Portal de Periódicos da CAPES

Functional Characterization of the Promoter of the X-linked Ectodermal Dysplasia Gene

1999; Elsevier BV; Volume: 274; Issue: 37 Linguagem: Inglês

10.1074/jbc.274.37.26477

1083-351X

Gina Pengue, Anand Srivastava, Juha Kere, David Schlessinger, Meredith C. Durmowicz,

Cancer-related gene regulation

Anhidrotic ectodermal dysplasia (EDA) is a disorder characterized by poor development of hair, teeth, and sweat glands, and results from lesions in the X-linked EDA gene. We have cloned a 1.6-kilobase 5′-flanking region of the human EDA gene and used it to analyze features of transcriptional regulation. Primer extension analysis located a single transcription initiation site 264 base pairs (bp) upstream of the translation start site. When the intact cloned fragment or truncated derivatives were placed upstream of a reporter luciferase gene and transfected into a series of cultured cells, expression comparable with that conferred by an SV40 promoter-enhancer was observed. The region lacks a TATA box sequence, and basal transcription from the unique start site is dependent on two binding sites for the Sp1 transcription factor. One site lies 38 bp 5′ to the transcription start site, in a 71-bp sequence that is sufficient to support up to 35% of maximal transcription. The functional importance of the Sp1 sites was demonstrated when cotransfection of an Sp1 expression vector transactivated the EDA promoter in the SL2 Drosophila cell line that otherwise lacks endogenous Sp1. Also, both Sp1 binding sites were active in footprinting and gel shift assays in the presence of either crude HeLa cell nuclear extract or purified Sp1 and lost activity when the binding sites were mutated. A second region involved in positive control was localized to a 40-bp sequence between −673 and −633 bp. This region activated an SV40 minimal promoter 4- to 5-fold in an orientation-independent manner and is thus inferred to contain an enhancer region. Anhidrotic ectodermal dysplasia (EDA) is a disorder characterized by poor development of hair, teeth, and sweat glands, and results from lesions in the X-linked EDA gene. We have cloned a 1.6-kilobase 5′-flanking region of the human EDA gene and used it to analyze features of transcriptional regulation. Primer extension analysis located a single transcription initiation site 264 base pairs (bp) upstream of the translation start site. When the intact cloned fragment or truncated derivatives were placed upstream of a reporter luciferase gene and transfected into a series of cultured cells, expression comparable with that conferred by an SV40 promoter-enhancer was observed. The region lacks a TATA box sequence, and basal transcription from the unique start site is dependent on two binding sites for the Sp1 transcription factor. One site lies 38 bp 5′ to the transcription start site, in a 71-bp sequence that is sufficient to support up to 35% of maximal transcription. The functional importance of the Sp1 sites was demonstrated when cotransfection of an Sp1 expression vector transactivated the EDA promoter in the SL2 Drosophila cell line that otherwise lacks endogenous Sp1. Also, both Sp1 binding sites were active in footprinting and gel shift assays in the presence of either crude HeLa cell nuclear extract or purified Sp1 and lost activity when the binding sites were mutated. A second region involved in positive control was localized to a 40-bp sequence between −673 and −633 bp. This region activated an SV40 minimal promoter 4- to 5-fold in an orientation-independent manner and is thus inferred to contain an enhancer region. anhidrotic ectodermal dysplasia nucleotide(s) base pair(s) polymerase chain reaction Anhidrotic ectodermal dysplasia (EDA)1 is an X-linked recessive disorder that affects the development of ectodermal structures (1Clarke A. J. Med. Genet. 1987; 24: 653-659Crossref Scopus (107) Google Scholar). The gene responsible for the disorder was originally isolated by positional cloning (2Kere J. Srivastava A.K. Montonen O. Zonana J. Thomas N. Ferguson B. Munoz F. Morgan D. Clarke A. Baybayan P. Chen E., Y. Ezer S. Saarialho-Kere U. de la Chapelle A. Schlessinger D. Nat. Genet. 1996; 13: 409-416Crossref PubMed Scopus (576) Google Scholar). Additional exons of the EDA gene have recently been identified, bringing the total number of exons in the gene to twelve (3Bayes M. Hartung A.J. Ezer S. Pispa J. Thesleff I. Srivastava A.K. Kere J. Hum. Mol. Genet. 1998; 7: 1661-1669Crossref PubMed Scopus (178) Google Scholar). Mutations in affected individuals have been characterized (2Kere J. Srivastava A.K. Montonen O. Zonana J. Thomas N. Ferguson B. Munoz F. Morgan D. Clarke A. Baybayan P. Chen E., Y. Ezer S. Saarialho-Kere U. de la Chapelle A. Schlessinger D. Nat. Genet. 1996; 13: 409-416Crossref PubMed Scopus (576) Google Scholar, 3Bayes M. Hartung A.J. Ezer S. Pispa J. Thesleff I. Srivastava A.K. Kere J. Hum. Mol. Genet. 1998; 7: 1661-1669Crossref PubMed Scopus (178) Google Scholar, 4Monreal A.W. Zonana J. Ferguson B. Am. J. Hum. Genet. 1998; 63: 380-389Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), and interruption of the orthologous gene in mouse leads to the Tabby phenotype (5Srivastava A.K. Pispa J. Du Y. Hartung A., P. Ezer S. Jenks T. Shimada T. Pekkannen M. Ko M.S.H. Thesleff I. Kere J. Schlessinger D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13069-13074Crossref PubMed Scopus (247) Google Scholar, 6Ferguson B.M. Brockdorff N. Formstone E. Ngyuen T. Kronmiller J.E. Zonana J. Hum. Mol. Genet. 1997; 6: 1589-1594Crossref PubMed Scopus (142) Google Scholar). Because affected individuals have sparse hair, rudimentary teeth, and no sweat glands, and Tabby mice show similar defects, the gene is believed to function at an early stage in ectodermal development, possibly at a branch point. Some hints as to the function of the EDA protein have been gained by findings that it associates with the cell membrane and may participate in the regulation of cell-cell or cell-matrix interactions (3Bayes M. Hartung A.J. Ezer S. Pispa J. Thesleff I. Srivastava A.K. Kere J. Hum. Mol. Genet. 1998; 7: 1661-1669Crossref PubMed Scopus (178) Google Scholar, 7Ezer S. Schlessinger D. Srivastava A. Kere J. Hum. Mol. Genet. 1997; 6: 1581-1587Crossref PubMed Scopus (21) Google Scholar). Consistent with a role in such interactions, exons of the gene encode collagen-like repeat motifs (2Kere J. Srivastava A.K. Montonen O. Zonana J. Thomas N. Ferguson B. Munoz F. Morgan D. Clarke A. Baybayan P. Chen E., Y. Ezer S. Saarialho-Kere U. de la Chapelle A. Schlessinger D. Nat. Genet. 1996; 13: 409-416Crossref PubMed Scopus (576) Google Scholar) that have been shown to form collagenous trimers in the extracellular domain of the EDA protein. 2S. Ezer, M. Bayes, O. Elomaa, D. Schlessinger, and J. Kere, submitted for publication.2S. Ezer, M. Bayes, O. Elomaa, D. Schlessinger, and J. Kere, submitted for publication. Studies of EDA gene expression and protein function have been complicated by the fact that the EDA transcript undergoes alternative splicing and is capable of forming eight distinct isoforms, many of which can be detected by reverse transcriptase-polymerase chain reaction (PCR) in a variety of tissues (3Bayes M. Hartung A.J. Ezer S. Pispa J. Thesleff I. Srivastava A.K. Kere J. Hum. Mol. Genet. 1998; 7: 1661-1669Crossref PubMed Scopus (178) Google Scholar). In addition, in situ hybridization and immunohistochemical analysis of various human embryonic, fetal, and adult tissues have demonstrated that the EDA gene and protein are expressed at low levels in several tissues unaffected in EDA as well as in the ectodermal tissues that develop abnormally (2Kere J. Srivastava A.K. Montonen O. Zonana J. Thomas N. Ferguson B. Munoz F. Morgan D. Clarke A. Baybayan P. Chen E., Y. Ezer S. Saarialho-Kere U. de la Chapelle A. Schlessinger D. Nat. Genet. 1996; 13: 409-416Crossref PubMed Scopus (576) Google Scholar, 9Montonen O. Ezer S. Saarialho-Kere U.K. Herva R. Karjalainen-Lindsberg M.-L. Kaitila I. Schlessinger D. Srivastava A.K. Thesleff I. Kere J. J. Histochem. Cytochem. 1998; 46: 281-289Crossref PubMed Scopus (40) Google Scholar). In this work, we have initiated studies to analyze the regulation of EDA gene expression. The minimal promoter region, including two Sp1 sites important for promoter function, has been defined. In addition, an enhancer region centered at 653 bp upstream of the transcription start site has been identified. The enhancer segment includes putative binding sites for two transcription factors known to regulate tissue- and developmental stage-specific expression of certain cardiac genes. HeLa, 293, and HaCaT cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfections were done with 10 μg of reporter plasmids. The 293 and HeLa cell lines were transfected in subconfluent cultures by the calcium phosphate method. The HaCaT cell line was transfected using liposomes (DOTAP, from Roche Molecular Biochemicals) with 7 μl of DOTAP per microgram of DNA. To normalize transfection efficiencies, a plasmid expressing β-galactosidase (pSV-β-Gal plasmid, Promega) was cotransfected with the test plasmid in each experiment. Promoter activity was normalized to protein concentration and β-galactosidase activity.Drosophila melanogaster SL2 cells were grown in Schneider's medium supplemented with 10% heat-inactivated fetal calf serum and transfected using calcium phosphate precipitation. Cells were harvested 48 h after transfection, and extracts were assayed for luciferase activity according to the Promega protocol. To create the p-1625 plasmid, the p2A5 genomic clone (10Srivastava A., K. Montonen O. Saarialho-Kere U. Chen E. Baybayan P. Pispa J. Limon J. Schlessinger D. Kere J. Am. J. Hum. Genet. 1996; 58: 126-132PubMed Google Scholar) was digested with Eco RI (position −1608) and Sma I (position +258), and the single-stranded ends of the released fragment were made double-stranded using the Klenow fragment of DNA polymerase I. The fragment was then blunt-end ligated into the Sma I site of the promoterless pGL2-Basic vector (Promega). Other constructs included: plasmid p-785, constructed by digestion of p2A5 with Acc I (position −768) and Sma I (position +258). The Acc I site was filled in as above, and the resulting fragment was cloned into the Sma I site of pGL2-Basic. p-338 was constructed by digestion of p-785 with Pst I (positions −321 and +168); the fragment was rendered blunt-ended with T4 DNA polymerase and cloned into the Sma I site of pGL2-Basic. p-88 was created using PCR. The promoter fragment (nt −71 to nt +63) was amplified using the upstream primer 5′-TCCCCCGGGTGGAGGCCCGGCT-3′ and the downstream primer 5′-GAAGATCTCCCGCCGAGGGAAT, with Sma I and Bgl II sites (underlined), respectively, incorporated into the primers. The PCR product was then cloned between the Sma I and Bgl II sites of pGL2-Basic. p-53 was constructed by digestion of p-785 with Nar I (position −35) and Sma I (position +258), filled in with Klenow fragment, and cloned into the Sma I site of plasmid pGL2-Basic. p125(5′−3′), p103(5′−3′), p83(5′−3′), and p63(5′−3′) were constructed by PCR, using the primers listed below. A Sma I site (underlined) was included in each of the 5′-primers, and a Bgl II site was included in each of the 3′-primers. The amplified DNA fragments were subcloned into the Sma I/Bgl II sites of the plasmid pGL2-Promoter (Promega). p125(5′−3′) and p103(5′−3′) were constructed using the 5′-primers 5′-TCCCCCGGGTACAGGGATCGATAG-3′ and 5′-TCCCCCGGGTGTTGAATTAATTAA-3′ and the same 3′-primer, 5′-GAAGATCTAGTAACAGAGAAGC-3′. p83(5′−3′) and p63(5′−3′) were constructed using the 5′-primer 5′-TCCCCCGGGCAAGAAATCCTAGGA-3′ and the 3′-primers 5′-GAAGATCTAGTAACAGAGAAGC-3′ and 5′-GAAGATCTATGCCAAGCGGAACTG-3′, respectively. p125(3′−5′), p103(3′−5′), p83(3′−5′), and p63(3′−5′) were similarly constructed by PCR, using the primers listed below. For these constructs, the Bgl II site was included in each of the 5′-primers, and the Sma I site was included in each of the 3′-primers. The DNA fragments were again subcloned into the Sma I/Bgl II sites of the plasmid pGL2-Promoter. p125(3′−5′), p103(3′−5′), and p83(3′−5′) were made using the 3′-primer 5′-TCCCCCGGGAGTAACAGAGAAGC-3′ and the 5′-primers, 5′-GAAGATCTTACAGGGATCGATAG-3′, 5′-GAAGATCTTGTTGAATTAATTAA-3′, and 5′-GAAGATCTCAAGAAATCCTAGGA-3′, respectively. p63(3′−5′) was constructed using the 3′-primer, 5′TCCCCCGGGATGCCAAGCGGAACTG-3′, and the 5′-primer, 5′-GAAGATCTCAAGAAATCCTAGGA-3′. pmBox1 and pmBox2 mutant constructs were created by PCR-mediated site-directed mutagenesis (11Aiyar A. Leis J. BioTechniques. 1993; 11: 366-368Google Scholar). The mutant plasmids contain GG to TT substitutions in both of the putative Sp1 boxes as follows: GGTTCGGGG (Box B1) and GGTTCGGAC (Box B2). The mutated bases are underlined. All plasmid constructs were analyzed by DNA sequencing to confirm that the constructions were correct. Plasmids pPac and pPac-Sp1 were kindly provided by Dr. Luigi Lania. HeLa cells were transfected as described, and RNA was isolated from these cells using the SV Total RNA isolation system from Promega. An EDA-specific primer, PE1625–22 (5′-GCAGCTCTACTCCGAGGGGTGG-3′), was end-labeled with 32P using T4 polynucleotide kinase, and primer extension reactions were carried out as described by Ordahl, et al. (12Ordahl C.P. Evans G.L. Cooper T.A. Kunz G. Perriard J.C. J. Biol. Chem. 1984; 259: 15224-15227Abstract Full Text PDF PubMed Google Scholar). The same primer was used in sequencing reactions that were done with the 33P Thermo Sequenase radiolabeled cycle sequencing terminator kit (Amersham Pharmacia Biotech). All samples were electrophoresed on a 6% polyacrylamide, 8 m urea sequencing gel in 1× TBE (0.1m Tris-HCl, 90 mm boric acid, 1 mmEDTA, pH 8.0), dried, and exposed to x-ray film with an intensifying screen. Gel shift assays were performed with radiolabeled double-stranded oligonucleotides from the EDA gene, nt −52 to nt −22 (Box B1) and nt −197 to nt −167 (Box B2). The oligonucleotides were labeled with 32P using T4 polynucleotide kinase. Nuclear extracts were prepared from 293, HeLa, and HaCaT cells, and DNA binding assays were performed as described (13Majello B. Arcone R. Toniatti C. Ciliberto G. EMBO J. 1990; 9: 457-465Crossref PubMed Scopus (144) Google Scholar). In competition experiments, unlabeled competitor was included in the preincubation reaction with the nuclear extract. Oligonucleotides containing the consensus sequence and mutated sequence for Sp1 binding (5′-ATTCGATCGGGGCGGGGCGAGC-3′ and 5′-ATTCGATCGG TT CGGGGGAC-3′, respectively) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz). For antibody “supershift” experiments, 0.5 and 1μg of polyclonal antisera specific for Sp1 and AP2 (Santa Cruz Biotechnology, Inc.) were added to the binding assay reactions and incubated on ice for 30 min before the radiolabeled oligonucleotides were added. Plasmid F1 (containing FragmentI) was constructed using PCR amplification of the EDA sequence from nt −102 to nt +63 with primers containing a Sma I site in the 5′-primer (5′-TCCCCCGGGGGCGAACCC-3′) and a Bgl II site in the 3′-primer (5′-GAAGATCTCCCGCCGAGGGAAT-3′). The amplified fragment was again inserted into the Sma I/Bgl II sites of pGL2-Basic. Plasmid F1 was cleaved with Bgl II restriction endonuclease, labeled by filling in ends with [α-32P]dCTP using the Klenow fragment of DNA polymerase I, and digested with Sma I. Plasmid p-785 (FragmentII), described above, was digested with Nar I, labeled by end-filling with [α-32P]dCTP and Klenow DNA polymerase, and then digested with Pst I. Each labeled fragment was gel purified. Binding reactions were carried out using 30–50 μg of nuclear extract or 1–2 footprinting units of purified Sp1 fragment (Promega) and 30,000 cpm of probe. After DNase digestion, DNA fragments were analyzed on 6% polyacrylamide sequencing gels containing 8m urea. The sequences of the binding sites were confirmed by Maxam-Gilbert G + A sequencing reactions performed on each fragment. The primary findings are that, although the level of EDA mRNA in tissues is low (2Kere J. Srivastava A.K. Montonen O. Zonana J. Thomas N. Ferguson B. Munoz F. Morgan D. Clarke A. Baybayan P. Chen E., Y. Ezer S. Saarialho-Kere U. de la Chapelle A. Schlessinger D. Nat. Genet. 1996; 13: 409-416Crossref PubMed Scopus (576) Google Scholar, 7Ezer S. Schlessinger D. Srivastava A. Kere J. Hum. Mol. Genet. 1997; 6: 1581-1587Crossref PubMed Scopus (21) Google Scholar, 9Montonen O. Ezer S. Saarialho-Kere U.K. Herva R. Karjalainen-Lindsberg M.-L. Kaitila I. Schlessinger D. Srivastava A.K. Thesleff I. Kere J. J. Histochem. Cytochem. 1998; 46: 281-289Crossref PubMed Scopus (40) Google Scholar), the promoter contains basal Sp1 elements and an enhancer region that sustain RNA transcription at a high level from a single initiation site. Because endogenous levels of the EDA transcript are extremely low (2Kere J. Srivastava A.K. Montonen O. Zonana J. Thomas N. Ferguson B. Munoz F. Morgan D. Clarke A. Baybayan P. Chen E., Y. Ezer S. Saarialho-Kere U. de la Chapelle A. Schlessinger D. Nat. Genet. 1996; 13: 409-416Crossref PubMed Scopus (576) Google Scholar, 7Ezer S. Schlessinger D. Srivastava A. Kere J. Hum. Mol. Genet. 1997; 6: 1581-1587Crossref PubMed Scopus (21) Google Scholar, 9Montonen O. Ezer S. Saarialho-Kere U.K. Herva R. Karjalainen-Lindsberg M.-L. Kaitila I. Schlessinger D. Srivastava A.K. Thesleff I. Kere J. J. Histochem. Cytochem. 1998; 46: 281-289Crossref PubMed Scopus (40) Google Scholar), the studies here have been carried out with cells transiently transfected with EDA constructs to increase signal strength. Although EDA is very widely expressed, epithelial-derived cell lines have been used as they are likely to be more relevant for a gene involved in skin appendage formation. The transcription start site was determined by primer extension analysis carried out on RNA preparations from HeLa epithelial cells transfected with plasmid p-1625. This plasmid contains a 1.6-kilobase genomic fragment that includes 5′-upstream sequences and part of the known cDNA sequence and has been shown to direct high levels of transcription (see below). As shown in Fig.1, lane 2, a single transcription start site was observed at nucleotide 3453 (G) of the EDA genomic sequence. DNA sequencing has revealed that the EDA gene lacks basal elements like the TATA box or an initiator sequence. The absence of such sites and the presence of Sp1 sites, including one in the basal promoter (see below), might have been expected to result in initiation of transcription at several locations. However, a single initiation site was consistently detected in these assays and in RNase protection assays (data not shown). This analysis extends the 5′-end of the EDA transcript 47-bp further than the published cDNA sequence. To test for promoter activity, a genomic fragment of 1.6 kilobases, including 5′-upstream sequences and part of the known cDNA sequence, was placed 5′ to a luciferase reporter gene, and its capacity to direct luciferase synthesis in transfection experiments was compared with a series of deletion mutant constructs. Each truncated segment was cloned into the promoterless reporter plasmid pGL2-Basic and transfected into human epithelia-derived cell lines. Each of these cell lines (HeLa, 293, and HaCat) have been shown to exhibit low levels of endogenous EDA gene expression, as detected by reverse transcriptase-PCR, similar to levels found in other cell types and tissues (2Kere J. Srivastava A.K. Montonen O. Zonana J. Thomas N. Ferguson B. Munoz F. Morgan D. Clarke A. Baybayan P. Chen E., Y. Ezer S. Saarialho-Kere U. de la Chapelle A. Schlessinger D. Nat. Genet. 1996; 13: 409-416Crossref PubMed Scopus (576) Google Scholar, 3Bayes M. Hartung A.J. Ezer S. Pispa J. Thesleff I. Srivastava A.K. Kere J. Hum. Mol. Genet. 1998; 7: 1661-1669Crossref PubMed Scopus (178) Google Scholar, 9Montonen O. Ezer S. Saarialho-Kere U.K. Herva R. Karjalainen-Lindsberg M.-L. Kaitila I. Schlessinger D. Srivastava A.K. Thesleff I. Kere J. J. Histochem. Cytochem. 1998; 46: 281-289Crossref PubMed Scopus (40) Google Scholar). Cells were transfected as described under “Experimental Procedures,” and after 48 h, cell extracts were prepared and luciferase activity was measured (Fig.2). This index of promoter strength was normalized to the activity of a cotransfected β-galactosidase reporter gene under SV40 promoter control. Promoter constructs retaining 1.6 kilobases to 35 bp of DNA upstream of the transcription initiation site varied in potency by more than 90%. Promoter activity as strong as an SV40 control promoter-enhancer (Fig.2) was maintained in constructs deleted up to −767 bp. Removal of the region between −767 and −321 bp, however, decreased promoter activity in HeLa cells and HaCaT cells by 50%, and in 293 cells by 40%. A further drop of the remaining activity was obtained after removing sequence extending from −321 to −71 bp (by 50% in HeLa and in HaCaT, and by 40% in 293 cells). As is often seen in transient expression assays of promoter activity, each of the promoter constructs exhibits a rather higher level of activity in one cell type rather than another (here, 293 cells compared with HeLa or HaCat cells). However, in all cell types, the subfragment of 71 bp immediately proximal to the transcription start site provides enough DNA sequence for basal transcription of the human EDA gene. It contains a single Sp1 site. In contrast, constructs further shortened to include only 35 bp upstream of the transcription initiation site, which eliminates the last Sp1 site, show no activity over the background from the promoterless luciferase vector pGL2 in HeLa and HaCaT cells. (The −35-bp construct transfected into 293 cells retained activity about 10% higher than the promoterless pGL2, probably because of a higher transfection efficiency.) Because the Sp1 binding sites (“GC boxes”) bind transcription factor Sp1 and other members of the Sp1 multigene family (14Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar), these results provide an indication that Sp1 or similar proteins may be important in the regulation of EDA expression. Based on the transfection experiments of deletion mutant constructs (Fig. 2), the region between +63 and −321 bp was chosen for further analysis. DNase I footprinting analysis was used to map binding sites of nuclear factors, using nuclear extracts prepared from HeLa cells. Similar results were obtained from the HaCaT and 293 cell lines (data not shown). Each of two fragments of the promoter was 5′-end-labeled to generate a single-strand-labeled fragment for the assays. Fragment I (−102 to +63 bp) showed one protected region (−45 to −31 bp; Fig. 3, lane 2), containing a canonical GC box (Box B1), i.e. a putative binding site for Sp1. It seemed likely that Sp1 might indeed bind to this region because it functions as an essential factor for several viral and cellular promoters (15Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1243) Google Scholar, 16Courey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1071) Google Scholar, 17Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 18Pugh B., F. Tjian R. Cell. 1990; 61: 1187-1197Abstract Full Text PDF PubMed Scopus (732) Google Scholar, 19Majello B. De Luca P. Hagen G. Suske G. Lania L. Nucleic Acids Res. 1994; 22: 4914-4921Crossref PubMed Scopus (202) Google Scholar). To establish whether this GC box is protected by Sp1, DNase I footprinting analysis was carried out with purified Sp1 protein. As shown in Fig. 3, lane 3, recombinant Sp1 protected the same GC-rich sequence noted in the experiment with HeLa cell nuclear extract, suggesting that Box B1 of the EDA promoter region can specifically interact with the transcription factor Sp1. In repeated comparable experiments using the second probe (-37 to −321 bp), a second potential binding site for Sp1 was detected between nt −177 and nt −189 (Box B2). However, a higher background was observed, possibly because of nonspecific binding, which resulted in a significantly weaker signal (data not shown). Stronger evidence that Box B2 also specifically interacts with Sp1 was derived from further experiments using gel shift assays and transactivation studies with an Sp1 expression vector (see below). To characterize further the functionality of the Sp1 sites, we performed electrophoretic mobility shift assays. We used 30-bp double-stranded oligonucleotides from positions −52 to −22 bp (Box B1) and −197 to −167 bp (Box B2). Incubation of the Box B1 probe with the HeLa nuclear extract resulted in the formation of three complexes (complex I, complex II, and a less consistently observed very weak complex III (Fig.4 A, lane 1)). Similar binding patterns were observed for the Sp1 binding sites in comparable assays with crude nuclear extract (14Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar). Complexes I and II were competed away by unlabeled oligonucleotide, verifying the specificity of the binding to DNA. The third complex was constitutively expressed and was not competed away by unlabeled probe; it is inferred to be a nonspecific binding site. To establish that binding was specific for Sp1, increasing amounts of an antibody to Sp1 were added to the binding reaction, resulting in a “supershift” (a shift to lower mobility) of Complex I (Fig.4 B, lanes 2 and 3). The inability of Sp1 to supershift the complex fully may be because of the comigration of a complex with another member of the Sp1-family, e.g. Sp3 (14Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 20Williams T. Admon A. Luscher B. Tjian R. Genes and Dev. 1988; 2: 1557-1569Crossref PubMed Scopus (449) Google Scholar). A control antibody to the transcription factor AP-2, which is expressed in HeLa cells and can also bind some GC-rich sequences (20Williams T. Admon A. Luscher B. Tjian R. Genes and Dev. 1988; 2: 1557-1569Crossref PubMed Scopus (449) Google Scholar), had no effect on the mobility of the bound complex (Fig. 4 B, lanes 4 and 5). To substantiate further that Sp1 bound to Box B1, competition experiments were performed in which unlabeled Box B1 oligonucleotides and double-stranded oligonucleotides containing the consensus Sp1 binding site or containing mutated Sp1 consensus sequence were added to the reactions and gel mobility shifts were again assessed. Complexes I and II were both competed for in a dose-dependent manner by Box B1 and Sp1 consensus double-stranded oligonucleotides (Fig. 4 C, lanes 2–5) but not by increasing amounts of double-stranded oligonucleotides containing a mutated Sp1 binding site (Fig.4 C, lanes 6–9). Similar results were obtained using as a probe the putative Box B2 Sp1 site (Fig.5). As shown in Fig. 5 A, lane 1, two other minor nonspecific complexes were also observed. Taken together, the results further demonstrate directly that Sp1 binds to both the Box B1 and Box B2 GC sequences. As another confirmation of Sp1 function, we tested the extent of its transactivation of the EDA promoter in the SL2 cell line. D. melanogaster Schneider cell line SL2 is known to be devoid of endogenous Sp1-like activity and thus serves as a useful cell line to test Sp1 effects in vivo (16Courey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1071) Google Scholar, 17Courey A.J. Holtzman D.A. Jackson S.P. Tjian R. Cell. 1989; 59: 827-836Abstract Full Text PDF PubMed Scopus (387) Google Scholar). When reporter plasmids p-1625 and p-88, which contain two and one Sp1 binding sites, respectively, were cotransfected with increasing amounts of the eukaryotic expression vector pPAC-Sp1 (16Courey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1071) Google Scholar), we observed a strong enhancement of transcription compared with the reporter vector pGL2-Basic (Fig. 6). Note that higher levels of added pPAC-Sp1 resulted in some decrease of transcription activity, perhaps because Sp1 cofactors were squelched; but the results confirm the ability of co-expressed Sp1 to induce expression from the EDA promoter, further supporting the functionality of the Sp1 sites. To assess the importance of each Sp1 site for EDA gene expression, we assayed the effects of point mutations in each of them. Reporter constructs were created that differed by 2 bp from the wild type promoter (−548 to +63 bp; see “Experimental Procedures”) and were assayed for luciferase activity in HeLa cells. The promoter activity from the mutant constructs pmBox1 and pmBox2 was decreased to 40 and 10% of the wild type promoter, respectively (Fig. 7). These results suggest that Box B2 may be functionally more important than Box B1; however, both sites were required for maximal promoter activity. Another positive regulatory sequence extending from −673 to −550 bp was detected using constructs with this fragment cloned in both orientations into the pGL2-Promoter vector. This vector contains an SV40 minimal promoter that drives the expression of the luciferase reporter gene. When constructs were transfected into HeLa cells, the EDA promoter fragment induced the production of luciferase by 4- to 5-fold compared with the level observed for the vector alone. Consistent with the notion that this DNA segment contains an enhancer binding site, the activity of the cloned region was very similar in both cloned orientations (Fig.8 A). To define the core region that still gave enhancer activity, smaller fragments of the region were tested by repeated transfection experiments. As shown in Fig. 8 A, enhancer activity was slightly reduced when the region between −673 and −653 bp was removed and completely abolished when the fragment was truncated further to −633 bp upstream of the transcription start site. From these results, we concluded that