ABSTRACT
The gut-enriched Krüppel-like factor (GKLF) is a recently identified eukaryotic transcription factor that contains three C2H2 zinc fingers. The amino acid sequence of the zinc finger portion of GKLF is closely related to several Krüppel proteins, including the lung Krüppel-like factor (LKLF), the erythroid Krüppel-like factor (EKLF) and the basic transcription element binding protein 2 (BTEB2). The DNA sequence to which GKLF binds has not been definitively established. In the present study we determined the DNA binding sequence of GKLF using highly purified recombinant GKLF in a target detection assay of an oligonucleotide library consisting of random sequences. Upon repeated rounds of selection and subsequent characterization of the selected sequences by base-specific mutagenesis a DNA with the sequence 5'-G/AG/AGGC/TGC/T-3' was found to contain the minimal essential binding site for GKLF. This sequence is present in the promoters of two previously characterized genes: the CACCC element of the [beta]-globin gene, which interacts with EKLF, and the basic transcription element (BTE) of the CYP1A1 gene, which interacts with Sp1 and several Sp1-like transcription factors. Moreover, the selected GKLF binding sequence was capable of mediating transactivation of a linked reporter gene by GKLF in co-transfection experiments. Our results establish GKLF as a sequence-specific transcription factor likely involved in regulation of expression of endogenous genes.
The zinc finger is a common structural motif used by transcription factors to bind DNA (1,2). The Cys2His2 (C2H2) type zinc fingers, initially identified in the Xenopus laevis transcription factor TFIIIA (3), represent the most abundant DNA binding motif of zinc finger-containing proteins (4). A characteristic three-dimensional structure of a C2H2 zinc finger consists of two antiparallel [beta]-strands followed by an [alpha]-helix (5). This structure in turn contacts 3 bp of DNA (2). Most zinc finger transcription factors contain multiple tandem repeats of the fingers, which confer the sequence specificity of the DNA that the individual factor recognizes.
A subset of C2H2 zinc finger proteins contains additional homology to the Drosophila segmentation gene product Krüppel (6). This homology is present in the region between two adjacent fingers and contains the highly conserved sequence TGEKPY/FX. Examples of Krüppel-related proteins include Sp1 (7), zif268/Egr-1 (8), WT-1 (9) and EKLF (10). These proteins are collectively involved in diverse aspects of eukaryotic gene regulation during growth, development and differentiation.
We recently identified a novel C2H2 zinc finger protein with Krüppel homology, which we named the gut-enriched Krüppel-like factor (GKLF) (11). Expression of GKLF is enriched in epithelial cells of the gastrointestinal tract (11,12) and in the epidermal layer of the skin (12). In cultured cells expression of GKLF is induced under conditions that promote growth arrest, such as serum deprivation and contact inhibition (11). In addition, enforced expression of GKLF in transfected cells results in inhibition of DNA synthesis (11). Taken together, these findings suggest that GKLF may have an important function in regulating proliferation of epithelial tissues.
Although the amino acid sequence of GKLF outside the zinc finger region is unique, that of its three zinc fingers is closely related to several Krüppel proteins, including LKLF (13), EKLF (10) and BTEB2 (14). Recently, by comparing the amino acid sequences necessary for nuclear localization, we observed that GKLF is more closely related to LKLF and EKLF than to BTEB2 (15). The DNA sequences with which these transcription factors interact also appear to share some degree of similarity; GKLF was shown to interact with the CACCC sequence that binds EKLF and the basic transcription element (BTE) that binds BTEB2 (12). Nevertheless, no methodical analysis of the DNA recognition sequence for GKLF has been performed to date. We therefore undertook the task of empirically determining the GKLF binding sequence, since we deemed this information essential for eventual deciphering of the biological functions of GKLF.
cDNA encoding the C-terminus of GKLF between amino acids 350 and 483, which contains the three zinc fingers, was subcloned into the XhoI-BamHI sites of the bacterial expression vector PET-16b (Novagen; Madison, WI) to produce a GKLF fusion protein tagged with 10 histidine residues at the N-terminus. This protein was named His-GKLFZn. Esherichia coli strain BL21(DE3)pLysS (Novagen) was transformed with the recombinant plasmid and induced with 2 mM isopropyl-[beta]-D-thiogalactopyranoside (IPTG) to produce recombinant His-GKLFZn protein. Five hundred milliliters of logarithmically growing bacteria were pelleted by centrifugation and resuspended in 20 ml buffer containing 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 6 M urea and 5 mM imidazole. The suspension was sonicated with a Fisher Scientific 550 Sonic Dismembrator at a setting of 50% for 20 s at a time for a total of 20 times, chilling to 4°C between sonications. The sample was then flowed through a Ni-NTA-agarose column (Qiagen, Santa Clarita, CA) equilibrated with the above buffer. After washing the column with a buffer containing 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 6 M urea and 60 mM imidazole the bound protein was eluted with the same buffer with the exception that the concentration of imidazole was raised to 1 M. The eluted fractions containing His-GKLFZn were dialyzed exhaustively against a solution of 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 µM ZnCl2 and 10% glycerol. The protein was stored at -70°C at an approximate concentration of 2 mg/ml.
The target detection assay (TDA) was performed based on a protocol by Thiesen and Bach (16), with some modifications. A library of single-stranded oligonucleotides containing the sequences 5'-CAAGCTTACTGCAGATGC(N)14CGTAGGATCCATCTAGAGT-3' (N is any nucleotide) was generated. The invariable 5'- and 3'-flanking sequences contained unique restriction sites. Fifteen micrograms of the library were made double-stranded with 5 µg reverse primer of sequence 5'-ACTCTAGATGGATCCTACG-3' in a reaction that contained 200 µM each of the four deoxynucleoside triphosphates, 25 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol (DTT) and 20 U Taq DNA polymerase (Boehringer Mannheim, Indiannoplis, IN) for one cycle of 98°C for 3 min, 94°C for 1 min, 47°C for 2 min, 72°C for 1 min and 72°C for 30 min in a Perkin Elmer thermocycler. Fifty nanograms of the double-stranded library were end-labeled with T4 polynucleotide kinase and 100 µCi [[gamma]-32P]ATP in a reaction containing 70 mM Tris-HCl, pH 7.6, 10 mM MgCl2 and 5 mM DTT at 37°C for 30 min. Four hundred nanograms of purified His-GKLFZn were added to the labeled library in a binding buffer (BB) of 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM KCl, 10 mM MgCl2 and 5 µM ZnCl2, at 4°C for 20 min, following which the mixture was electrophoresed in a 7% non-denaturing polyacrylamide gel in 0.5× TBE (1× TBE is Tris-HCl, pH 8.0, 89 mM boric acid and 2 mM EDTA). After autoradiography the region of the gel above the free probe was excised and the DNA eluted electrophorectically and concentrated by ethanol precipitation. The eluted DNA was then amplified by PCR in a reaction containing 10 ng end-labeled reverse primer, 10 ng forward primer with sequence 5'-ACAAGCTTACTGCAGATGC-3', 25 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 1 mM DTT and 10 U Taq DNA polymerase for 30 PCR cycles of 96°C for 5 min, 96°C for 1 min, 50°C for 1 min and 72°C for 1 min. Two hundred nanograms of purified His-GKLFZn were then used in a gel shift reaction with the newly created probe as described above. The same procedure was repeated for four additional rounds, at which time shifted oligonucleotides were digested with PstI and BamHI and subcloned into pBluescript digested with the same enzymes. After transformation of competent host cells DNA from individual clones was isolated and the insert within each clone sequenced.
EMSA of synthetic oligonucleotides was performed in BB using 1 pmol end-labeled double-stranded oligonucleotide and 100 ng purified His-GKLFZn per reaction. Control reactions contained 100 ng bovine serum albumin. In reactions that included unlabeled oligonucleotides as competitors a 1- to 20-fold molar excess of unlabeled DNA was added to the protein in BB at 4°C for 10 min before addition of the probe. In reactions in which antiserum or preimmune serum was used the serum was incubated with the protein in BB at 4°C for 10 min before addition of the probe.
Transient transfection of COS-1 cells with the eukaryotic expression vector containing the full-length GKLF, PMT3-GKLF, was performed by the lipofection method as previously described (11). Twenty four to 48 h following transfection whole cell extracts were prepared by resuspending centrifuged cell pellets in BB containing 0.05% NP40 and sonincating the cell suspensions at 20% intensity for 20 s in a 550 Sonic Dismembrator. After a brief centrifugation glycerol was added to 10% to the supernatant and the extracts were stored at -70°C. Twenty micrograms of crude extracts were used for each EMSA reaction.
A lucifierase reporter plasmid driven by a minimal TATA box, pGL2-TATA-Luc, was constructed by ligating the adenovirus E1b TATA box derived from plasmid E1b-CAT (17) into the XhoI and BglII sites of the pGL2-Basic vector (Promega, Madison, WI). Two tandem copies of either the wild-type GKLF binding sequence as determined by TDA or a mutated sequence which failed to bind GKLF (M6; Fig. 3) were subcloned into pGL2-TATA-Luc, giving rise to TDA(WT)×2-pGL2-TATA-Luc or TDA(M6-Mut)×2-pGL2-TATA-Luc. Co-transfection experiments were performed with 5 µg/10 cm dish each of the luciferase reporter and PMT3 expression constructs containing GKLF, together with 1 µg/dish internal standard pCMV-SPORT-[beta]-galactosidase (Life Technologies, Gaithersburg, MD). In addition to full-length GKLF, two truncated GKLF constructs in expression vector PMT3 were used in the experiments. They included PMT3-GKLF(1-401), which had all three zinc fingers deleted, and PMT3-GKLF(350-483), which contained the zinc finger region only (15). Lucifierase activity was determined as recommended by Promega (Madison, WI). Transfected cells were lysed with Cell Culture Lysis Reagent (Promgega) and the lysates cleared by microcentrifugation. Assays were performed on cell lysates using Luciferase Assay Substrate and Luciferase Assay Buffer (Promega) in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). [beta]-Galactosidase activity was determined by chemiluminescent assay (18) using Lumi-Gal 530 (Lumigen Inc., Southfield, MI). All luciferase activities were standardized against [beta]-galactosidase activities in transfected cells.
To empirically determine the DNA sequence to which GKLF binds we performed TDA on a library of oligonucleotides with randomized sequences at 14 nt positions. Multiple rounds of EMSA using highly purified recombinant protein containing the zinc finger portion of GKLF and amplification by PCR of the oligonucleotides shifted by the recombinant protein were performed to enrich for GKLF recognition sequences. After a total of five rounds the shifted oligonucleotides were subcloned into the multiple cloning sites of the pBluescript plasmid and the sequence of the 14 nt insert in individual clones was determined. Inserted sequences from 80 clones were analyzed and the ability of each insert to bind GKLF was verified by EMSA. Figure 1 shows the inserted sequences from 49 clones, each of which was able to bind GKLF with high affinity. With the exception of two invariable guanine residues at the 3'-most location of 46 of the 49 oligonucleotide inserts, the sequences selected by GKLF were fairly relaxed, although adenine and guanine residues were evidently preferred over thymidine and cytosine residues. It would appear that the core binding sequence for GKLF may include at least an additional 2 nt, CG, situated at the beginning of the 3'-flanking sequence (the shaded area on the right hand side of Fig. 1). This assertion was substantiated by the observation that the same invariable sequence 5'-GGCG-3' was also present independent of the 5'- and 3'-flanking sequences in three individual clones (#30, #32 and #54; Fig. 1). Shown at the bottom of Figure 1 is the relative frequency of appearance of each nucleotide in the 14 positions of the 46 clones and a compiled sequence of the 14 nt.
We next examined the specificity of the selected sequence in binding to GKLF by performing competition experiments. As a probe we selected the insert sequence from clone 47 (5'-AGGAGAAAGAAGGG-3'), which represented a high affinity binding site for GKLF. A double-stranded oligonucleotide containing this sequence, dubbed the TDA sequence, was synthesized, labeled to high specific activity and analyzed by EMSA. One picomole of probe and 100 ng recombinant GKLF were used in each reaction, which also contained variable amounts of unlabeled specific or non-specific competing DNA. As shown in Figure 2, incubation of GKLF with the probe alone without any competitors resulted in formation of a single DNA-protein complex (C, lanes 1 and 5). Addition of increasing amounts of unlabeled probe (WT, Fig. 1) resulted in a gradual diminution of complex formation (lanes 2-4). In contrast, the addition of an unlabeled non-specific oligonucleotide, poly(dI·dC), failed to compete for formation of the DNA-protein complex (lanes 6-8). The specificity of binding was evident from the fact that at a 5-fold molar excess the wild-type oligonucleotide was able to compete for the majority of binding (lane 4), yet at a molar ratio as high as 200-fold (lane 8) poly(dI·dC) failed to compete for binding entirely. These results indicate that the sequence selected by TDA binds to GKLF in a highly specific manner.
Although each insert of the 49 clones shown in Figure 1 was capable of binding to GKLF with high affinity, it was unclear whether each of the 14 nt selected was necessary for binding. To determine the minimal essential nucleotide sequence required for binding a series of mutant oligonucleotides, each with base substitution at two positions, were synthesized and analyzed by competition experiments. In all, 10 mutant oligonucleotides were obtained, which extended from the beginning of the 14 nt insert to eight bases into the 3'-flanking sequence. Figure 3 shows the result of one such competition experiment. When present the amount of competitor DNA was in 10-fold molar excess over the probe. As seen, the unlabeled wild-type oligonucleotide (lane 2) competed efficiently for formation of the DNA-protein complex, as were mutants M1-M4, M9 and M10, suggesting that the mutations within these oligonucleotides were not essential for binding. In contrast, mutants M5-M7 failed to compete (lanes 7-9). Mutant M8 (lane 10) competed to some extent, although not as efficiently as the wild-type sequence. This partial competition by the M8 mutant oligonucleotide has been repeatedly observed. The results from Figure 3 indicate that the sequence altered in M5-M7 and in part in M8, i.e. 5'-AGGGCGTA-3', contains the minimal essential binding site for GKLF.
The minimal essential binding sequence for GKLF established by the mutagenesis experiments is similar to a number of previously established DNA sequences which interact with known transcription factors. One example is BTE (basal transcription element; 19), which is present in the promoters of and is essential for basal transcription of a number of genes encoding the superfamily of cytochrome P450 (CYP) enzymes, including CYP1A1 (19,20). A second example is the CACCC sequence within the promoter of the [beta]-globin gene (21) with which EKLF interacts (10). To determine whether GKLF binds to these two sites competition experiments were performed using synthetic oligonucleotides containing published sequences. As shown in Figure 5, BTE (lanes 5-7) competed as efficiently for binding as the wild-type GKLF TDA sequence (lanes 2-4). Although to a somewhat lesser degree, the CACCC sequence (EKLF, lanes 8-10) also competed. The slightly lower efficiency of binding of the CACCC sequence to GKLF is consistent with the result in Figure 3, when the M7A1 mutant oligonucleotide, which also contains an inverted CACCC sequence, was used as competitor (Fig. 4, lane 4). In contrast, a synthetic oligonucleotide containing the binding site for Sp1 (lanes 11-13) or AP2 (lanes 14-16) competed poorly for binding. Of note is that a major difference in the binding sites for GKLF/BTE/EKLF and for Sp1/AP2 is the presence of a T residue in the 3'-most position in the minimal essential binding sequence (bold, Fig. 5) in the former group. These results therefore establish that this T is an important residue for binding to GKLF. Lastly, the first two nucleotides in the minimal essential binding sequence of GKLF (AG) are interchangeable with GA, as demonstrated by the equal binding affinity of GKLF to the TDA sequence and to BTE (Fig. 5).
The TDA used to select the GKLF binding sequence involved a truncated form of GKLF, a portion that contained only the zinc fingers. To determine whether full-length GKLF also binds to the same sequence extracts were prepared from COS-1 cells transfected with an expression vector containing full-length GKLF (PMT3-GKLF) and analyzed by EMSA using the GKLF TDA oligonucleotide as probe. As a control extracts were prepared from similarly transfected cells with vector (PMT3) alone. Figure 6 shows that a DNA-protein complex was formed between the probe and extracts from PMT3-GKLF-transfected cells (lane 1). This complex was completely disrupted when the reaction was performed in the presence of an anti-GKLF serum (lane 3), but not in the presence of a preimmune serum (lane 2). In contrast, no complexes were apparent when extracts from PMT3-transfected cells were analyzed (lanes 4-6). These results indicate that full-length GKLF is capable of binding to the selected GKLF binding sequence and that anti-GKLF serum is able to interact with the formed complex and disrupt its formation.
To demonstrate that the sequence selected by TDA is important for GKLF-mediated transcriptional activity co-transfection experiments were performed in CHO cells using an expression vector containing full-length or truncated GKLF and a reporter gene driven by two tandem copies of the wild-type or a mutated site that no longer binds GKLF (M6, Fig. 3). As shown in Figure 7, full-length GKLF was able to activate the reporter gene driven by the wild-type GKLF binding site (shaded bar, lane B) but not by the mutated GKLF binding site (open bar, lane B). In contrast, neither empty vector (lane A) nor a truncated GKLF construct that lacked either the zinc fingers (lane C) or the region outside the zinc fingers which contains the putative transactivation domain (lane D) of GKLF was able to activate the reporter gene driven by either the wild-type (shaded bars) or mutated (open bars) sequence. These results indicate that full-length GKLF alone is capable of activating transcription mediated by the selected binding sequence.
Within the last decade molecular techniques such as TDA (16) and other closely related methods, including SAAB (selected and amplified binding sites; 22) and CASTing (cyclic amplification and selection of targets; 23), have proven to be potent tools in identifying the binding sequences for transcription factors. In particular, a considerable number of publications have utilized this principal of target site selection and established the putative binding sites for a diverse group of zinc finger-containing transcription factors (16,21,24-27). Similarly, in the present study we were able to establish a binding site for GKLF using highly purified recombinant GKLF that contained the three zinc fingers. The identified binding sequence should facilitate characterization of the biological functions of GKLF, including the target genes that it regulates.
This work was in part supported by grants from the National Institutes of Health (to V.W.Y.). J.M.S. was supported by a National Research Service Award from the National Institutes of Health.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Production of recombinant GKLF
Target detection assay
Electrophoretic mobility shift assay (EMSA)
Transfection
Reporter assay
Results
TDA of GKLF binding sequence
Specificity of binding of GKLF to the selected sequences
Identifying the minimal essential binding sequence for GKLF
Binding of GKLF to previously established DNA sequences that interact with known transcription factors
Full-length GKLF also binds to the sequence selected by TDA
GKLF transactivates a reporter gene driven by the TDA-selected sequence
Discussion
Acknowledgements
References
REFERENCES
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 Q. XIE and D. H. ALPERS The two isozymes of rat intestinal alkaline phosphatase are products of two distinct genes Physiol Genomics, June 29, 2000; 3(1): 1 - 8. [Abstract] [Full Text] [PDF]
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D. E. Geiman, H. Ton-That, J. M. Johnson, and V. W. Yang Transactivation and growth suppression by the gut-enriched Kruppel-like factor (Kruppel-like factor 4) are dependent on acidic amino acid residues and protein-protein interaction Nucleic Acids Res., March 1, 2000; 28(5): 1106 - 1113. [Abstract] [Full Text] [PDF]
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L.-G. Guy, N. Delvoye, and L. Wall Expression of a Human beta -Globin Transgene in Mice with the CACC Motif and Upstream Sequences Deleted from the Promoter Still Depends on Erythroid Kruppel-like Factor J. Biol. Chem., February 4, 2000; 275(5): 3675 - 3680. [Abstract] [Full Text] [PDF]
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 L Ma, J Merenmies, and L. Parada Molecular characterization of the TrkA/NGF receptor minimal enhancer reveals regulation by multiple cis elements to drive embryonic neuron expression Development, January 9, 2000; 127(17): 3777 - 3788. [Abstract] [PDF]
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 K. W. Foster, S. Ren, I. D. Louro, S. M. Lobo-Ruppert, P. McKie-Bell, W. Grizzle, M. R. Hayes, T. R. Broker, L. T. Chow, and J. M. Ruppert Oncogene Expression Cloning by Retroviral Transduction of Adenovirus E1A-immortalized Rat Kidney RK3E Cells: Transformation of a Host with Epithelial Features by c-MYC and the Zinc Finger Protein GKLF Cell Growth Differ., June 1, 1999; 10(6): 423 - 434. [Abstract] [Full Text]
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S. Kuo, S. E. Chesrown, J. K. Mellott, R. J. Rogers, J.-L. Hsu, and H. S. Nick In Vivo Architecture of the Manganese Superoxide Dismutase Promoter J. Biol. Chem., February 5, 1999; 274(6): 3345 - 3354. [Abstract] [Full Text] [PDF]
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N. Matsumoto, F. Laub, R. Aldabe, W. Zhang, F. Ramirez, T. Yoshida, and M. Terada Cloning the cDNA for a New Human Zinc Finger Protein Defines a Group of Closely Related Kruppel-like Transcription Factors J. Biol. Chem., October 23, 1998; 273(43): 28229 - 28237. [Abstract] [Full Text] [PDF]
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W. Zhang, J. M. Shields, K. Sogawa, Y. Fujii-Kuriyama, and V. W. Yang The Gut-enriched Kruppel-like Factor Suppresses the Activity of the CYP1A1 Promoter in an Sp1-dependent Fashion J. Biol. Chem., July 10, 1998; 273(28): 17917 - 17925. [Abstract] [Full Text] [PDF]
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W. Zhang, D. E. Geiman, J. M. Shields, D. T. Dang, C. S. Mahatan, K. H. Kaestner, J. R. Biggs, A. S. Kraft, and V. W. Yang The Gut-enriched Kruppel-like Factor (Kruppel-like Factor 4) Mediates the Transactivating Effect of p53 on the p21WAF1/Cip1 Promoter J. Biol. Chem., June 9, 2000; 275(24): 18391 - 18398. [Abstract] [Full Text] [PDF]
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F. H. Brembeck and A. K. Rustgi The Tissue-dependent Keratin 19 Gene Transcription Is Regulated by GKLF/KLF4 and Sp1 J. Biol. Chem., September 1, 2000; 275(36): 28230 - 28239. [Abstract] [Full Text] [PDF]
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P. J. Adam, C. P. Regan, M. B. Hautmann, and G. K. Owens Positive- and Negative-acting Kruppel-like Transcription Factors Bind a Transforming Growth Factor beta Control Element Required for Expression of the Smooth Muscle Cell Differentiation Marker SM22alpha in Vivo J. Biol. Chem., November 22, 2000; 275(48): 37798 - 37806. [Abstract] [Full Text] [PDF]
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