; EMSA, electrophoretic
mobility shift assay; BisTris propane, 1,3-bis]tris(hydroxymethyl)methylamino[propane; CBP, cAMP-response element-binding protein-
binding protein.
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small internal deletion of the sequence between nt −122 and −61 of the p21WAF1/Cip1 promoter
that removed the first four of the six Sp1 sites from the proximal promoter (33). Reporter
constructs containing the proximal promoter region of the p21WAF1/Cip1 gene with various 5′-
end points as well as internal deletions or point mutations affecting the various Sp1 sites in the
proximal promoters have all been described (23). The WWP-Luc and DM-Luc constructs are
two luciferase reporters that contain 2.4 and 2.2 kb, respectively, of the 5′-flanking sequence
of the p21WAF1/Cip1 gene and were kindly provided by B. Vogelstein and K. Kinzler (7). The
DM-Luc construct lacks the upstream p53-binding site at nt −2301 in the p21WAF1/Cip1
promoter (7).
The polyclonal rabbit anti-GKLF serum was described (12). Anti-p53 serum was purchased
from Santa Cruz Biotechnology (sc-6243), and the monoclonal antibody against
p21WAF1/Cip1 was purchased from Pharmingen (SXM30). The p53−/− and p53+/+ mouse
embryo fibroblasts (MEFs) were generously provided by L. Donehower (34). The 10(1)-p53
val135 cell line was provided by A. Levine (35). This cell line, which was derived from the
parental 10(1) cell line (36), is an immortalized murine embryo fibroblast line that lacks
endogenous p53 expression, but contains a stably transfected temperature-sensitive p53
protein, val135 (37). At the nonpermissive temperature of 38.5 °C, p53 val135 is
transcriptionally inactive, whereas at the permissive temperature of 31.5 °C, it is
transcriptionally competent (35). The sense and antisense oligonucleotides to GKLF contain
nucleotide sequences corresponding to amino acid codons 7–13 of GKLF in the sense and
antisense orientations, respectively. At the center of this sequence (amino acid 10) is the second
of two initiation methionine codons of GKLF, which was felt to be in a translationally more
favorable context than the first (14). The nucleotide sequence of the antisense oligonucleotide
is 5′-GCT GAC AGC CAT GTC AGA CTC-3′, and that of the sense oligonucleotide is 5′-
GAG TCT GAC ATG GCT GTC AGC-3′. Note that the underlined sequence represents the
initiation methionine codon at amino acid 10 (12).
Conditions of Cell Treatments and Northern and Western Blot Analyses
For the serum deprivation experiments, the content of fetal calf serum in the cell medium was
reduced from 10 to 0.5% to induce a growth-arrested state (12). To cause DNA damage, methyl
methanesulfonate (MMS) was added to cells at a concentration of 100 μg/ml, which has
previously been shown to result in cell cycle arrest (38). After various treatment periods, total
RNA was isolated from cells using Triazol (Life Technologies, Inc.). Twenty μg of RNA from
each sample were studied by Northern blot analyses using conditions previously described
(12). Blots were probed with a full-length cDNA encoding GKLF (12), p21WAF1/Cip1 (7), or
glyceraldehyde-3-phosphate dehydrogenase (CLONTECH). The conditions for Western blot
analysis were also previously described, using a 1:1000 dilution of an affinity-purified
polyclonal anti-GKLF serum (12).
Transfection and Luciferase and CAT Assays
All transfections were performed by lipofection as described (20,21,30,31). Unless otherwise
specified, all reactions contained 5 μg each of the reporter and effector constructs/10-cm dish.
Luciferase and CAT assays were performed as described (20,21).
Reverse Transcription-Polymerase Chain Reactions
RNA was extracted from human embryonic kidney (HEK) 293 cells and human colonic
carcinoma HT29 cells (39). The content of Gklf transcript from each cell line was determined
using reverse transcription-PCR. The content of the β-actin transcript was similarly determined
as a control. One μg of RNA was reverse-transcribed in an 80-μl volume containing 50 mM
Tris-HCl, pH 8.3, 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, 0.5 mM dGTP, 0.5 mM dATP,
0.5 mM dTTP, 0.5 mM dCTP, 80 units of RNase inhibitor, 100 pmol of random primer, pd
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(N)6, and 200 units of Moloney murine leukemia virus reverse transcriptase (Life
Technologies, Inc.) at 42 °C for 1 h. The cDNA was then amplified in a 50-μl reaction that
contained 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 10 mM MgCl2, 0.1% gelatin, 2.5 units of
REDTaq DNA polymerase (Sigma), 0.2 mM dGTP, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dCTP,
and 40 pM each of the forward and reverse primers (see below) at the following settings: 94 °
C for 45 s, 45 °C for 1 min, and 72 °C for 1.5 min for a total of 40 cycles. The PCR products
were then visualized on a 1.5% agarose gel stained with ethidium bromide.
The primers used in the PCR were synthesized according to the published cDNA sequences
encoding human GKLF and β-actin (Gen-Bank™ Data Bank accession numbers AF105036
and X00351, respectively). The forward primer sequence for GKLF is 5′-AGGTCGGAC-
CACCTCGCCTTACACATG-3′, and the reverse primer sequence is 5′-
AAGGTAAAGAGAATACAAGGTGATCTTTTATGC-3′. The length of the expected PCR
product was 345 bp. The forward and reverse primer sequences for β-actin are 5′-
TACGCCAACACAGTGCTGTCTGG-3′ and 5′-TACTCCTGCTTGCTGATCCACAT-3′,
respectively, with the expected PCR product measuring 206 bp.
Electrophoretic Mobility Shift Assays
EMSAs were performed as described (20). Preparation of nuclear extracts from COS-1 cells
transfected with PMT3 expression constructs containing full-length GKLF, truncated GKLF
containing only the zinc fingers or lacking the zinc fingers, or PMT3 vector alone was as
described previously (20,21). Purified p53 containing the DBD was kindly provided by N.
Pavletich (40). This domain contains the core portion of p53 between amino acids 102 and
292, which binds with high affinity to a p53 recognition site (40,41). The purification of
recombinant p53 DBD expressed from the pET3d bacterial expression vector (Novagen) in
transformed Escherichia coli BL21(D3) cells was as described previously (40). The protein
was supplied at a concentration of 14 mg/ml in a solution of 50 mM BisTris propane HCl, pH
6.8, 200 mM sodium phosphate, and 5 mM dithiothreitol and had a >98% purity of the core
domain.
The wild-type p21 oligonucleotide used in EMSA contains the sequence between nt −129 and
−99 of the p21WAF1/Cip1 promoter, which includes both Sp1-1 and Sp1-2 sites (27). The mutant
p21 oligonucleotide contains a 3-bp substitution in the Sp1-1 site. The sequences in the sense
orientation for the two oligonucleotides are shown below.
The oligonucleotide probe containing the binding site for p53 was derived from the p53-
response sequence in the promoter of the human GADD45 gene (42) and has the sequence 5′-
TACAGAACATGTCTAAGCATGCTGGGG-3′ in the sense orientation. When indicated,
unlabeled competitor oligonucleotides were added in 10-, 20-, or 50-fold molar excess of the
probe to the reaction.
In Vitro Synthesis of p53 and Immunoprecipitation
[35S]Methionine-labeled p53 was synthesized by the TNT Coupled Reticulocyte Lysate system
(Promega) using a full-length cDNA encoding p53 cloned in pBluescript (provided by B.
Vogelstein). Ten μl of the translation product were mixed with 50 μg of nuclear extracts
prepared from transfected COS-1 cells in a final volume of 100 μl containing 20 mM HEPES,
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pH 7.5, 40 mM KCl, 3 mM MgCl2, 1 mM dithiothreitol, and 5% glycerol at 4 °C for 2 h. At the
completion of the incubation, 15 μg of affinity-purified anti-GKLF serum or preimmune serum
were added to the reaction, which was gently rotated overnight at 4 °C. Fifty μl of packed
protein A-Sepharose beads (Amersham Pharmacia Biotech) were then added to each reaction,
and the incubation was continued for 1 h at 4 °C. The beads were subsequently collected by
centrifugation, washed three times with the incubation buffer, and resuspended in sample buffer
before electrophoresis.
RESULTS
Both Gklf and p21WAF1/Cip1 Are Induced during Growth Arrest—
Previously, we showed that the levels of the Gklf transcript were low in actively proliferating
cells, but were increased in cells that had been deprived of serum (12). Results of the Northern
blot analysis in Fig. 1A recapitulate this event. Fig. 1A also shows that upon serum deprivation,
the levels of the p21WAF1/Cip1 transcript rose concomitantly with those of Gklf. To determine
whether Gklf is induced during growth arrest under a different condition, we treated NIH 3T3
cells with MMS, which causes DNA damage and subsequently cell cycle arrest (38). As shown
in Fig. 1B, the levels of Gklf mRNA were increased 2 h after the addition of MMS, as were
those of p21WAF1/Cip1 mRNA. When normalized to the expression of the control
glyceraldehyde-3-phosphate dehydrogenase gene, which was not affected by the treatment, the
degree of induction of p21WAF1/Cip1 was higher than that of Gklf between 2 and 8 h of MMS
treatment (Fig. 1B, bar graph). This contrasts with the changes in mRNA levels of the two
genes during the initial 30 min of treatment, in which the rise in Gklf preceded that in
p21WAF1/Cip1 (Fig. 1C). These results suggest that both Gklf and p21WAF1/Cip1 respond
similarly to signals elicited during growth arrest due to DNA damage. However, the induction
of Gklf begins slightly earlier than that of p21WAF1/Cip1 during the initial phase of DNA damage.
Induction of GKLF and p21WAF1/Cip1 by MMS Is Dependent on p53
To determine whether the inductive responses of Gklf and p21WAF1/Cip1 to MMS treatment are
dependent on p53, we compared the expression of the two genes in MEFs isolated from mice
that contained (p53+/+) or lacked (p53−/−) p53 created by homologous recombination (34). As
shown in Fig. 2 (lanes 1 and 2), neither fibroblasts contained appreciable amounts of GKLF
and p21WAF1/Cip1 in the untreated state, despite a relative abundance of p53 in the p53+/+ cells.
Upon the addition of MMS, there was a dramatic increase in the levels of GKLF and
p21WAF1/Cip1 beginning at 2 h but, only in p53+/+ MEFs (lanes 4, 6, and 8). In contrast, although
there was a p53-independent response in GKLF and p21WAF1/Cip1 production to MMS in
p53−/− cells (lanes 3, 5, and 7), this component appeared minor compared with the p53-
proficient cells. We conclude that the increase in expression of Gklf in response to MMS-
induced DNA damage, like that of p21WAF1/Cip1, is dependent on the presence of p53.
Both GKLF and p53 Transactivate the p21WAF1/Cip1 Proximal Promoter through an Identical
cis-Element
The sequential pattern of expression in Gklf followed by p21WAF1/Cip1 immediately after the
addition of MMS raised the intriguing question of whether GKLF might be responsible in part
for the induction of p21WAF1/Cip1. We considered this plausible, as the promoter of the
p21WAF1/Cip1 gene contains a number of GC-rich cis-elements that resemble Sp1-binding sites
(7,23–29), and GKLF has been shown to bind to a GC-rich DNA sequence with which Sp1
also interacts (20,21).
To determine whether GKLF regulates the p21WAF1/Cip1 promoter, we performed
cotransfection experiments in HEK 293 cells using a series of p21WAF1/Cip1 promoter-reporter
constructs (23) and an expression construct containing either wild-type GKLF or mutant GKLF
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with its zinc fingers deleted (Fig. 3A, effectors 2 and 3, respectively) (12,20,30,31). Since p53
has been shown to transcriptionally activate a reporter gene when linked to the 5′-flanking
sequence of p21WAF1/Cip1 (7,23), expression constructs containing wild-type p53 or mutant
p53 that lost its ability to bind DNA (Fig. 3A, effectors 4 and 5, respectively) (32) were also
included in the analysis. Consistent with a previous report (13), HEK 293 cells contained a
negligible amount of Gklf transcript at the base-line level as determined by reverse
transcription-PCR relative to a human colon cancer carcinoma cell line, HT29 (Fig. 3L). This
low level of base-line Gklf expression in HEK 293 cells allowed a better delineation of the
effects of GKLF on the p21WAF1/Cip1 promoter activity.
Fig. 3B shows that both wild-type GKLF and p53 (effectors 2 and 4, respectively), but not
mutant GKLF and p53 (effectors 3 and 5, respectively), transactivated the CAT reporter gene
linked to nt −2320 to +16 of the p21WAF1/Cip1 promoter sequence. However, neither wild-type
GKLF nor wild-type p53 was able to transactivate the same promoter that had a small internal
deletion in the sequence between nt −122 and −61 (Fig. 3C). These results indicate that GKLF,
like p53, is capable of activating the p21WAF1/Cip1 promoter and that in order for both proteins
to act on the promoter, the sequence between nt −122 and −61 is essential. The dependence of
p53 on this proximal region of the p21WAF1/Cip1 promoter was unexpected since the binding
sites for p53 in 2320 nt of the promoter were previously localized to sequences much farther
upstream from the immediate flanking region of the p21WAF1/Cip1 gene (43).
The sequence between nt −122 and −61 of the p21WAF1/Cip1 promoter contains four GC-rich
elements that resemble Sp1-binding sites, which have previously been designated Sp1-1 to
Sp1-4 sites in the 5′ to 3′ direction (27). To precisely define the cis-element(s) within this
sequence that mediates the activating effect of GKLF and p53 on the p21WAF1/Cip1 promoter,
we performed additional cotransfection experiments in which the CAT reporter gene was
linked to either −154 to +16 bp of the promoter (Fig. 3D) or to one that contained various 5′-
and internal deletions or point mutations (Fig. 3, E–K). It is clear from the results of these
experiments that the transactivating effects of GKLF and p53 were co-localized to an identical
cis-element, which was the first Sp1-binding site (Sp1-1 site) beginning at nt −116 in the
p21WAF1/Cip1 promoter.
GKLF, but Not p53, Binds to the Sp1-1 Element in the p21WAF1/Cip1 Promoter
To determine whether GKLF or p53 binds to the Sp1-1 sequence identified above, we
performed EMSAs between GKLF and a labeled oligonucleotide containing the sequence
between nt −129 and −99 of the p21WAF1/Cip1 promoter (Fig. 4A, p21 (wt) Probe) or between
the DBD of p53 (40,41) and an established p53-binding sequence (Fig. 4B, p53 Probe). Nuclear
extracts prepared from COS-1 cells transfected with the PMT3 expression vector containing
either full-length (FL) GKLF (Fig. 4A, lane 1) or the zinc finger (ZF) portion of GKLF (lane
9) bound to the wild-type p21 probe. The resulting DNA-protein complexes (C1 and C2) were
competed away by an unlabeled wild-type p21WAF1/Cip1 sequence (Fig. 4A, lanes 2–4 and
10–12, respectively), but not by a mutated competitor in which the Sp1-1 site was destroyed
due to a 3-bp substitution (lanes 5–7 and 13–15, respectively). As controls, nuclear extracts
prepared from either vector alone-transfected cells (C, lane 16) or cells transfected with a
mutant GKLF construct lacking the zinc fingers (ΔZF, lane 17) did not exhibit any appreciable
binding to the wild-type p21 probe. The results in Fig. 4A therefore provide strong evidence
that GKLF interacts directly with the Sp1-1 site of the p21WAF1/Cip1 promoter. In contrast, the
DNA-binding domain of p53, although clearly capable of binding to an established p53-binding
sequence (Fig. 4B, lane 2), failed to interact with the p21WAF1/Cip1 sequence since an unlabeled
wild-type p21 probe did not compete at all (lanes 6–8). Moreover, p53 DBD failed to form a
complex with a labeled wild-type p21 oligonucleotide (data not shown). These results suggest
that although the transactivating effect of p53 on the p21WAF1/Cip1 proximal promoter depends
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on the Sp1-1 site, as does GKLF, it is mediated by a mechanism that does not involve the direct
binding of p53 to the DNA.
p53 Physically Interacts with GKLF and Transcriptionally Activates the GKLF Promoter,
Resulting in a Synergistic Activation of the p21WAF1/Cip1 Promoter by p53 and GKLF
One potential method by which p53 may accomplish its indirect effect on the p21WAF1/Cip1
proximal promoter is by forming a physical complex with GKLF, thus gaining access to the
promoter. To test this hypothesis, we performed co-immunoprecipitation experiments using
in vitro synthesized p53 and GKLF produced in transfected cells. As shown in Fig. 4C, anti-
GKLF serum (α) specifically coprecipitated p53 when p53 was combined with nuclear extracts
from cells transfected with either the zinc finger region of GKLF (lane 6) or full-length GKLF
(lane 7), but not with those transfected with the PMT3 vector alone (lane 8). No p53 was
detected in any of the reactions incubated with preimmune (PI) serum (Fig. 4C, lanes 3–5).
These findings provide strong evidence for a physical interaction between p53 and GKLF in
a region that includes the zinc fingers of GKLF. It is of interest to note that only full-length
p53, but not an internally initiated p53 with an estimated molecular mass of 40 kDa (Fig. 4C,
lane 1), was recovered in the immunoprecipitates (lanes 6 and 7). This suggests that the N-
terminal portion of p53 may be necessary for the interaction with GKLF.
The dependence of Gklf induction on p53 as shown in Fig. 2 suggests that Gklf, like
p21WAF1/Cip1, is regulated by p53 during DNA damage. Indeed, the results in Fig. 4D show
that p53 transcriptionally activated Gklf since a luciferase reporter linked to 5.0 kb (bar 1), but
not 1.0 kb (bar 3), of the mouse Gklf promoter was transactivated by wild-type p53. In contrast,
a mutant p53 failed to activate either reporter (Fig. 4D, bars 2 and 4). The degree of induction
of the −5.0-kb Gklf promoter activity by p53 was comparable to that seen for 2.4 kb of the
p21WAF1/Cip1 promoter (p21 WWP-Luc) (Fig. 4D, compare bars 1 and 5). These results
therefore suggest that there is a p53-response element(s) between −5.0 and −1.0 kb of the
Gklf promoter. The exact location of this element(s) has not been determined since only the
first kb of the Gklf promoter has been sequenced so far (22) and does not contain a p53-binding
site.
To determine whether the physical interaction between GKLF and p53 and the transcriptional
induction of Gklf by p53 are physiologically relevant to the regulation of the p21WAF1/Cip1
promoter, we performed cotransfection experiments using subsaturating concentrations of
expression vectors containing GKLF, p53, or both and a luciferase reporter gene containing
2.4 kb (WWP-Luc) or 2.2 kb (DM-Luc) of the p21WAF1/Cip1 promoter sequence (7). The two
reporters differed from each other in that WWP-Luc included an upstream p53-binding site
located at nt −2301 (33). As shown in Fig. 5, the combination of GKLF and p53 resulted in a
synergistic induction of the p21WAF1/Cip1 promoter either in the presence (bar 3) or absence
(bar 6) of the upstream p53-binding sequence. These results suggest that GKLF and p53 act
in a cooperative manner to activate p21WAF1/Cip1 gene expression by a mechanism that does
not require the upstream p53-binding site of the p21WAF1/Cip1 promoter.
GKLF Is Necessary for the Inductive Effect of p53 on the p21WAF1/Cip1 Promoter
The sequential induction of Gklf and p21WAF1/Cip1 during the early phase of DNA damage and
the physical dependence of p53 on GKLF in activating the p21WAF1/Cip1 proximal promoter
raised the possibility that GKLF may be important in mediating the effect of p53 on stimulating
p21WAF1/Cip1 gene expression. To address this possibility, we examined a system in which
activation of p53 is inducible due to a temperature-sensitive mutation. As shown in Fig. 6A,
induction of wild-type p53 activity by shifting 10(1) cells stably transfected with the
temperature-sensitive p53 val135 (35,37) from the nonpermissive (38.5 °C) to the permissive
(31.5 °C) temperature resulted in a considerable accumulation of GKLF as well as
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p21WAF1/Cip1 to a degree similar to that observed in MMS-treated p53+/+ MEFs (Fig. 2). Next,
to assess the role of GKLF in mediating the inductive effect of p53 on p21WAF1/Cip1 expression,
we treated cells with a 21-nt anti-sense oligonucleotide containing the sequence that surrounds
the initiation methionine codon of GKLF in an attempt to block the translation of Gklf mRNA.
We then determined the effects of such treatments on the production of p21WAF1/Cip1 at the
permissive temperature. A sense oligonucleotide with the complementary sequence to the
antisense oligonucleotide was used as a control. As shown in Fig. 6B (lanes 3, 5, and 7), cells
treated with increasing concentrations of the antisense oligonucleotide contained progressively
lower levels of GKLF. Importantly, the same cells produced correspondingly lower levels of
p21WAF1/Cip1 as well. Although there was some decrease in the levels of GKLF (and
consequently, p21WAF1/Cip1) in cells treated with the highest concentration (3.0 μM) of the
control sense oligonucleotide (Fig. 6B, lane 6), this was probably due to a nonspecific, perhaps
toxic side effect of the high oligonucleotide concentration. Be that as it may, the results in Fig.
6B indicate that a decreased production of GKLF leads to an inhibition of p21WAF1/Cip1
synthesis. We therefore conclude that GKLF is an important mediator of the action of p53 in
inducing p21WAF1/Cip1 gene expression.
DISCUSSION
This study reveals a novel mechanism by which expression of the p21WAF1/Cip1 gene is
modulated during cellular stress induced by DNA damage. At lease five lines of evidence in
the study suggest that GKLF plays a physiologically relevant and possibly crucial role in
mediating the activating effect of p53 on p21WAF1/Cip1 expression: 1) the sequential manner
in which Gklf and p21WAF1/Cip1 are expressed in the immediate period following DNA damage
(Fig. 1C); 2) the requirement of the GKLF-response element in the p21WAF1/Cip1 promoter
(i.e. the Sp1-1 site) for the transactivating effect of p53, despite the presence of other bona
fide p53-response elements in the same promoter (Fig. 3, B and C); 3) the physical interaction
between p53 and GKLF (Fig. 4C) and the transcriptional induction of Gklf by p53 (Fig. 4D);
4) the cooperative manner in which GKLF and p53 activate the p21WAF1/Cip1 promoter (Fig.
5); and 5) the ability of antisense GKLF oligonucleotides to inhibit p21WAF1/Cip1 synthesis
upon p53 activation (Fig. 6). These findings demonstrate that p53 may depend on GKLF to
activate the p21WAF1/Cip1 promoter, thus implicating GKLF as an important component of the
p53 network of cell cycle regulators.
Based on the observations of this study, we propose a model that portrays the regulation of the
p21WAF1/Cip1 proximal promoter by GKLF and p53 during cellular stress elicited by DNA
damage. In this model, activation of p53 represents an immediate response to DNA damage
as depicted by numerous previous studies (reviewed in Refs. 1–3). The activated p53 causes
an increase in the quantity of GKLF, which is mediated, at least in part, at the level of
transcription (Fig. 4D). In addition, p53 physically interacts with GKLF and consequently
allows it to gain access to the p21WAF1/Cip1 proximal promoter through the Sp1-1 site to which
GKLF alone binds. A result of this complex relationship is the synergistic induction of the
activity of the p21WAF1/Cip1 promoter (Fig. 5). This model would therefore predict an
immediate and possibly maximal induction of expression of the gene encoding p21WAF1/Cip1
following DNA damage. This would assure the immediate cessation of cell cycle progression
due to the potent inhibitory effect of p21WAF1/Cip1 on cyclin-dependent kinases (48). It is of
note that despite the involvement of the various Sp1 elements in the p21WAF1/Cip1 promoter in
mediating the responses of the promoter to numerous other physiological stimuli (Fig. 7), the
lone utilization of the Sp1-1 site by GKLF and consequently by p53 has not previously been
documented.
The mechanism by which GKLF participates in the regulation of the p21WAF1/Cip1 promoter
by p53 is reminiscent of that for another growth arrest-associated gene, GADD45 (49). Like
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Gklf and p21WAF1/Cip1, expression of GADD45 is induced by genotoxic stresses such as DNA
damage (50). In addition to a strong p53-binding element in an intronic sequence of
GADD45 (42), p53 was shown to contribute to the stress response of the GADD45 promoter
(50). Much of this stress responsiveness was localized to a GC-rich motif of the proximal
promoter to which the tumor suppressor WT1 (Wilms’ tumor 1) (52) binds, but p53 does not.
The mechanism by which p53 activates the promoter is thought to be mediated by its ability
to physically interact with WT1 (50). This resulted in a strong and cooperative induction of
the GADD45 promoter when p53 and WT1 were concurrently introduced (50). Finally,
abrogation of WT1 function by an antisense vector markedly reduced the induction of the
GADD45 promoter (50). Similar to the conclusion of the present study, it was concluded that
p53 contributes to the positive regulation of the GADD45 promoter primarily by protein-protein
interactions.
Recent literature provides another example in which the p21WAF1/Cip1 promoter can be
cooperatively regulated by multiple proteins with important functions in cell cycle control. In
a previous study (45), BRCA1 was shown to transactivate the p21WAF1/Cip1 proximal promoter
through the region between nt −117 and −93, which contains both Sp1-1 and Sp1-2 sites. This
resulted in an inhibition of progression into the S phase in cells that overexpressed BRCA1
(45). Importantly, p53 potentiated the BRCA1-dependent activation of the p21WAF1/Cip1
promoter by physically interacting with BRCA1 (53). Thus, it appears that p53 activates
expression of its target genes such as p21WAF1/Cip1 and GADD45 by multiple but perhaps
interrelated mechanisms. These mechanisms include direct binding of p53 to the classical p53-
response elements and indirect interaction with non-consensus binding sites through physical
contacts with other regulatory proteins, including GKLF, WT1, and BRCA1.
Another potential mechanism responsible for the synergistic induction of the p21WAF1/Cip1
promoter by p53 and GKLF may involve the participation of other regulatory proteins. In this
regard, both p53 (54,55) and GKLF (31) have been shown to interact with a group of
transcriptional coactivators, including p300 and CBP (56–59). In fact, the ability of GKLF to
activate transcription is dependent on its interaction with p300/CBP (31). Thus, it is possible
to modify the model proposed in Fig. 7 to include p300/CBP, which can serve as a bridge
between p53/GKLF and the basal transcriptional machinery such as the TATA-binding factor
and RNA polymerase II (60,61). It is of interest to note that p300 and CBP are enzymes that
display histone acetylase activity (62,63) and that the activity of the p21WAF1/Cip1 promoter is
subject to regulation by compounds that alter chromatin structure due to acetylation such as
butyrate, trichostatin A, and trapoxin (Fig. 7). Moreover, the Sp1-like cis-elements responsible
for the action of these compounds appear to differ among one another (Fig. 7). It is formally
possible that the targets of regulation by these compounds may be unique transcription factors
that recognize the different Sp1-like elements in the p21WAF1/Cip1 promoter.
The results in Fig. 1 indicate that both Gklf and p21WAF1/Cip1 are induced by serum deprivation
and by DNA damage. However, the kinetics in which the two genes are induced by the two
conditions are distinctly different from each other. During the course of serum deprivation, the
extent of induction for both Gklf and p21WAF1/Cip1 is quite similar (Fig. 1A). However, the time
course of induction for Gklf and p21WAF1/Cip1 in cells treated with MMS is different from that
of serum deprivation. Thus, with the exception of an earlier induction of Gklf during the initial
30 min of MMS treatment (Fig. 1C), the level of induction of p21WAF1/Cip1 after 1 h of MMS
treatment is significantly higher than that of Gklf (Fig. 1, B and C). These results suggest that
factors in addition to GKLF may be involved in the rise in p21WAF1/Cip1 transcript level after
the immediate phase of DNA damage. However, the parallel rise in the levels of both Gklf and
p21WAF1/Cip1 transcripts during serum deprivation suggests a potentially more uniform
mechanism of induction of the two genes, perhaps including a mechanism that is independent
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of p53, as has been demonstrated in other systems (64). Experiments are in progress to address
this potentially p53-independent component of Gklf and p21WAF1/Cip1 activation.
In the intestinal tract, the Gklf transcript is detected primarily in terminally differentiated, post-
mitotic epithelial cells (12–14). Interestingly, the p21WAF1/Cip1 transcript is also distributed in
the same cell population (65). Moreover, the intestinal expression of p21WAF1/Cip1 both during
development and in the adult mouse has been shown to be independent of p53 under basal
conditions (51). Whether the in vivo expression of Gklf is also independent of p53 is unclear
at this point. However, it is clear that the induction of both Gklf and p21WAF1/Cip1 in response
to genotoxic stress is highly dependent on p53 (Fig. 2). Moreover, this inductive response is
not limited to the intestinal cell lineage and includes fibroblasts such as NIH 3T3 and MEFs.
Thus, the in vitro behavior of Gklf as modulated by stress is more ubiquitous than its in vivo
tissue distribution. This may be viewed as additional evidence for the potentially broader
significance of GKLF in mediating the “guardian” function of p53.
Acknowledgements
We thank B. Vogelstein, K. Kinzler, A. Levine, L. Donehower, and N. Palvetich for providing plasmids, reagents,
and cell lines.
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Fig. 1. Northern blot analysis of Gklf and p21WAF1/Cip1 in NIH 3T3 cells during growth arrest
Growth arrest was induced in actively proliferating NIH 3T3 cells maintained in a medium
containing 10% fetal calf serum (FCS) by the reduction of serum content to 0.5% (A) or by the
addition of 100 μg/ml MMS to the medium (B and C). RNA was isolated at the indicated time
points, and 20 μg were loaded in each lane and analyzed for the message content of Gklf,
p21WAF1/Cip1, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The bar graphs show
the quantitative information of -fold induction of Gklf (open bars) and p21WAF1/Cip1 (closed
bars) at each treatment time point over the untreated (Basal) value for each experiment. The
calculation was performed first by normalizing the band intensity of the Gklf or
p21WAF1/Cip1 transcript to that of glyceraldehyde-3-phosphate dehydrogenase at each time
point and then comparing the normalized value of Gklf or p21WAF1/Cip1 at each treatment time
point with that of untreated cells (time 0).
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Fig. 2. Western blot analysis of GKLF and p21WAF1/Cip1 in MEFs proficient or deficient in p53
MEFs were prepared from p53-deficient (−/−) mouse embryos (34) or their wild-type littermate
control (+/+) and treated with 100 μg/ml MMS for the time periods indicated. Proteins were
isolated and analyzed for the content of p53, GKLF, or p21WAF1/Cip1 by Western blot analysis.
Load represents a portion of the gel stained with Coomassie Blue before electrophoretic
transfer.
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Fig. 3. GKLF and p53 transactivate the p21WAF1/Cip1 promoter
A depicts the five effectors used throughout the cotransfection studies. Effector 1 is the PMT3
expression vector alone. Effectors 2 and 4 are expression constructs of wild-type GKLF and
p53, respectively. Effector 3 is a mutant GKLF that does not have its zinc fingers (ZF) (30).
Effector 5 is a mutant p53 with a missense mutation at codon 143 (X) in the DBD of p53
(32). Various regions of the p21WAF1/Cip1 promoter were linked to the CAT reporter (B–K)
and cotransfected with an equivalent quantity of the various effectors into HEK 293 cells. The
four Sp1-binding sites (27) between nt −122 and −61 of the promoter are represented by the
four arrowheads. The locations for Sp1-1 and Sp1-2 are identified in D. The × in J and K
represents a 3-bp mutation in the first and second Sp1-binding sites, respectively. The
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numbers on the x axis in D–K correspond to the five effectors shown in A. C is the substrate
chloramphenicol, and AC represents the acetylated product. % Conversion = (AC/(AC + C))
× 100. Shown in D–K are the means of three independent experiments. Bars represent S.D.
L shows the results of reverse transcription-PCR of the mRNA levels of Gklf and β-actin in
HT29 and HEK 293 cells.
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Fig. 4. Relationship among p53, GKLF, and the Sp1-1 site of the p21WAF1/Cip1 promoter
A, GKLF binds to the Sp1-1 site. EMSAs were performed using nuclear extracts prepared from
COS-1 cells transfected with an expression construct containing the full-length (FL) GKLF
(lanes 1–7) or the zinc finger (ZF) region of GKLF (lanes 9–15) and a radiolabeled
oligonucleotide probe containing the sequence between nt −129 and −99 of the p21WAF1/Cip1
promoter, which includes both Sp1-1 and Sp1-2 sites (27). Where indicated, increasing
amounts of unlabeled oligonucleotides representing either the wild-type (wt) sequence or a
mutated (mut) sequence that contains a 3-bp substitution in the Sp1-1 site were included. Lane
8 contains the probe alone without added proteins. Lanes 16 and 17 contain nuclear extracts
obtained from COS-1 cells transfected with the PMT3 vector alone (C) and the GKLF construct
that lacks the zinc fingers (ΔZF) as in Fig. 3A, respectively. C1 is the complex formed between
full-length GKLF and the probe, and C2 is formed between the zinc fingers and the probe. F
is free probe. B, p53 does not interact with the sequence between nt −129 and −99 of the
p21WAF1/Cip1 promoter. EMSAs were performed with the purified DBD of p53 (40) and a
labeled probe representing an established p53-binding site. Competitors include unlabeled p53-
binding sequence (lanes 3–5) and unlabeled wild-type p21WAF1/Cip1 sequence between nt −129
and −99 (lanes 6–8). C3 indicates the complex between p53 DBD and the probe. C, GKLF
interacts with p53. 35S-Labeled p53 synthesized by in vitro transcription and translation was
mixed with nuclear extracts from COS-1 cells transfected with PMT3-GKLF(ZF), PMT3-
GKLF(FL), or PMT3 vector alone and precipitated with either preimmune (PI) serum or anti-
GKLF serum (α). Lane 1 (*) contains the input p53, and lane 2 is p53 mixed with protein A-
Sepharose beads without added serum. The precipitated materials were resolved by denaturing
polyacrylamide gel electrophoresis and visualized by autoradiography. D, p53 transactivates
the Gklf promoter. Either 5.0 or 1.0 kb of the 5′-flanking sequence of the mouse Gklf gene was
linked to a luciferase reporter and cotransfected into Chinese hamster ovary cells with an
expression construct containing either wild-type p53 or mutant p53 that no longer binds DNA
(see Fig. 3A). Included was a p21 WWP-Luc construct containing 2.4 kb of the
p21WAF1/Cip1 promoter sequence linked to the luciferase reporter as a control (7). Shown are
the means of four experiments. Bars are S.D.
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Fig. 5. Synergistic activation of the p21WAF1/Cip1 promoter by GKLF and p53
Cotransfection experiments were performed in HEK 293 cells with a luciferase reporter linked
to 2.4 or 2.2 kb of the p21WAF1/Cip1 promoter sequence (WWP-Luc, which contains an
upstream p53-binding site at nt −2301, or DM-Luc, which does not, respectively (7 and 33))
and subsaturating amounts of expression constructs containing GKLF, p53, or both. Shown
are the means of four independent experiments. Bars represent S.D.
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Fig. 6. GKLF mediates the inductive effect of p53 on p21WAF1/Cip1
A, induction of Gklf and p21WAF1/Cip1 in 10(1) cells (36) containing a temperature-sensitive
mutant p53 protein. 10(1) cells stably expressing the temperature-sensitive p53 val135 mutant
(35,37) were propagated at either the nonpermissive temperature of 38.5 °C or the permissive
temperature of 31.5 °C for the time periods indicated. Proteins were harvested and analyzed
for p53, GKLF, or p21WAF1/Cip1 by Western blot analysis. Both GKLF and p21WAF1/Cip1 were
absent at time 0 when cells were maintained at 38.5 °C (data not shown). B, inhibition of
p21WAF1/Cip1 formation in the 10(1)-p53 val135 cell line by antisense oligonucleotides to
GKLF. Cells were transfected by lipofection with increasing amounts of sense (S) or antisense
(AS) oligonucleotides to GKLF at 38.5 °C for 5 h and shifted to 31.5 °C for an additional 24
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h before being harvested for quantification of p53, GKLF, or p21WAF1/Cip1 by Western blot
analysis.
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Fig. 7. Model for the regulation of the p21WAF1/Cip1 proximal promoter by p53 and GKLF
The locations of the six Sp1-like elements within −154 bp of the p21WAF1/Cip1 promoter are
designated according to a previous report (27). The model illustrates that the activation of p53
by DNA damage leads to both an increase in GKLF synthesis and an interaction between p53
and GKLF (double arrow), which cumulates in the binding of GKLF to the Sp1-1 element of
the p21WAF1/Cip1 promoter. The various Sp1 cis-elements that mediate the functions of other
physiological stimuli are also indicated. They include the phorbol ester phorbol 12-myristate
13-acetate (PMA) and okadaic acid (OA) (23); trapoxin (TPX), a histone deacetylase inhibitor
(44); BRCA1, the breast cancer tumor suppressor gene (45); transforming growth factor-β
(TGF-β) (24); Ca2+, which is important in keratinocyte differentiation (28); a
geranylgeranyltransferase I (GGTI) inhibitor (46); butyrate (27) and trichostatin A (TSA)
(29), both also histone deacetylase inhibitors; levostatin, a 3-hydroxy-3-methylglutaryl-
coenzyme A reductase inhibitor (47); and progesterone (43).
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