p.1-THE GUT-ENRICHED KRÜPPEL-LIKE FACTOR (KRÜPPEL-LIKE FACTOR 4) MEDIATES THE TRANSACTIVATING EFFECT OF p53 ON THE p21 WAF1/Cip1 PROMOTER

p21 , gene. We show that the gene encoding gut-enriched Krüppel-like factor (GKLF; KLF4) is concurrently induced with p21 WAF1/Cip1 during serum deprivation and DNA damage elicited by methyl methane sulfonate (MMS). The increases in expression of both GKLF and p21 WAF1/Cip1 due to DNA damage are dependent on p53. Moreover, during the first 30 minutes of MMS treatment, the rise in GKLF mRNA level precedes that in p21 WAF1/Cip1 , suggesting that GKLF may be involved in the induction of p21 WAF1/Cip1 . Indeed, GKLF activates p21 WAF1/Cip1 through a specific Sp1-like cis -element in the proximal p21 WAF1/Cip1 promoter. The same element is also required by p53 to activate the p21 WAF1/Cip1 promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21 WAF1/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 in the p21 WAF1/Cip1 promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21 WAF1/Cip1 is demonstrated by the ability of antisense GKLF oligonucleotides to block the production of p21 WAF1/Cip1 in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21 WAF1/Cip1 and may be part of a novel pathway by which cellular responses to stress are modulated. The established binding site for GKLF is rich in GC content and overlaps with that for the transcription factor Sp1 By coincidence, the proximal promoter of the p21 WAF1/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 p21 WAF1/Cip1 in several physiologically diverse processes, including the gene’s responsiveness to phorbol ester (23), transforming growth factor- β (TGF- β ) (24-26), and sodium butyrate (27), and in keratinocyte differentiation (28). As both GKLF and p21 WAF1/Cip1 are growth arrest-associated genes, we sought to determine whether GKLF is involved in regulating p21 WAF1/Cip1 expression. We demonstrate that GKLF not only transactivates the p21 WAF1/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. effect of p53 p21 p.35-The locations of the 6 Sp1-like elements within –154 bp of the p21 WAF1/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 p21 WAF1/Cip1 promoter. The various Sp1 cis -elements that mediate the functions of other physiological stimuli are also indicated. They include the phorbol ester, 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); Ca ++ , which is important in keratinocyte differentiation (28); GGTI , a geranylgeranyltransferase I inhibitor (46); butyrate (27) and trichostatin A (TSA) (29), both also histone deacetylase inhibitors; levostatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (47); and progesterone (43).


INTRODUCTION
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)(2)(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, an important consequence of p53 activation is the transcriptional induction of the gene encoding the cyclin-dependent kinase (Cdk) inhibitor p21 (also called WAF1 or Cip1) (7,8). p21 WAF1/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 or KLF4; reference 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)(13)(14), skin (14,15), and thymus (16), and in vascular endothelial cells (17). Both the in vivo (12)(13)(14)(15)(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)(13)(14)(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 p21 WAF1/Cip1 gene contains a number of GC-rich elements (7), some of which have been shown to bind Sp1 (23)(24)(25)(26)(27)(28)(29). These Sp1-binding sites have been shown to be important in controlling expression of p21 WAF1/Cip1 in several physiologically diverse processes, including the gene's responsiveness to phorbol ester (23), transforming growth factor-β (TGF-β) (24)(25)(26), and sodium butyrate (27), and in keratinocyte differentiation (28). As both GKLF and p21 WAF1/Cip1 are growth arrest-associated genes, we sought to determine whether GKLF is involved in regulating p21 WAF1/Cip1 expression. We demonstrate that GKLF not only transactivates the p21 WAF1/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.

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 included full-length GKLF [PMT3-GKLF-(1-483)], truncated GKLF that lacked the three zinc fingers [PMT3-GKLF-(1-401)], and truncated GKLF that contained the zinc fingers only [PMT3-GKLF-(350-483)]. pC53-SN3 and pC53-SX3, two CMV-based expression constructs containing wild-type p53 and mutated p53 with a missense mutation at codon 143 (m143) 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 p21 WAF1/Cip1 promoter to chloramphenicol acetyl transferase (CAT) have previously been described (23). They included the CAT reporter linked to either 2,320 nt 5'-flanking sequence of the p21 WAF1/Cip1 gene that contained an upstream p53-binding site at nt -2301 (33) or the same 2,320 nt 5'-flanking sequence with a small internal deletion of the sequence between nt -122 and -61 of the p21 WAF1/Cip1 promoter which removed the first 4 of the 6 Sp1 sites from the proximal promoter (33). Reporter constructs containing the proximal promoter region of the p21 WAF1/Cip1 gene with various 5' endpoints 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 were two luciferase reporters that contained 2.4 and 2.2 kb, respectively, of the 5'-flanking sequence of the p21 WAF1/Cip1 gene and were kindly provided by Vogelstein and Kinzler (7). The DM-Luc construct lacked the upstream p53binding site at nt -2301 in the p21 WAF1/Cip1 promoter (7). The polyclonal rabbit anti-GKLF serum was described (12). Anti-p53 serum was purchased from Santa Cruz (#sc-6243) and the monoclonal antibody against p21 WAF1/Cip1 was purchased from Pharmingen (SXM30). The p53 -/-and +/+ mouse embryonic 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 lacked endogenous p53 expression but contained a stably transfected temperature-sensitive p53 protein, val135 (37). At the nonpermissive temperature of 38.5 o C, p53 val135 is transcriptionally inactive; whereas at the permissive temperature of 31.5 o C, it is transcriptionally competent (35). The sense and antisense oligonucleotides to GKLF contained nucleotide sequences corresponding to aa codons 7 to 13 of GKLF in the sense and antisense orientation, respectively. At the center of this sequence, aa position 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 was 5'-GCT GAC AGC CAT GTC AGA CTC -3' and that of the sense oligonucleotide was 5'-GAG TCT GAC ATG GCT GTC AGC -3'. Note that the underlined sequence represents the initiation methionine codon at aa position 10 (12).

Conditions of cell treatments, and Northern and Western blot analyses
For the serum deprivation experiments, the content of fetal calf serum (FCS) in the cell media was reduced from 10% to 0.5% to induce a growth-arrested state (12). To cause DNA damage, methyl methane sulfonate (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 Technology). Twenty µg 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), p21 WAF1/Cip1 (7), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Clontech). The conditions for Western blot analysis were also previously described, using a 1:1,000 dilution of an affinity-purified polyclonal anti-GKLF serum (12).

Reverse transcription-polymerase chain reactions (RT-PCR)
RNA was extracted from human embryonic kidney (HEK) 293 cells and the human colonic carcinoma HT29 cells (39). The content of GKLF transcript from each cell line was determined using RT-PCR. The content of the β-actin transcript was similarly determined as a control. One µg RNA was reverse transcribed in an 80 µl volume containing 50 mM Tris-HCl,

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 (FL) GKLF, truncated GKLF containing only the zinc fingers (ZF) or lacking the zinc fingers (∆ZF), or PMT3 vector alone was previously described (20,21). Purified p53 containing the DNA-binding domain (DBD) was kindly provided by N. Pavletich (40). This domain contained the core portion of p53 between aa residues 102-292, which bound 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 E. coli BL21(D3) cells was previously described (40). The protein was supplied at a concentration of 14 mg/ml in a solution of 50 mM bistrispropane-HCl, pH 6.8, 200 mM Na phosphate, and 5 mM DTT and had a >98% purity of the core domain. The wild-type p21 oligonucleotide used in EMSA contained the sequence between nt -129 and -99 of the p21 WAF1/Cip1 promoter, which included both Sp1-1 and Sp1-2 sites (27). The mutant p21 oligonucleotide contained 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 p53response sequence in the promoter of the human GADD45 gene (42)

Both GKLF and p21 WAF1/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 Figure 1A recapitulate this event. Figure 1A also shows that upon serum deprivation, the levels of the p21 WAF1/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 methyl methane sulfonate (MMS), which causes DNA damage and subsequently cell cycle arrest (38). As seen in Figure 1B, the levels of GKLF mRNA were increased 2 hr after the addition of MMS, as were those of p21 WAF1/Cip1 mRNA. When normalized to the expression of the control GAPDH gene, which was not affected by the treatment, the degree of induction in p21 WAF1/Cip1 was higher (between 1.6-and 2.8-fold) than that in GKLF between 2 and 8 hr of MMS treatment ( Figure 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 p21 WAF1/Cip1 ( Figure 1C). These results suggest that both GKLF and p21 WAF1/Cip1 respond similarly to signals elicited during growth arrest due to DNA damage.
However, the induction of GKLF begins slightly earlier than that of p21 WAF1/Cip1 during the initial phase of DNA damage.

The induction of GKLF and p21 WAF1/Cip1 by MMS is dependent on p53
To determine whether the inductive responses of GKLF and p21 WAF1/Cip1 to MMS treatment are dependent on p53, we compared the expression of the two genes in mouse embryonic fibroblasts (MEFs) isolated from mice that contained (p53 +/+) and lacked p53 (p53 - Both GKLF and p53 transactivate the p21 WAF1/Cip1 proximal promoter through an identical ciselement The sequential pattern of expression in GKLF followed by p21 WAF1/Cip1 immediately after the addition of MMS raised the intriguing question whether GKLF might be responsible in part for the induction of p21 WAF1/Cip1 . We considered this plausible as the promoter of the p21 WAF1/Cip1 gene contains a number of GC-rich cis-elements that resemble Sp1-binding sites (7,(23)(24)(25)(26)(27)(28)(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 p21 WAF1/Cip1 promoter, we performed cotransfection experiments in human embryonic kidney (HEK) 293 cells using a series of p21 WAF1/Cip1 promoter-reporter constructs (23) and an expression construct containing either the wild-type or a mutated GKLF that had its zinc fingers deleted ( Figure 3A, effectors 2 and 3,  (12,20,30,31). Since p53 has been shown to transcriptionally activate a reporter gene when linked to the 5' flanking sequence of p21 WAF1/Cip1 (7,23), expression constructs containing the wild-type or a mutated p53 that lost its ability to bind DNA ( Figure 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 baseline level as determined by RT-PCR, relative to a human colon cancer carcinoma cell line, HT29 ( Figure 3L).
This low level of baseline GKLF expression in HEK 293 cells allowed a better delineation of the effects of GKLF on the p21 WAF1/Cip1 promoter activity. Figure 3B shows that both wild-type GKLF and p53 (effectors 2 and 4, respectively), but not mutated GKLF and p53 (effectors 3 and 5, respectively), transactivated the CAT reporter gene linked to nt -2,320 to +16 of the p21 WAF1/Cip1 promoter sequence. However, neither wildtype 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 ( Figure 3C). These results indicate that GKLF, like p53, is capable of activating the p21 WAF1/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 p21 WAF1/Cip1 promoter was unexpected since the binding sites for p53 in 2,320 nt of the promoter were previously localized to sequences much further upstream from the immediate flanking region of the p21 WAF1/Cip1 gene (43).
The sequence between nt -122 and -61 of the p21 WAF1/Cip1 promoter contains 4 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 +16 bp of the promoter ( Figure 3D) or to one that contained various 5' and internal deletions, or point mutations (Figures 3E-3K). 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 p21 WAF1/Cip1 promoter.

GKLF but not p53 binds to the Sp1-1 element in the p21 WAF1/Cip1 promoter
To determine whether GKLF or p53 binds to the Sp1-1 sequence identified above, we performed electrophoretic mobility shift assays (EMSAs) between GKLF and a labeled oligonucleotide containing the sequence between nt -129 and -99 of the p21 WAF1/Cip1 promoter [p21 (wt) Probe; Figure 4A], or between the DNA-binding core domain (DBD) of p53 (40,41) and an established p53-binding sequence (p53 Probe; Figure 4B). Nuclear extracts prepared The dependence of GKLF induction on p53 as seen in Figure 2 suggests that GKLF, like p21 WAF1/Cip1 , is regulated by p53 during DNA damage. Indeed, the results in Figure 4D show that p53 transcriptionally activates GKLF since a luciferase reporter linked to 5.0 ( Figure 4D The two reporters differed from each other in that WWP-Luc included an upstream p53-binding site located at nt -2,301 (33). As shown in Figure 5 promoter raised the possibility that GKLF may be important in mediating the effect of p53 on stimulating p21 WAF1/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 Figure   6A, induction of wild-type p53 activity by shifting 10 (1) Figure 6B, lane 6), this was probably due to a non-specific, perhaps toxic side-effect of the high oligonucleotide concentration. Be that as it may, the results in Figure 6B indicate that a decreased production of GKLF leads to an inhibition of p21 WAF1/Cip1 synthesis.
We, therefore, conclude that GKLF is an important mediator of the action of p53 in inducing p21 WAF1/Cip1 gene expression. following DNA damage. This would assure the immediate cessation of cell cycle progression due to the potent inhibitory effect of p21 WAF1/Cip1 on cyclin-dependent kinases (48). It is of note that despite the involvement of the various Sp1 elements in the p21 WAF1/Cip1 promoter in mediating the responses of the promoter to numerous other physiological stimuli (Figure 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 p21 WAF1/Cip1 promoter by p53 is reminiscent of that for another growth arrest-associated gene, GADD45 (49).
Like GKLF and p21 WAF1/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 (51), 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, Wilm's Tumor 1 (WT1; 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, the authors concluded that p53 Importantly, p53 potentiated the BRCA1-dependent activation of the p21 WAF1/Cip1 promoter by physically interacting with BRCA1 (53). Thus, it appears that p53 activates expression of its target genes such as p21 WAF1/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 p21 WAF1/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 co-activators, including p300 and CBP (56)(57)(58)(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 Figure 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/CBP are enzymes that display histone acetylase activity (62,63) and that the activity of the p21 WAF1/Cip1 promoter is subject to regulation by compounds that alter chromatin structure due to acetylation such as butyrate, trichostatin A (TSA), and trapoxin (TPX) (Figure 7). Moreover, the Sp1-like cis-elements responsible for the action of these compounds appear to differ among one another (Figure 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 p21 WAF1/Cip1 promoter.
Results in Figure 1  In the intestinal tract, GKLF transcript is detected primarily in the terminally differentiated, post-mitotic epithelial cells (12)(13)(14). Interestingly, p21 WAF1/Cip1 transcript is also distributed in the same cell population (65). Moreover, the intestinal expression of p21 WAF1/Cip1 both during development and in the adult mouse has been shown to be independent of p53 at basal condition (66). 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 p21 WAF1/Cip1 in response to genotoxic stress are highly dependent on p53 ( Figure 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 an additional evidence for the potentially broader significance of GKLF in mediating the "guardian" function of p53.    were performed with nuclear extracts obtained from COS-1 cells transfected with the PMT3 vector alone (C) and the GKLF construct that lacked the zinc fingers (∆ZF) as in Figure 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. with an expression construct containing either wild-type (wt) or a mutated (mut) p53 that no longer binds DNA (see Figure 3A). Included was a p21 WWP-Luc construct that contained 2.