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Transcriptional Regulation of the Differentiation-linked Human K4 Promoter Is Dependent upon Esophageal-specific Nuclear Factors

1998; Elsevier BV; Volume: 273; Issue: 37 Linguagem: Inglês

10.1074/jbc.273.37.23912

1083-351X

Oliver G. Opitz, Timothy D. Jenkins, Anil K. Rustgi,

Hair Growth and Disorders

The stratified squamous epithelium comprises actively proliferating basal cells that undergo a program of differentiation accompanied by morphological, biochemical, and genetic changes. The transcriptional regulatory signals and the genes that orchestrate this switch from proliferation to differentiation can be studied through the keratin gene family. Given the localization of keratin 4 (K4) to the early differentiated suprabasal compartment and having previously demonstrated that targeted disruption of this gene in murine embryonic stem cells results in impairment of the normal differentiation program in esophageal and corneal epithelial cells, we studied the transcriptional regulation of the human K4 promoter. A panel of K4 promoter deletions were found in transient transfection assays to be predominantly active in esophageal and corneal cell lines. A critical cis-regulatory element resides between −163 and −140 bp and contains an inverted CACACCT motif. A site-directed mutated version of this motif within the K4 promoter renders it inactive, whereas the wild-type version is active in a heterologous promoter system. It specifically binds esophageal-specific zinc-dependent transcriptional factors. Our studies demonstrate that regulation of the human K4 promoter is in part mediated through tissue-specific transcriptional factors. The stratified squamous epithelium comprises actively proliferating basal cells that undergo a program of differentiation accompanied by morphological, biochemical, and genetic changes. The transcriptional regulatory signals and the genes that orchestrate this switch from proliferation to differentiation can be studied through the keratin gene family. Given the localization of keratin 4 (K4) to the early differentiated suprabasal compartment and having previously demonstrated that targeted disruption of this gene in murine embryonic stem cells results in impairment of the normal differentiation program in esophageal and corneal epithelial cells, we studied the transcriptional regulation of the human K4 promoter. A panel of K4 promoter deletions were found in transient transfection assays to be predominantly active in esophageal and corneal cell lines. A critical cis-regulatory element resides between −163 and −140 bp and contains an inverted CACACCT motif. A site-directed mutated version of this motif within the K4 promoter renders it inactive, whereas the wild-type version is active in a heterologous promoter system. It specifically binds esophageal-specific zinc-dependent transcriptional factors. Our studies demonstrate that regulation of the human K4 promoter is in part mediated through tissue-specific transcriptional factors. keratin epidermal bullosa simplex epidermolytic hyperkeratosis base pair(s) Epstein-Barr virus polymerase chain reaction untranslated region electrophoretic mobility shift assay N-tris(hydroxymethyl)methylglycine keratinocyte-specific factor Gut-enriched Krüppel-like factor cAMP-response element-binding protein. The esophagus is lined by a stratified squamous epithelium that is composed of proliferating basal cells and differentiated suprabasal, intermediate, and superficial squamous cells. Other sites sharing the stratified squamous epithelium include the cornea, oropharynx, larynx, skin, and anogenital tract. Basal cells undergo an exquisite program of differentiation accompanied by a series of morphological, biochemical, and genetic changes as they migrate to the luminal surface with the eventual production of flattened superficial squamous cells. Squamous cells are sloughed from the surface, which may in part by governed by processes such as senescence and apoptosis. There is constant renewal of inner basal cells that differentiate in the suprabasal layer. Insights into the underlying molecular mechanisms can be gained through an appreciation of the genes, as exemplified by the keratins, that govern this process and their transcriptional regulation. For example, keratins 5 and 14 are present in proliferating basal cells. Early differentiation genes in suprabasal cells include keratins 1 and 10, whereas late differentiation genes in superficial squamous cells include loricrin, profillagrin, and transglutaminase (1Byrne C. Tainsky M. Fuchs E. Development. 1994; 120: 2369-2383Crossref PubMed Google Scholar). The keratins are classified as the type I and type II intermediate filaments (2Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1278) Google Scholar). The type I keratins are acidic, range from 40 to 60 kDa and consist of at least 17 distinct proteins: 12 epithelial type I keratins (K91 through K20), and 5 hair keratins (3Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4528) Google Scholar). The type II keratins are neutral to basic, range from 50 to 70 kDa, and consist of at least 15 distinct proteins: 10 epithelial (K1 through K8; with multiple forms of several of these) and 5 hair keratins (3Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4528) Google Scholar). The type I keratins in humans are found in a cluster on chromosome 17, and the type II keratins are on chromosome 12. A similar situation exists in mouse with the type I keratins on chromosome 11 and the type II on chromosome 15. Keratins are obligate heterodimers of one type I molecule and one type II molecule (4Hatzfeld M. Weber K. J. Cell Biol. 1990; 110: 1199-1210Crossref PubMed Scopus (198) Google Scholar). In vivo specific pairing is apparently due to the tissue-specific expression patterns of the keratins (3Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4528) Google Scholar). For example, K5 and K14 heterodimerize in basal cells and K1 and K10 heterodimerize in differentiating suprabasal cells. Although keratins are critical for providing structural strength for epithelial cells, further clues about keratin function have been gained through the discovery of mutations associated with diseases transmitted in an autosomal dominant fashion which have indicated that keratins are important in cellular organization and growth (5Fuchs E. J. Cell Biol. 1994; 125: 511-516Crossref PubMed Scopus (129) Google Scholar). For example, epidermal bullosa simplex (EBS) is characterized by intraepidermal blistering due to cell lysis within the basal layer (6Bonifas J.M. Rothman A.I. Epstein E.H. Science. 1991; 254: 1202-1205Crossref PubMed Scopus (344) Google Scholar). Mutations have been identified in K5 and K14 genes that act in a dominant-negative fashion to cause EBS (6Bonifas J.M. Rothman A.I. Epstein E.H. Science. 1991; 254: 1202-1205Crossref PubMed Scopus (344) Google Scholar). Epidermolytic hyperkeratosis (EH) involves blistering of suprabasal layers of the skin due to cell lysis and degeneration, basal cell hyperplasia, and a thickened granular layer and stratum corneum (skin ridges or scales). Mutations in K1 and K10 are found in EH (7Cheng J. Syder A.J., Yu, Q.C. Letai A. Paller A.S. Fuchs E. Cell. 1992; 70: 811-819Abstract Full Text PDF PubMed Scopus (289) Google Scholar, 8Rothnagel J.A. Dominey A.M. Dempsey L.D. Longley M.A. Greenhalgh D.A. Gagne T.A. Huber M. Frenk E. Hohl D. Roop D.R. Science. 1992; 257: 1128-1130Crossref PubMed Scopus (324) Google Scholar). EBS and EH have been recapitulated with murine models in which the keratins are disrupted by homologous recombination in embryonic stem cells (9Chan Y. Anton-Lamprecht I., Yu, Q.C. Jackel A. Zabel B. Ernst J.P. Fuchs E. Genes Dev. 1994; 8: 2563-2573Crossref PubMed Scopus (159) Google Scholar, 10Rugg E.L. McLean W.H.I. Lane E.B. Pitera R. McMillan J.R. Dopping-Hepenstal P.J.C. Navsaria H.A. Leigh I.M. Eady R.A.J. Genes Dev. 1994; 8: 1563-1573Crossref Scopus (150) Google Scholar) or are aberrantly expressed in transgenic mice (11Vassar R. Rosenberg M. Ross S. Tyner A. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1563-1567Crossref PubMed Scopus (298) Google Scholar, 12Fuchs E. Esteves R.A. Coulombe P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6906-6910Crossref PubMed Scopus (166) Google Scholar, 13Byrne C. Fuchs E. Mol. Cell. Biol. 1993; 13: 3176-3190Crossref PubMed Scopus (131) Google Scholar). K4 and K13 are found in suprabasal cells of nonkeratinizing stratified squamous epithelia (14Takahashi T. Shikata N. Senzaki H. Shintaku M. Tsubura A. Histopathology. 1995; 26: 45-50Crossref PubMed Scopus (52) Google Scholar). K4 is a type II keratin (59 kDa) the type I partner of which is K13. K4 has highest expression in the esophagus and cornea, but to a lesser extent is also found in the tongue, pharynx, larynx, and the anus (15van Muijen G.N.P. Ruiter D.J. Franke W.W. Achtstatter T. Saasnoot W.H.B. Ponec M. Warnaar S.O. Exp. Cell Res. 1986; 162: 97-113Crossref PubMed Scopus (257) Google Scholar). It appears during fetal development while these tissues differentiate. White sponge nevus is a benign autosomal dominant disorder characterized by thickened, white opalescent spongy-fold mucosa, primarily in the mouth but also in the esophageal epithelium, and has been shown recently to be due to a mutation in K4 (16Rugg E.L. McLean W.H. Allison W.E. Lunny D.P. Macleod R.I. Felix D.H. Lane E.B. Mundro C.S. Nat. Genet. 1995; 11: 450-452Crossref PubMed Scopus (112) Google Scholar). Recently, we have developed a model in which the mouse K4 gene has been disrupted by homologous recombination in embryonic stem cells (17Ness S. Edelmann W. Jenkins T. Liedtke W. Rustgi A.K. Kucherlapati R. J. Biol. Chem. 1998; 273: 23904-23911Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). K4 homozygous null mice have a phenotype largely restricted to the esophagus and cornea. There are severe disturbances in esophageal epithelial differentiation manifest by loss of the maturation sequence from the basal to the superficial squamous layers (17Ness S. Edelmann W. Jenkins T. Liedtke W. Rustgi A.K. Kucherlapati R. J. Biol. Chem. 1998; 273: 23904-23911Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). There is also evidence of basal cell hyperplasia in the cornea (17Ness S. Edelmann W. Jenkins T. Liedtke W. Rustgi A.K. Kucherlapati R. J. Biol. Chem. 1998; 273: 23904-23911Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Thus, K4 appears important in maintaining the differentiation phenotype in a highly tissue-specific fashion, and these findings provide additional basis for studying the molecular mechanisms underlying the transcriptional regulation of the K4 promoter. It appears that a particular combination of nuclear factors in a tissue that leads to the transcription of specific keratins. Illustrative of the regulation of basal cell promoters, AP2 binds the KER1 element in the K14 promoter (18Leask A. Rosenberg M. Vassar R. Fuchs E. Genes Dev. 1990; 4: 1985-1998Crossref PubMed Scopus (109) Google Scholar, 19Leask A. Byrne C. Fuchs E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7948-7952Crossref PubMed Scopus (204) Google Scholar) and a non-phorbol ester-responsive AP1 regulates the bovine K5 promoter (20Casatorres J. Navarro J.M. Blessing M. Jorcano J.L. J. Biol. Chem. 1994; 269: 20489-20496Abstract Full Text PDF PubMed Google Scholar). Suprabasal cell promoters that have been studied include the K1 promoter, where a 3′ element is keratinocyte-specific and Ca+ dependent (21Lu B. Rothnagel J.A. Langley M.A. Tsai S. Roop D.R. J. Biol. Chem. 1994; 269: 7443-7449Abstract Full Text PDF PubMed Google Scholar). Additionally, the K3 promoter has two cis-regulatory elements within 300 bp of the transcription start site that are regulated by Sp-1-related and NF-κB-related factors (22Wu R.L. Galvin S. Wu S.K. Xu C. Blumenberg M. Sun T-T. J. Cell Sci. 1993; 105: 303-316PubMed Google Scholar, 23Wu R.L. Chen T.T. Sun T.T. J. Biol. Chem. 1994; 269: 28450-28459Abstract Full Text PDF PubMed Google Scholar). Apart from the regulation of keratin cellular promoters, viral gene expression is mediated through overlapping mechanisms in the stratified squamous epithelium. The human papillomavirus-18 C-enhancer is regulated by an apparent tissue-restricted factor designated KRF-1 (24Mack D.H. Laimins L.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9102-9106Crossref PubMed Scopus (77) Google Scholar) as well as the ubiquitous Oct-1 and AP1. Our previous studies have demonstrated that the Epstein-Barr virus (EBV) ED-L2 promoter is active in suprabasal cells of the tongue and esophagus in transgenic mice (25Nakagawa H. Wang T. Zukerberg L. Odze R. Togawa K. May G.W. H Wilson J. Rustgi A.K. Oncogene. 1997; 14: 1185-1190Crossref PubMed Scopus (113) Google Scholar). Furthermore, the basal ED-L2 promoter activity is attributable to a 70-kDa keratinocyte-specific factor (KSF) that is zinc-dependent and binds a CACACCT motif (26Nakagawa H. Inomoto T. Rustgi A.K. J. Biol. Chem. 1997; 272: 16688-16699Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The ED-L2 promoter is also regulated by phorbol ester, a factor that activates the differentiation program in stratified squamous epithelial cells (27Jenkins T.D. Nakagawa H. Rustgi A.K. J. Biol. Chem. 1997; 272: 24433-24442Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Phorbol ester induces ED-L2 promoter activity in part through USF and Gut-enriched Krüppel-like factor (GKLF) (27Jenkins T.D. Nakagawa H. Rustgi A.K. J. Biol. Chem. 1997; 272: 24433-24442Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 28Jenkins T.D. Opitz O.G. Okano J.-I Rustgi A.K. J. Biol. Chem. 1998; 273: 10747-10754Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). We have now characterized the human K4 promoter forcis-regulatory elements and key trans-acting factors. Importantly, there is a region from −163 to −140 bp that contains an inverted CACACCT motif that contributes significantly to K4 promoter activity. When mutated, endogenous K4 promoter activity is abrogated. This cis-regulatory element binds esophageal-specific transcriptional factors, indicating that the suprabasal K4 promoter is in part regulated in a highly tissue-restricted fashion. The human K4 promoter was cloned with a PCR-based method that allows for the identification of genomic sequence from cDNA sequence (CLONTECH promoter finder kit). The human keratin 4 mRNA sequence was obtained from GenBank accession no. X67683. Briefly, a special adapter is ligated to the ends of DNA fragments generated by digestion of human genomic DNA with EcoRV,ScaI, DraI, PvuII, and SspI separately. Following adapter ligation, a small amount of the DNA from each “library” is used as a template for a primary PCR reaction using an outer adapter primer (AP1) (CLONTECH) and an outer gene-specific primer (GSP1) (Table I, part A). The primary PCR reaction mixture is then diluted and used as a template for a secondary PCR reaction using the nested adapter primer (AP2) (CLONTECH) and a nested gene-specific primer (GSP2) (Table I, part A). GSP1 and GSP2 are designed to anneal at the 5′ end of the known keratin 4 cDNA. PCR amplifications were performed using Tth polymerase mix (CLONTECH), which includes the Tth-Start antibody for automatic hot start. Primary PCR reactions were performed in 50-μl volumes containing 1 μl of ligated and diluted DNA, 40 mmTris-HCl, pH 9.3, 85 mm KOAc, 1.1 mm MgOAc, 10 μm adapter primer 1 (AP1) and GSP1, 10 mmdNTP, and 1 μl of Tth polymerase mix. The cycle parameters were as follows: 7 cycles of denaturation at 94 °C for 25 s and annealing/extension at 72 °C for 4 min, followed by 32 cycles of denaturation at 94 °C for 25 s and annealing/extension at 67 °C for 4 min and a final annealing/extension time for 4 min. A secondary PCR reaction was performed with 1 μl of a 1:100 dilution of the primary PCR reaction using AP2 and GSP2. The same reaction composition and cycle parameters were used except 5 and 20 thermocycles were performed, respectively. PCR products were examined on 1% agarose gels. PCR products from each library were cloned into a TA type vector (Novagen). DNA sequencing of the 5′ and 3′ ends was by the dideoxy-mediated chain termination method using the Sequenase version 2.0 DNA sequencing kit (U.S. Biochemical Corp.), vector primers T7 sense and M13 antisense (Novagen), and sequence-specific primers. Sequence analysis was performed using GenBank.Table IOligonucleotide primer sequences for PCR-based cloning of the human K4 promoter (A), construction of K4 promoter 5′ deletion constructs (B), and K4–163 promoter mutant constructs (C)A. Cloning the human keratin 4 promoter GSP15′-AGGCACCTCTCTTGCCACCGCCTACAAT-3′ GSP25′-CTCGGACACACTGCTGTCTGGCAATCAT-3′B. Human keratin 4 promoter plasmid constructs K4–10405′-GCGTCGACAAAAACATTAGCTCTGAGAA-3′ K4–9405′-GCGTCGACACCATTACAGAAAACCAGAA-3′ K4–5405′-GCGTCGACTACAGAGCACTCCATTAATC-3′ K4–3405′-GCGTCGACGTGATGGGGTGTCTTTTCTG-3′ K4–1635′-GCGTCGACCAGATGACTTCTGTAGGTGT-3′ K4–1405′-GCGTCGACTAGTGACATGCTCAACGGGT-3′ K4+595′-GAAGATCTGGCTGCAGAGAGCGAGCTG-3′C. Human keratin 4 mutant promoter constructs K4–163 KMT1S5′-TGTGCCCTCGTCGACCAGAGTTGAAAGGTAGGTGTG-3′ K4–163 KMT1AS5′-GAGCATGTCACTAAACACACCTACCTTTCAACTCTG-3′ K4–163 KMT2S5′-GCCCTCGTCGACCAGATGACTTCTGTCTAACATTTTAGT-3′ K4–163 KMT2AS5′-CCCGTTGAGCATGTCACTAAAATGTTAGACAGAAGTCAT-3′A, gene-specific primers 1 and 2 (GSP 1 and 2) are designed as non-overlapping, nested PCR primers at the 5′ end of the human keratin 4 cDNA. Adapter ligated primer 1 and 2 (AP1 and 2) were provided by the manufacturer. B, the PCR primers for 5′ deletion constructs with the corresponding nucleotide position within the K4 promoter are indicated. A SalI restriction enzyme site at the 5′ end of each construct and a BglII restriction enzyme site at position +59 were added to facilitate cloning. C, the overlapping PCR primers for the K4–163 mutant constructs were introduced using the overlap extension site directed mutagenesis technique (30Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6823) Google Scholar). ASalI site was introduced at position −163 bp to facilitate cloning. Open table in a new tab A, gene-specific primers 1 and 2 (GSP 1 and 2) are designed as non-overlapping, nested PCR primers at the 5′ end of the human keratin 4 cDNA. Adapter ligated primer 1 and 2 (AP1 and 2) were provided by the manufacturer. B, the PCR primers for 5′ deletion constructs with the corresponding nucleotide position within the K4 promoter are indicated. A SalI restriction enzyme site at the 5′ end of each construct and a BglII restriction enzyme site at position +59 were added to facilitate cloning. C, the overlapping PCR primers for the K4–163 mutant constructs were introduced using the overlap extension site directed mutagenesis technique (30Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6823) Google Scholar). ASalI site was introduced at position −163 bp to facilitate cloning. A 1.1-kilobase pair human K4 5′-UTR fragment was isolated by PCR amplification to remove its putative endogenous translation start site (+59) with denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min for 30 cycles. After confirmatory DNA sequencing, the reaction product was digested with SalI and BglII, agarose gel-purified, and ligated into the luciferase reporter gene promoterless vector, pXP2 (29Nordeen S.K. BioTechniques. 1988; 6: 454-457PubMed Google Scholar), to generate the keratin 4 full-length promoter construct, designated as K4 −1040. This plasmid contained 1100 bp (−1040 bp to +59 bp) of the human keratin 4 promoter. A subsequent series of K4 promoter 5′ deletion constructs (K4 −940, K4 −540, K4 −340, K4 −163, and K4 −140) were generated in a similar fashion using K4 −1040 as a template for PCR with sense primers designed at the different positions of the promoter 5′ to the putative transcription start site and the antisense primer at position +59 (Table IB). The K4 −740 and K4 −185 constructs were generated by digestion of K4 −1040 with KpnI and BglII orBamHI and BglII, respectively, deleting 300 or 855 bp, respectively. K4 −163 promoter constructs containing mutant nucleotides spanning region −163 to −140 bp, designated as region K, were generated by site-directed mutagenesis using overlap extension PCR as described by Ho et al. (30Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6823) Google Scholar). Complementary oligonucleotide primers were used to generate two DNA fragments having overlapping ends. These fragments were combined in a subsequent “fusion” reaction, in which the overlapping ends anneal, allowing the 3′ overlap of each strand to serve as a primer for the 3′ extension of the complementary strand. The resulting fusion product was amplified further by PCR. Specific alterations in the nucleotide sequence were introduced by incorporating nucleotide changes into overlapping oligonucleotide primers. Primer sequences for overlapping mutant primers are shown in Table IC. Primers K4 −540 and K4 +59 were used to amplify the fusion product, which was finally digested using a SalI site introduced at position −163 and a BglII site at the 3′ end with subsequent subcloning. Two mutant versions of region K were introduced to obtain the promoter constructs K4 −163 KMT1 and K4 −163 KMT2. Minimal promoter DNA constructs containing wild-type or mutant nucleotides of region K were generated by ligation of kinased double-stranded synthetic oligonucleotides (Table II) into aBglII site of the pGL3-promoter vector containing the minimal SV40 promoter fused to the luciferase reporter gene (Promega). All constructs were verified with DNA sequencing (U.S. Biochemical Corp.). Plasmids were purified by a modified alkaline lysis method (Qiagen).Table IISense strand sequences of double-stranded oligonucleotides of the human keratin 4 promoter used for EMSAs and minimal promoter constructsK4-K (wild-type)5′-GATCTCAGATGACTTCTGTAGGTGTGTTA-3′K4-KMT15′-GATCTCAGAGTTGAAAGGTAGGTGTGTTA-3′K4-KMT25′-GATCTCAGATGACTTCTGTCTAACATTTA-3′K4-B (wild-type)5′-AGCTTGTAGGTGTGTTC-3′K4-BMT15′-AGCTTGTAGGTGCGTTC-3′K4-BMT25′-AGCTTGTAGGTGTTTTC-3′K4-BMT35′-AGCTTGTATATGTGTTC-3′ED-L2-F (wild-type)5′-AGCCACACCTAA-3′ED-L2-FMT5′-AGCAACACCTAA-3′Nucleotide positions in the human keratin 4 promoter are −163 to −140 bp for probe K and −151 to −140 bp for probe B. Nucleotide position of probe F is −215 to −204 bp within the EBV ED-L2 promoter. At the 5′ end of each K4 oligonucleotide, BglII (probes K) orHindIII (probes B) restriction enzyme sites were added to facilitate Klenow fill-in labeling and cloning. Sequences of interest are depicted in bold, and mutations are underlined. Open table in a new tab Nucleotide positions in the human keratin 4 promoter are −163 to −140 bp for probe K and −151 to −140 bp for probe B. Nucleotide position of probe F is −215 to −204 bp within the EBV ED-L2 promoter. At the 5′ end of each K4 oligonucleotide, BglII (probes K) orHindIII (probes B) restriction enzyme sites were added to facilitate Klenow fill-in labeling and cloning. Sequences of interest are depicted in bold, and mutations are underlined. Esophageal squamous carcinoma cell lines TE-12, TE-11, T.Tn, and T.T, pancreatic cancer cell line Panc-1 (ATCC, Rockville, MD), skin cancer cell line SCC-13, cervical cancer cell line HeLa (ATCC), colon cancer cell line HT-29 (ATCC), embryonic kidney cell line 293 (ATCC), and fibroblast cell line SL 68 (ATCC), all of human origin, were cultured under standard conditions with Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Sigma), 100 units/ml penicillin, 100 mg/ml streptomycin (Sigma), andl-glutamine (Sigma). A rabbit normal corneal cell line SIRC (ATCC) was grown in Eagle's minimal essential medium with 10% serum and antibiotics, as indicated. Transient transfection of all DNA constructs was carried out using the calcium phosphate precipitation technique (5 Prime → 3 Prime, Inc.). Cells were plated at a density of 1 × 106 cells/35-mm well and transfected 24 h later with 2 μg of the luciferase reporter plasmid and 2 μg of pGreen Lantern-1, a reporter plasmid encoding a green fluorescent protein (Life Technologies, Inc.). Cotransfections were carried out, adding to the luciferase reporter plasmid 0.5 or 1 μg of expression plasmids pRC/RSV-empty, pCMV-12S.E.1A, pCMV-12SRG2, CMVβ-p300, or pRC/RSV-mCBP. The transfectant mixture consisted of a 250-μl solution of 125 mm CaCl2, 25 mm Hepes, pH 7.05, 0.75 mm Na2HPO4, 5 mmKCl, 140 mm NaCl, and 6 mm glucose. After a 12-h incubation, cells were washed twice with phosphate-buffered saline and fresh 10% serum containing medium was exchanged. Luciferin assays were performed using luciferin, ATP, and coenzyme A (Promega) with a Monolight luminometer (Analytical Luminescence Laboratory) as described previously (26Nakagawa H. Inomoto T. Rustgi A.K. J. Biol. Chem. 1997; 272: 16688-16699Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 27Jenkins T.D. Nakagawa H. Rustgi A.K. J. Biol. Chem. 1997; 272: 24433-24442Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 28Jenkins T.D. Opitz O.G. Okano J.-I Rustgi A.K. J. Biol. Chem. 1998; 273: 10747-10754Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Cells were harvested 36 h after transfection, washed twice with phosphate-buffered saline, lysed in 200 μl of 1× cell culture lysis reagent (Promega), and 40 μl of the lysate was mixed with 100 μl of luciferase assay reagent consisting of 20 mm Tricine, 1.07 mmMgCO3, 2.67 mm MgSO4, 0.1 mm EDTA, 33.3 mm dithiothreitol, 270 μm coenzyme A, 530 μm ATP, and 470 μm luciferin. Incubations were performed in triplicate, and results were calculated as the mean ± S.E. values for luciferase activity. Values were then expressed as a -fold increase or decrease compared with the control for each set of experiments. Activities were expressed as the mean of at least three independent transfection experiments. In a subset of the transfection experiments, cells were examined under fluorescence microscopy (475 nm excitation peak, 490 nm emission peak) to assess green fluorescent protein production. The percentage of fluorescing cells was determined in each well and found not to vary within a given transfection experiment, indicating that transfection efficiency was uniform. Nuclear extracts from different cell lines were prepared as described previously (26Nakagawa H. Inomoto T. Rustgi A.K. J. Biol. Chem. 1997; 272: 16688-16699Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 27Jenkins T.D. Nakagawa H. Rustgi A.K. J. Biol. Chem. 1997; 272: 24433-24442Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 28Jenkins T.D. Opitz O.G. Okano J.-I Rustgi A.K. J. Biol. Chem. 1998; 273: 10747-10754Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The protein concentration was determined by a colorimetric method (Bio-Rad protein assay). α-32P-Labeled oligonucleotide DNA probes were constructed with 5 pmol of double-stranded oligonucleotides (Table II), synthesized by the phosphoramidite procedure (Applied Biosystems), and purified by gel electrophoresis. Radiolabeling was done by a 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 μmdTTP, 0.33 μm [α-32P]dCTP (NEN Life Science Products), 1 unit of DNA polymerase I Klenow fragment (Amersham Pharmacia Biotech), and followed by polyacrylamide gel-purification. EMSAs were carried out by incubating 5 μg of nuclear extract with 5 fmol of the α-32P-labeled oligonucleotide DNA probe (20,000 cpm) in a 20-μl binding reaction containing 10 mmTris-HCl, 50 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, 10% glycerol, and 1.0 μg of poly(dA-dT) (Amersham Pharmacia Biotech). After incubation at room temperature for 15 min, the samples were loaded on a 6% polyacrylamide, 0.25× Tris borate gel and electrophoresed at 10 V/cm for 2 h. The gels were dried and exposed to x-ray film (Kodak X-AR) at −80 °C for 12 h. For competition experiments, the nuclear extract was preincubated with 100-fold excess of unlabeled double-stranded oligonucleotides (Table II) prior to the addition of the α-32P-labeled oligonucleotide probe. All oligonucleotides were synthesized by the phosphoramidite procedure (Applied Biosystems) and purified by gel electrophoresis. Immune supershift assays were performed using a polyclonal anti-AP2 antibody (AP2α (C-18)) and polyclonal anti-CBP antibodies (CBP1 (451), CBP2 (A-22)) (all from Santa Cruz Biotechnology). The antibody was preincubated with the nuclear extract at 4 °C for 1–12 h prior to the addition of the α-32P-labeled oligonucleotide DNA probe. Other conditions for EMSAs are described above. The B and BMT2 single-stranded oligonucleotides corresponding to the wild-type and mutant sequences of the human keratin 4 promoter (Table II) were labeled by annealing corresponding 7-mers (oligonucleotides) followed by the Klenow fill-in reaction, as described above, except that 5-bromodeoxyuridine was substituted for dTTP. Binding reactions were performed in an identical fashion, except that the reaction was for 30 min at 4 °C to inhibit protein degradation. Samples were exposed to a medium wave (312 nm) UV transilluminator (UVP Inc.) for 30 min on ice at a distance of 3 cm. Samples were then mixed with 2× SDS-sample buffer and boiled for 5 min. Electrophoresis was carried out on a 10% SDS-polyacrylamide gel. Gels were then dried and exposed to x-ray film (Kodak X-AR) at −80 °C for 12 h. To obtain the 5′-untr