From the Gastrointestinal Unit and
§ Hematology-Oncology Unit, Massachusetts General Hospital,
Harvard Medical School, Boston, Massachusetts 02114
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ABSTRACT |
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The Krüppel-like family of transcription factors comprises genes that appear to have tissue-restricted functions. Expression of gut-enriched Krüppel-like factor (GKLF) may be important in the switch from proliferation to differentiation in the squamous epithelium. We sought to determine transcriptionally mediated effects of GKLF on two promoters active in the esophageal squamous epithelium, namely the Epstein-Barr virus ED-L2 and human keratin 4 promoters. Both promoters contain a CACCC-like motif previously shown to bind GKLF. To determine whether GKLF regulates genes containing this element, we first demonstrated expression and then cloned the full-length human GKLF from an esophageal squamous carcinoma cell line. In a transient transfection system, GKLF increased the activity of both promoters >25-fold, localized to regions containing the CACCC-like element. Recombinant GKLF specifically binds the CACCC-like motif in both promoters. GKLF epitope-tagged protein leads to the formation of two proteins of 65 and 34 kDa. The chromatographically purified 65-kDa protein binds the CACCC-like element from both Epstein-Barr virus ED-L2 and keratin 4 promoters, which is not attenuated by the 34-kDa protein. In summary, GKLF is expressed in esophageal squamous epithelial cells and transcriptionally activates two esophageal epithelial promoters important at the transition toward differentiation.
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INTRODUCTION |
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Zinc finger transcription factors bind DNA through motifs that
contain a zinc atom bound to 4 amino acids, either cysteine or
histidine (1, 2). While encompassing a broad and diverse group of
proteins, zinc finger transcription factors also can be further
subclassified based upon their homology to the Drosophila Krüppel protein (3). There is compelling evidence that a certain group of Krüppel-like transcription factors share structural and
functional similarities and include erythroid Krüppel-like factor
(EKLF)1 (4), lung
Krüppel-like factor (LKLF) (3), gut-enriched Krüppel-like
factor (GKLF) (5, 6) also referred to as epithelial zinc finger or EZF
(7), basic transcription element binding-protein 2 (8), and basic
Krüppel-like factor (9). These factors share zinc finger domains
at the carboxyl terminus and transactivation domains at the amino
terminus. Expression is relatively tissue-restricted based upon
Northern blot analysis and RNA in situ hybridization studies. For example, EKLF is found predominantly in erythroid cells of
the bone marrow and spleen (4); LKLF is found in the lung epithelium,
hematopoeitic organs, and testis (3); and GKLF is found in epithelial
cells of the gastrointestinal tract (intestine and esophagus), lung,
testis, and skin (5, 7). These factors have DNA binding domains that
bind the cognate CACCC motif or CACCC-like variants, including CACACCC
(3, 7, 10). EKLF binds the CCACACCCT site in the mouse and
human -globin promoters (10). A pSG5-EKLF expression vector
transactivates a CACCC site-containing reporter in CV-1 cells 13-fold
(10). Similarly, LKLF can transactivate a human
-globin promoter
(3).
An emerging theme among the family of Krüppel-like transcription factors is their involvement in potentiating cell differentiation or quiescence. Targeted disruption of LKLF in mouse embryonic stem cells supports the notion that LKLF is critical in maintaining single positive T cells in a state of quiescence (11). Expression of GKLF is high in growth-arrested fibroblasts and nearly absent in cells in an exponential phase of proliferation (5). Constitutive expression of GKLF leads to inhibition of DNA synthesis (5). The role of GKLF in regulation of cellular growth is further supported by cellular localization studies which indicate that GKLF mRNA is abundant in the middle to upper crypt region of the colonic mucosa, an area in which proliferating cells make a commitment to early differentiation (5). Additionally, GKLF localizes to suprabasal cells in skin, tongue, and esophageal squamous epithelial cells (7). Proliferating basal cells in these epithelia lose this capacity and begin to differentiate in the suprabasal layer where GKLF is expressed. The localization of the GKLF gene on chromosome 9q22 has led to the speculation that dysregulation of GKLF gene expression may play a role in the pathogenesis of squamous epithelium-derived neoplasms which have been shown to have abnormalities in this chromosomal region (12-14).
While it has been demonstrated that EKLF and LKLF transactivate the
-globin promoter, gene targets for GKLF transactivation have yet to
be identified. Given the localization of GKLF in the suprabasal
esophageal squamous epithelium and the role of the Krüppel-like
transcription factors in regulating cellular differentiation and
quiescence, we postulated that genes expressed during the transition
toward early differentiation may be transactivated by GKLF. Two such
candidates are the Epstein-Barr virus ED-L2 (15, 16) and the keratin 4 promoters.
Previous work has shown that the ED-L2 promoter is active in esophageal squamous suprabasal cells of transgenic mice (17, 18). The ED-L2 promoter is basally regulated by a novel zinc-dependent nuclear protein that binds a CACCC-like motif (19). Phorbol ester leads to enhanced activation of the ED-L2 promoter through the binding of an E-box by upstream stimulatory factor by USF and a zinc-dependent factor that interacts specifically with CACACCC (20). Keratin 4 is also highly expressed in esophageal squamous suprabasal cells, and its expression is associated with the switch to differentiation that occurs as cells exit the proliferation zone (21, 22). Sequence analysis of the keratin 4 promoter (GenBankTM accession number X97566) reveals a GTGTGGG or inverted CACACCC motif in the proximal 5'-untranslated region. We investigated whether ED-L2 and K4, expressed in suprabasal squamous epithelial cells of the esophagus, are transactivated by GKLF, thereby providing a basis for GKLF's role in vivo.
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EXPERIMENTAL PROCEDURES |
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Cloning and Analysis of the Human GKLF cDNA from the Human
Esophageal Squamous Cancer Cell Line TE-11--
Oligonucleotide
primers were designed to amplify the zinc finger-encoding region of the
transcription factor GKLF, yielding an 840-base pair (bp) polymerase
chain reaction (PCR) product. The oligonucleotide primer sequences are
as follows: GKLF-S, 5'-AGTTGGACCCAGTATACATTCCGCCACAGCAGCCT-3'; GKLF-AS, 5'-TTAAAAGTGCCTCTTCATGTGTAAGGCAAGGTGGT-3'. Total RNA was
extracted from lysates of subconfluent 293, TE-11, and 3T3 cells with a
denaturing solution consisting of 4 M guanidinium thiocyanate, 0.1 M -mercaptoethanol, 0.5% sarcosyl,
25 mM sodium citrate (pH 7.0), and 10% volume of 2 M sodium acetate (pH 4.0). The cell lysate was mixed
with a 5:1 ratio of water-saturated phenol and chloroform/isoamyl
alcohol mixture (49:1), and incubated at 4 °C for 15 min. After
centrifugation, the aqueous phase was precipitated with ethanol, and
RNA was dissolved in denaturing solution. It was then reprecipitated
with ethanol, washed with 80% ethanol, and redissolved in water
treated with 0.1% diethyl pyrocarbonate. An aliquot of total RNA was
poly(A)-selected (polyAT tract mRNA isolation system III; Promega
Corp.).
Construction of the GKLF Epitope-tagged Expression Vector-- The pBK-CMV plasmid containing the full-length GKLF cDNA was digested with BamHI to yield a 1.4-kb fragment containing the full-length open reading frame of GKLF. This fragment was ligated into pcDNA3.1/His B (Invitrogen) in frame and 3' to the HIS6 and Anti-Xpress epitope tag coding sequence. The GKLF expression construct is represented in the schematic shown in Fig. 1A. The GKLF expression plasmid as well as pcDNA3.1/His B and pcDNA3.1/His-lacZ plasmids (Invitrogen) were purified by a modified alkaline lysis method (Qiagen).
Northern and Western Blot Analysis of GKLF Expression in the 293 Cell Line-- GKLF-nonexpressing 293 cells were transiently transfected with the different pcDNA3.1/His plasmids, and total RNA was harvested 24 h after transfection, using methods previously described (20). RNA concentration was determined by spectrophotometry. 20 µg of total RNA was resuspended in sample buffer consisting of 50% deionized formamide, 6.7% formaldehyde, 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA (pH 8.0) and heated at 60 °C for 10 min. Electrophoresis was performed at 20 V for 2 h followed by 40 V for 4 h with buffer consisting of 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA (pH 8.0). The RNA was transferred onto a Hybond N nylon membrane (Amersham Pharmacia Biotech) followed by UV cross-linking (Stratalinker, Stratagene). The 840-bp probe used for GKLF cloning was used for Northern blot analysis and was labeled using a random primed labeling method (Amersham Pharmacia Biotech). To assess equivalent loading of RNA samples, equal amounts of 18 S and 28 S RNA were identified in each lane. Northern blot hybridization was carried out using Rapid-hyb buffer (Amersham Pharmacia Biotech) and the conditions described above.
For Western blot analysis, lysates from transfected 293 cells were prepared in a lysis buffer with protease inhibitors as described previously (25). 100 µg of total protein from each sample was separated on a 10% SDS-polyacrylamide gel. Following electrophoresis, the protein was transferred to an Immobilon membrane (Millipore Corp.) at 10 V for 12 h at 4 °C. The membrane was treated with 5% milk, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% Tween 20 for 1 h. The primary antibody (Anti-Xpress, Invitrogen) was used at a 1:4000 dilution, and the secondary antibody, horseradish peroxidase-conjugated goat anti-mouse (Amersham Pharmacia Biotech), was used at a 1:2500 dilution. Horseradish peroxidase activity was detected with a chemiluminescence system (ECL system, Amersham Pharmacia Biotech).Tissue Culture Cell Lines and Transient Transfection Studies-- The human esophageal squamous carcinoma TE-11, 3T3 fibroblast (ATCC), and human embryonic kidney 293 cell lines (ATCC) were cultured under standard conditions with Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum (Sigma), 100 units/ml penicillin, 100 µg/ml streptomycin (Sigma), and 2 mM L-glutamine (Sigma).
After PCR amplification with primers specific to the ED-L2 and K4 promoter sequences and confirmatory DNA sequencing of PCR products, fragments of the ED-L2 promoter and K4 promoter were agarose gel-purified and directionally ligated into the luciferase reporter gene promoterless vector pXP2 (26) to generate luciferase reporter constructs. The oligonucleotide sequences listed in Table I were used to generate promoter deletion constructs with PCR conditions previously described (20). The resulting luciferase reporter constructs (ED-L2Affinity and Bio-Gel P-60 Column Purification of Recombinant GKLF Fusion Protein-- Following transient transfection of 293 cells with 2 µg of the GKLF expression construct, total cellular protein was harvested after 24 h for purification of the GKLF histidine-tagged fusion protein. Cells were mechanically detached from the plates and resuspended in a total of 4 ml of native binding buffer (20 mM NaH2PO4, 500 mM NaCl, pH 7.8) with aprotinin (5 µg/ml) and phenylmethanesulfonyl fluoride (100 µg/ml). Cells were lysed with two cycles of freezing-thawing, followed by passage through an 18-gauge needle four times.
ProBond affinity resin columns (Invitrogen) were equilibrated for purification of the histidine-tagged protein according to the manufacturer's specifications. 293 cell lysate was added to the equilibrated affinity column in a volume of 4 ml of native binding buffer. The column was sealed at both ends and incubated at 4 °C for 1 h with gentle agitation. The column was then packed, and the supernatant was removed. Next, the column was washed twice in wash buffer (20 mM sodium phosphate, 500 mM NaCl, pH 6.0) with aprotinin (5 mg/ml) and phenylmethanesulfonyl fluoride (100 mg/ml). The recombinant GKLF fusion protein was eluted by adding 4 ml of elution buffer (20 mM sodium phosphate, 500 mM NaCl, pH 4.0) with aprotinin (5 µg/ml) and phenylmethanesulfonyl fluoride (100 µg/ml) and incubating at 4 °C for 15 min with gentle agitation. The final elution volume of 4.0 ml was concentrated to 1.0 ml by vacuum centrifugation at 4 °C. Separation of the affinity column-purified 65- and 34-kDa GKLF fusion proteins was accomplished using gel filtration chromatography (27). Bio-Gel P-60 (Bio-Rad) was hydrated for 24 h in elution buffer, suspended in a 2-fold excess of buffer, and degassed for 15 min. A 50% slurry of gel was then allowed to settle in a 7.5-ml column. A 200-µl solution of bovine serum albumin (66 kDa) and ovalbumin (40 kDa) in elution buffer (1 mg/ml) was passed through the column to calibrate and confirm the efficiency of size separation of these two proteins. After clearing the column with 5 ml of elution buffer, 200 µl of recombinant GKLF solution was passed over the column and twenty 200-µl fractions were collected. Purified protein concentration was determined by a colorimetric method (Bio-Rad protein assay). Qualitative purity of protein was assessed by a silver stain method (Bio-Rad) after 5 µg of total protein from each fraction was separated on a 10% SDS-polyacrylamide gel (28).Electrophoretic Mobility Shift Assays (EMSAs)--
Purified
recombinant GKLF protein was prepared as described above for use in
EMSAs, except the buffers were supplemented with a mixture of 0.5 µg/ml protease inhibitors aprotinin, chymostatin, and pepstatin
(Boehringer Mannheim). 5 pmol of a double-stranded oligonucleotide,
synthesized by the phosphoramidite procedure (Applied Biosystems) and
purified by gel electrophoresis, was radiolabeled by the Klenow fill-in
reaction in a buffer consisting of 10 mM Tris-HCl (pH 7.5),
5 mM MgCl2, 7.5 mM dithiothreitol, 33 µM dATP, 33 µM dGTP, 33 µM
dTTP, 0.33 µM [-32P]dCTP (NEN Life
Science Products), and 1 unit of DNA polymerase I Klenow fragment
(Amersham Pharmacia Biotech). The oligonucleotide (sense) sequences
derived from the ED-L2 and K4 promoters are shown in Table I with the
putative GKLF binding motif in boldface type. The 5'-end of the ED-L2
sense oligonucleotide corresponds to ED-L2 promoter position
135, and
that of the K4 oligonucleotide corresponds to K4 promoter position
281. At the 5'-end of each oligonucleotide, a BamHI
restriction site was added to facilitate Klenow fill-in labeling.
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RESULTS |
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GKLF Is Expressed in the TE-11 Human Esophageal Squamous Cancer Cell Line and Not in the 293 Human Embryonic Kidney Cell Line-- Our previous studies suggest that tissue-restricted zinc-dependent nuclear factors are important in regulating gene expression in the esophageal squamous epithelium (19, 20). The Epstein-Barr virus ED-L2 promoter is highly active in cells derived from this tissue compartment, as a result of transactivation by a zinc-dependent factor designated keratinocyte-specific factor (19), and a phorbol ester-induced zinc-dependent factor (20). The recent description of GKLF led us to test the hypothesis that this factor may play a prominent role in the tissue-specific regulation of gene expression in the esophageal squamous epithelium.
Prior studies demonstrated that GKLF is expressed in the normal adult murine squamous epithelium (7). We initially determined whether TE-11 cells expressed GKLF using RT-PCR. In addition, we tested whether 293 cells and 3T3 fibroblasts express GKLF. As previous studies have shown, 3T3 fibroblasts express GKLF as determined by RT-PCR designed to yield an 840-base pair PCR product encompassing the zinc finger domain of GKLF (7). Concurrent RT-PCR reactions with RNA template from TE-11 and 293 cells yielded positive and negative results, respectively (data not shown). Based on these results, we chose to pursue a cloning strategy designed to obtain the full-length human cDNA of GKLF and any other Krüppel-like factor genes that might share homology in the zinc finger domain. Using the 840-bp human GKLF sequence as a probe under relatively low stringency conditions, a TE-11 cDNA library was screened. Approximately 30 individual positively hybridizing clones were identified, 50% of which had sequence identical to the GKLF published sequence. No other genes were identified that shared homology with GKLF, a result consistent with previous attempts to identify closely related Krüppel-like factor genes (11). One full-length GKLF clone was selected for further analysis. A BamHI fragment containing the entire open reading frame of GKLF was placed in frame into the expression vector pcDNA3.1/His B. This vector contains a HIS6 and epitope tag 5' to the inserted GKLF cDNA (Fig. 1A). The GKLF fusion construct was transfected into 293 cells for determination of expression characteristics since these cells do not have endogenous GKLF. Separate aliquots of 293 cells were transfected with the native pcDNA3.1/His B vector and with a pcDNA vector containing the
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GKLF Transactivates the ED-L2 and K4 Promoters in the 293 Cell Line-- To assess transcriptional activation by GKLF, we selected the 293 cell line which does not express GKLF as determined by RT-PCR. This cell line is highly transfectable, and we have determined previously that promoters that are generally active in keratinocyte-derived cell lines are active in 293 cells, although with reduced activity (19). Thus, our 293 cell transient transfection system took advantage of a low level of basal promoter activity coupled with the absence of endogenous GKLF.
Different Epstein-Barr virus ED-L2 promoter-reporter constructs were used to test potential GKLF transactivation. These constructs had been previously shown by us to confer significant phorbol ester-induced promoter activity, attributable to the bp between
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Histidine-tagged 65-kDa GKLF Fusion Protein Is Isolated and Purified from Transiently Transfected 293 Cells-- To define the biochemical characteristics of the interaction of GKLF with the CACCC-like motif contained within the ED-L2 and K4 promoters, histidine-tagged GKLF was expressed and purified from 293 cells for use in electromobility shift assays. As indicated above, Western blot analysis of the expressed GKLF demonstrated that major (65-kDa) and minor (34-kDa) forms of the histidine-tagged recombinant GKLF were present in transiently transfected 293 cells. Previous analysis of the GKLF cDNA predicts that the open reading frame should yield a 58-kDa protein (5) in addition to the 7-kDa epitope and histidine tag. To investigate the DNA binding properties of GKLF, extracts were initially prepared that contained both 65- and 34-kDa forms of the fusion protein and that subsequently contained the 65-kDa protein size-fractionated from the 34-kDa protein using gel chromatography techniques.
The 293 cell lysate shown by Western blot to contain GKLF fusion protein contains several bands in addition to GKLF as demonstrated by silver staining (Fig. 3A). Using a nickel-based affinity column, the histidine-tagged proteins were purified from unrelated proteins until the final fraction contained predominantly 65-kDa GKLF with a lesser amount of the 34-kDa protein for use in EMSAs (Fig. 3A). Western blot analysis confirmed the identity of the histidine-tagged, purified proteins (data not shown).
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EMSAs of Recombinant Affinity Column-purified GKLF Demonstrate That the CACCC-like Motif Is Critical for Specific GKLF DNA Binding Activity-- The affinity column-purified GKLF containing both 65- and 34-kDa GKLF fusion protein was used in EMSAs. Two series of radiolabeled probes and competitor oligonucleotides were used, which represented the wild type ED-L2 and K4 promoter sequences containing the CACACCC and GTGTGGG motifs, respectively. In addition, mutant double-stranded oligonucleotides were used to determine the specificity of binding of the recombinant GKLF.
The CACACCC motif in the ED-L2 promoter was found to bind the affinity-purified GKLF in the EMSA (Fig. 4A). This binding is specific as shown by competition studies using wild type and mutant unlabeled excess competitor oligonucleotides (Table I). Importantly, the CACACCC motif is critical for binding GKLF, since mutant competitor does not eliminate the GKLF-labeled probe complex. Radiolabeled mutant probe does not bind GKLF, and affinity-purified extracts from untransfected or empty vector-transfected cells do not reconstitute the complex demonstrated with GKLF (data not shown).
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Purified 65-kDa GKLF Fusion Protein Is Sufficient to Reconstitute the EMSA Complex, and the 34-kDa Protein Has No Inhibitory Effect on Formation of the Specific Complex-- Since the affinity column-purified GKLF contained detectable amounts of a histidine-tagged 34-kDa protein, we were interested in determining whether this protein had relevance in the DNA protein interaction between the 65-kDa predicted form of GKLF and its DNA target. The 65-kDa protein was separated from the 34-kDa protein and used in the EMSA. This result was compared with results from EMSAs utilizing both forms of the protein. Because of the small amount of 34-kDa protein relative to excess 65-kDa GKLF, it was not possible to completely isolate the 34-kDa protein using conventional chromatography techniques. Nonetheless, the addition of the 34-kDa protein did not affect the specific complex formed with the ED-L2 and K4 motifs. Competitor studies as above confirmed that the complex required the CACCC-like element, and co-migration studies with affinity-purified GKLF indicated that these complexes were identical (data not shown). The 65-kDa GKLF fusion protein is sufficient to reconstitute the EMSA complex for both the CACACCC motif (Fig. 5A) and the GTGTGGG motif (Fig. 5B).
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DISCUSSION |
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Many epithelial cell types undergo defined programs of proliferation and differentiation. Such programs occur, for example, along the crypt-villus axis in the small and large intestine. In the squamous upper gastrointestinal tract, proliferating basal cells undergo a switch to become terminally differentiated superficial squamous cells in the stratified squamous epithelium. In this context, it is important to delineate both the genes responsible for this proliferation-differentiation gradient and transcription factors that orchestrate the pattern of gene expression in the switch from proliferation to differentiation. Other models of transcriptional regulation, particularly in the skin and hematopoietic systems, suggest that the process is mediated by both ubiquitous (29, 30) and tissue-specific (31-33) genes and transcription factors.
The Krüppel-like family of transcription factors, comprising GKLF, EKLF, LKLF, and basic transcription element binding-protein 2, may be essential tissue-restricted factors influencing the proliferation-differentiation gradient. GKLF is localized to gut epithelial compartments at the transition between proliferating and early differentiated cells, and previous studies have implicated a role for GKLF in differentiation (5, 7). To investigate mechanisms of gene regulation in the squamous upper gastrointestinal tract, we have focused attention on potential gene targets of GKLF, particularly because TE-11 cDNA library screening yielded GKLF as the only Krüppel-like factor in cells derived from this tissue compartment. This finding supports those observations of other studies, which have to date found only four members of this restricted family. A recent screening of a murine embryonic cDNA library with a probe for the zinc finger domain of EKLF also identified the previously described basic transcription element binding-protein 2, LKLF, and GKLF (11).
Our previous studies have shown that the Epstein-Barr virus ED-L2 (19,
20) and human keratin 4 promoters2 are active in suprabasal
cells of the esophageal squamous epithelium. Both promoters contain
multiple CACCC-like elements. ED-L2 contains a CACCC-like element
between bp 218 and
187, which binds a tissue specific,
zinc-dependent nuclear factor responsible for most of the
unstimulated activity of this promoter in esophageal squamous epithelial cells (19). The CACCC-like element between
144 and
114
binds a phorbol 12-myristate 13-acetate-inducible,
zinc-dependent factor whose molecular weight as estimated
by UV cross-linking is in the range of several of the
Krüppel-like factors (20). In addition, the human keratin 4 promoter contains a palindromic CACCC-like element at position
281.
Given the potential importance of GKLF in tissue-specific gene
regulation at the transition from proliferation to differentiation in
the suprabasal layer of the esophageal squamous epithelium, the
activity of these two representative promoters at this transition, and
the presence of CACCC-like motifs in both promoters, we investigated
whether GKLF is capable of specifically binding these sites in the two
promoters and activating gene expression.
After generation of an expression construct of the full-length human GKLF cDNA fused to coding sequence for a polyhistidine and epitope tag, transient transfection into 293 cells revealed a single 3.5-kb transcript. This is in contrast to previous findings which identified a second 1.9-kb transcript postulated to result from alternative splicing (7). The smaller transcript was only identified in tissues with the highest GKLF expression, namely newborn mouse skin and lung. It is possible that the absence of this transcript in 293 cells reflects the absence of alternative splicing or instability of the smaller transcript.
A smaller 34-kDa protein in addition to the 65-kDa protein predicted to result from the GKLF open reading frame was detected by Western blot analysis using an antibody to the epitope tag at the amino terminus. Sequence analysis revealed that there was no stop codon in the region predicted to result in a smaller protein, and the Northern blot suggested that the smaller protein was not the result of alternative splicing at the 3'-end of the RNA. Ultimately, further analysis of the different size GKLF transcripts in newborn mouse skin and the proteins encoded by these transcripts is needed to determine biological relevance. It is conceivable that the smaller protein may have functional consequences for in vivo GKLF DNA binding. Alternatively, proteolytic cleavage in the 293 cell line at a site unrelated to in vivo post-translational processing of GKLF is possible, although nonspecific proteolytic degradation of the 65-kDa GKLF appears unlikely, given a distinct and single 34-kDa band on Western blot analysis.
Our studies demonstrate that transiently expressed GKLF leads to
transactivation of the ED-L2 promoter dependent upon the presence of
sequence containing the CACACCC motif. Additionally, when the CACACCC
motif is mutated within the endogenous ED-L2 promoter, GKLF does not
transactivate the promoter, also suggesting that activation is mediated
through the CACACCC cis-regulatory motif. A similar
phenomenon was observed with the human keratin 4 promoter. Transient
transfection of 293 cells with the K4 promoter reporter constructs and
GKLF demonstrates that the region activated by GKLF resides between
340 and
140 of the promoter. There is a palindromic GTGTGGG motif
at position
281 of the keratin 4 promoter. These experiments also
suggest that a unique sense or antisense orientation of the CACCC-like
element may not be essential for GKLF-mediated transactivation.
EMSAs with affinity column and gel filtration chromatographically purified GKLF fusion protein confirm the DNA-GKLF interaction and the ability of GKLF to interact specifically with the CACCC-like motif as found in the ED-L2 and K4 promoters. Additionally, we found that the 65-kDa GKLF fusion protein is sufficient to reconstitute DNA binding in the EMSA. The mixture of the 34- and 65-kDa proteins had similar DNA binding characteristics with both the ED-L2 and K4 promoter elements. While it is tempting to speculate that these two proteins may have in vivo relevance in differential regulation of expression of different genes, the biochemical characteristics of the two proteins need to be established further.
The role of the Krüppel-like factors in the regulation of gene
expression is underscored by emerging evidence suggesting that these
factors may provide specific tissue compartments with a mechanism for
governing tissue-restricted development and cell-specific differentiation. The recent demonstration that LKLF is required to
maintain a quiescent state in single positive T cells and that absence
of LKLF leads to a spontaneously activated phenotype is evidence in
favor of this view (10). Additionally, LKLF was shown to play an
important role in vascular development, specifically in the formation
of the tunica media compartment and blood vessel stabilization during
murine embryogenesis (34). Targeted disruption of EKLF leads to
defective hematopoiesis in the fetal liver (35), as well as lethal
-thalassemia (36). Further study of the role GKLF has in governing
proliferation and differentiation should include identification of
additional gene targets in the squamous upper gastrointestinal tract
apart from ED-L2 and K4 and the role that GKLF overexpression or
absence has in control of these basic processes.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Ian Rosenberg and Vincent Yang for helpful discussions.
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FOOTNOTES |
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* This work was supported by the American Digestive Health Foundation of the American Gastroenterological Association Funderberg Awards (to A. R.); American Cancer Society Junior Faculty Research Award JFRA-649 (to A. R.); National Institutes of Health Grants R01-DK53377 (to A. R); 1P01 DE12467-01A1 (to A. R.), and DK07191 (to T. J.); a Glaxo Wellcome Institute for Digestive Health Basic Research Award (to T. J.); Center for the Study of Inflammatory Bowel Disease Award 5P30DK43357-08 (to T. J); and Deutsche Krebshilfe D/96/17197 (to O. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Gastrointestinal Unit, Jackson 904, Massachusetts General Hospital, 50 Blossom St., Boston, MA 02114. Tel.: 617-724-3740; Fax: 617-726-3673; E-mail: rustgi{at}helix.mgh.harvard.edu.
1 The abbreviations used are: EKLF, erythroid Krüppel-like factor; LKLF, lung Krüppel-like factor; GKLF, gut-enriched Krüppel-like factor; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; PIPES, 1,4-piperazinediethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; K4, keratin 4; EMSA, electrophoretic mobility shift assay.
2 O. G. Opitz, T. D. Jenkins, and A. K. Rustgi, unpublished observations.
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REFERENCES |
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