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The Dominant Role of Sp1 in Regulating the Cystathionine β-Synthase –1a and –1b Promoters Facilitates Potential Tissue-specific Regulation by Kruppel-like Factors

2004; Elsevier BV; Volume: 279; Issue: 10 Linguagem: Inglês

10.1074/jbc.m310211200

1083-351X

Kenneth N. Maclean, Eva Kraus, Jan P. Kraus,

Epigenetics and DNA Methylation

Cystathionine β-synthase (CBS) catalyzes the condensation of serine with homocysteine to form cystathionine and occupies a crucial regulatory position between the methionine cycle and transsulfuration. The human cystathionine β-synthase gene promoters –1a and –1b are expressed in a limited number of tissues and are coordinately regulated with proliferation through a redox-sensitive mechanism. Site-directed mutagenesis, DNase I footprinting and deletion analysis of 5276 bp of 5′ proximal –1b flanking sequence revealed that this region does not confer tissue-specific expression and that 210 bp of proximal sequence is sufficient for maximal promoter activity. As little as 32 bp of the –1b proximal promoter region is capable of driving transcription in HepG2 cells, and this activity is entirely dependent upon the presence of a single overlapping Sp1/Egr1 binding site. Co-transfection studies in Drosophila SL2 cells indicated that both promoters are transactivated by Sp1 and Sp3 but only the –1b promoter is subject to a site-specific synergistic regulatory interaction between Sp1 and Sp3. Sp1-deficient fibroblasts expressing both Sp3 and NF-Y were negative for CBS activity. Transfection of these cells with a mammalian Sp1 expression construct induced high levels of CBS activity indicating that Sp1 has a critical and indispensable role in the regulation of cystathionine β-synthase. Sp1 binding to both CBS promoters is sensitive to proliferation status and is negatively regulated by Kruppel-like factors in co-transfection experiments suggesting a possible mechanism for the tissue specific regulation of cystathionine β-synthase. Cystathionine β-synthase (CBS) catalyzes the condensation of serine with homocysteine to form cystathionine and occupies a crucial regulatory position between the methionine cycle and transsulfuration. The human cystathionine β-synthase gene promoters –1a and –1b are expressed in a limited number of tissues and are coordinately regulated with proliferation through a redox-sensitive mechanism. Site-directed mutagenesis, DNase I footprinting and deletion analysis of 5276 bp of 5′ proximal –1b flanking sequence revealed that this region does not confer tissue-specific expression and that 210 bp of proximal sequence is sufficient for maximal promoter activity. As little as 32 bp of the –1b proximal promoter region is capable of driving transcription in HepG2 cells, and this activity is entirely dependent upon the presence of a single overlapping Sp1/Egr1 binding site. Co-transfection studies in Drosophila SL2 cells indicated that both promoters are transactivated by Sp1 and Sp3 but only the –1b promoter is subject to a site-specific synergistic regulatory interaction between Sp1 and Sp3. Sp1-deficient fibroblasts expressing both Sp3 and NF-Y were negative for CBS activity. Transfection of these cells with a mammalian Sp1 expression construct induced high levels of CBS activity indicating that Sp1 has a critical and indispensable role in the regulation of cystathionine β-synthase. Sp1 binding to both CBS promoters is sensitive to proliferation status and is negatively regulated by Kruppel-like factors in co-transfection experiments suggesting a possible mechanism for the tissue specific regulation of cystathionine β-synthase. Cystathionine β-synthase (EC 4.2.1.22, CBS) 1The abbreviations used are: CBS, cystathionine β-synthase; AdoMet, S-adenosyl-l-methionine; EMSA, electrophoretic mobility shift assay; Hcy, homocysteine; tHcy, total homocysteine; KLF, Kruppel-like factor; LKLF, lung Kruppel-like factor; BKLF, basic Kruppel-like factor; CMV, cytomegalovirus. catalyzes a pyridoxal 5′-phosphate-dependent β-replacement reaction condensing homocysteine (Hcy) and serine to form cystathionine, which is subsequently converted to cysteine by the action of cystathionine γ-lyase. In addition to being essential for the synthesis of cysteine by transsulfuration, CBS is also a key regulator of plasma total Hcy (tHcy) levels (1Mudd S.H. Levy H.L. Kraus J.P. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 2007-2056Google Scholar). CBS deficiency is the most common cause of homocystinuria, an inherited autosomal recessive metabolic disease, which if untreated, causes skeletal abnormalities, dislocated optic lenses, mental retardation, and a dramatically increased incidence of vascular disorders particularly thromboembolic disease (1Mudd S.H. Levy H.L. Kraus J.P. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 2007-2056Google Scholar). Moderate elevation of plasma tHcy has been identified as a major risk factor for Alzheimer's disease (2Seshadri S. Beiser A. Selhub J. Jacques P.F. Rosenberg I.H. D'Agostino R.B. Wilson P.W. Wolf P.A. N. Engl. J. Med. 2002; 346: 476-483Crossref PubMed Scopus (2867) Google Scholar, 3Kruman I.I. Kumaravel T.S. Lohani A. Pedersen W.A. Cutler R.G. Kruman Y. Haughey N. Lee J. Evans M. Mattson M.P. J. Neurosci. 2002; 22: 1752-1762Crossref PubMed Google Scholar), neural tube defects (4Mills J.L. McPartlin J.M. Kirke P.N. Lee Y.J. Conley M.R. Weir D.G. Scott J.M. Lancet. 1995; 345: 149-151Abstract PubMed Scopus (500) Google Scholar), and cardiovascular disease (5Selhub J. Haematologica. 1997; 82: 129-132PubMed Google Scholar). Because of the key role of CBS in regulating plasma tHcy levels, there is a need for a greater understanding of the mechanisms and principles that govern its regulation. Previously, we have determined the complete genomic sequence of human CBS (6Kraus J.P. Oliveriusova J. Sokolova J. Kraus E. Vlcek C. de Franchis R. Maclean K.N. Bao L. Bukovska G. Patterson D. Paces V. Ansorge W. Kozich V. Genomics. 1998; 52: 312-324Crossref PubMed Scopus (102) Google Scholar) and mapped the transcriptional start sites of five human CBS mRNA isoforms, designated CBS –1a, –1b, –1c, –1d, and –1e, respectively (7Bao L. Vlcek C. Paces V. Kraus J.P. Arch. Biochem. Biophys. 1998; 350: 95-103Crossref PubMed Scopus (90) Google Scholar). Of these, we found that isoforms –1a and –1b form the vast majority of transcripts, whereas isoforms –1c, –1d, and –1e are relatively rare. We identified two promoter regions upstream of exons –1a and –1b and found that the –1b promoter has ∼10-fold greater promoter activity than that of the –1a promoter in both HepG2 and COS7 cells (6Kraus J.P. Oliveriusova J. Sokolova J. Kraus E. Vlcek C. de Franchis R. Maclean K.N. Bao L. Bukovska G. Patterson D. Paces V. Ansorge W. Kozich V. Genomics. 1998; 52: 312-324Crossref PubMed Scopus (102) Google Scholar). The CBS promoter regions constitute an interesting paradox in that they have multiple sites of transcriptional initiation, are GC-rich and lack the classic TATA box sequence. Yet, despite having all of the usual characteristics of a ubiquitously expressed "housekeeping" gene, CBS is expressed in a highly tissue-specific manner with a wide range of tissues completely negative for CBS activity. The temporal and spatial expression patterns of CBS appear to be developmentally regulated (8Mudd S.H. Finkelstein J.D. Irreverre F. Laster L. J. Biol. Chem. 1965; 240: 4382-4392Abstract Full Text PDF PubMed Google Scholar, 9Quere I. Paul V. Rouillac C. Janbon C. London J. Demaille J. Kamoun P. Dufier J.L. Abitbol M. Chasse J.F. Biochem. Biophys. Res. Commun. 1999; 254: 127-137Crossref PubMed Scopus (56) Google Scholar, 10Maclean K.N. Janosik M. Kraus E. Kozich V. Allen R.H. Raab B.K. Kraus J.P. J. Cell. Physiol. 2002; 192: 81-92Crossref PubMed Scopus (54) Google Scholar). Recently, we have shown that both human and yeast CBS are coordinately regulated with proliferation and that the human CBS –1b promoter is serum- and fibroblast growth factor-responsive and is down-regulated by growth arrest due to nutrient depletion or the action of differentiation-inducing reagents (10Maclean K.N. Janosik M. Kraus E. Kozich V. Allen R.H. Raab B.K. Kraus J.P. J. Cell. Physiol. 2002; 192: 81-92Crossref PubMed Scopus (54) Google Scholar, 11Maclean K.N. Janosik M. Oliveriusova J. Kery V. Kraus J.P. J. Inorg. Biochem. 2000; 81: 161-171Crossref PubMed Scopus (41) Google Scholar). Another group has recently investigated the CBS –1b promoter and defined the minimal promoter region as a 125-bp region of sequence proximal to exon –1b. These investigators also found that the –1b promoter is activated by a synergistic interaction between NF-Y and either Sp1 or Sp3 (12Ge Y. Konrad M.A. Matherly L.H. Taub J.W. Biochem. J. 2001; 357: 97-105Crossref PubMed Scopus (65) Google Scholar). CBS expression levels in HepG2 and HT1080 cells were directly correlated with differences in the level of Sp1/Sp3 binding to the CBS –1b promoter region in these cells possibly as a consequence of phosphorylation by protein kinase A. This finding was proposed as a possible mechanism for the tissue-specific regulation of CBS (13Ge Y. Matherly L.H. Taub J.W. J. Biol. Chem. 2001; 276: 43570-43579Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In this report, we examine ∼5 kbp of CBS 5′-flanking sequence for the ability to modulate CBS expression and to confer tissue-specific expression. We demonstrate that as little as 32 bp of the –1b proximal promoter region is capable of driving transcription in HepG2 cells and that a single Sp1/Egr1 binding site is essential for this activity. Both the CBS –1b and –1a promoters are regulated in a proliferation-sensitive manner by Sp1 and that, although this transcription factor is involved in a previously unreported synergistic interaction with Sp3 in specifically regulating the –1b promoter, neither Sp3 nor NF-Y are able to substitute for Sp1 in vivo. Additionally, we present evidence to show that members of the KLF family of transcription factors have the potential to play a role in the tissue-specific regulation of the CBS gene by acting to block transcriptional activation of the CBS promoters by Sp1. Chemicals and Reagents—Unless otherwise stated, all chemicals were obtained from Sigma (St. Louis, MO). Reagents for enhanced chemiluminescence (ECL) were obtained from Amersham Biosciences (Piscataway, NJ). Restriction and modifying enzymes were purchased from New England Biolabs (Beverly, MA). All reagents for mammalian tissue culture were obtained from Life Sciences Technologies (Rockville, MD) except for fetal calf serum, which was obtained from HyClone (Logan, UT). Synthetic oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). Media and Mammalian Cell Culture—HepG2 hepatocellular carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured as described previously (10Maclean K.N. Janosik M. Kraus E. Kozich V. Allen R.H. Raab B.K. Kraus J.P. J. Cell. Physiol. 2002; 192: 81-92Crossref PubMed Scopus (54) Google Scholar). Drosophila SL2 (Schneider) cells were a generous gift from Dr. Joan Hooper (Department of Cellular and Structural Biology, University of Colorado Health Sciences Center). SL2 cells were maintained in Schneider's insect medium supplemented in the dark at room temperature with heat-inactivated 10% fetal bovine serum and 2 mm glutamine plus 100 units of penicillin and 100 μg of streptomycin/ml. Sp1–/– fibroblasts were a generous gift from Dr. Jeremy Boss (Emory University) and were passaged as described previously (14Ping D. Boekhoudt G. Zhang F. Morris A. Philipsen S. Warren S.T. Boss J.M. J. Biol. Chem. 2000; 275: 1708-1714Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Plasmids, Transfections, and Luciferase Assays—The sequence and construction of the CBS –1b promoter luciferase reporter construct pCBS47 and the CBS –1a promoter luciferase reporter construct pCBS37 has been described previously (6Kraus J.P. Oliveriusova J. Sokolova J. Kraus E. Vlcek C. de Franchis R. Maclean K.N. Bao L. Bukovska G. Patterson D. Paces V. Ansorge W. Kozich V. Genomics. 1998; 52: 312-324Crossref PubMed Scopus (102) Google Scholar). A series of extended CBS –1b plasmid reporter constructs containing progressively larger regions of 5′-flanking sequence were generated by cleaving pCBS474 with SphI and either XbaI, SacI, BglII, BamHI, or XhoI and subsequently sub-cloning in the adjacent SphI-XbaI (pCBS478 –8236 to –3576), SphI-SacI (pCBS479 –7241 to –3576), SphI-BglII (pCBS4780 –5189 to –3576), SphI-BamHI (pCBS –4518 to –3576), or XhoI-SphI restriction fragments of 5′-flanking DNA that were isolated from a previously described P1 cosmid containing the CBS genomic DNA (6Kraus J.P. Oliveriusova J. Sokolova J. Kraus E. Vlcek C. de Franchis R. Maclean K.N. Bao L. Bukovska G. Patterson D. Paces V. Ansorge W. Kozich V. Genomics. 1998; 52: 312-324Crossref PubMed Scopus (102) Google Scholar). The largest reporter construct CBS476 (–8943 to –3576), was generated by subcloning the EcoRI-XbaI fragment from the P1 cosmid into pZERO-2 (Invitrogen). This region was then excised from this vector as a KpnI-XbaI fragment and ligated into pCBS478, which had been cut with the same enzymes. In this manner we generated a range of larger CBS promoter luciferase constructs with up to 5276 kbp of 5′-flanking sequence. To investigate the minimal sequence region of the –1b promoter capable of driving transcription, we made progressive deletion derivatives of our base construct pCBS47. Two constructs, pCBS474 (–3877 to –3576) and pCB475 (–3792 to –3576) were made by end filling and subsequent blunt-end ligating after digesting pCBS47 with KpnI and SphI or ApaI, respectively. The pCBS473 construct (–3737 to –3576) was generated by subcloning of a PCR fragment amplified from a pCBS 47 template using sense (5′-CGTAGGTACCTGCTCTGGCACGAGACAT-3′) and antisense (5′-TACGAAGCTTCTGGACGGATACATGGAAAAGAGG-3′) primers. Nucleotides shown in boldface represent KpnI (sense) and HindIII (antisense) sites introduced to facilitate directional cloning of the amplification product. The sequence of this clone was verified by DNA sequencing. The pCBS472 (–3699 to –3576) and pCBS471 (–3679 to –3576) were generated by sub-cloning double-stranded synthetic oligonucleotides containing the desired sequences. This strategy was also used to generate mutant forms of pCBS472 with the CCAAT sequence mutated to CGTTG (pCBS472-1) or the Sp1 core CGCC sequence mutated to CTTC (pCBS472-2). All of the pCBS47 –1b reporter construct derivatives tested had a common 3′ end that included the transcription start sites defined previously upstream of exon –1b (7Bao L. Vlcek C. Paces V. Kraus J.P. Arch. Biochem. Biophys. 1998; 350: 95-103Crossref PubMed Scopus (90) Google Scholar). The pPacNF-YA, pPacNF-YB, and pPacNF-YC plasmid constructs were kindly provided by Dr. T. F. Osborne (University of California, Irvine, CA). The mammalian expression construct D4 NF-YA-m29 expressing the dominant negative NF-YA29 subunit, capable of forming an inactive NF-Y complex by trimerization with NF-YB and NF-YC, was obtained from Dr. Roberto Mantovani (University of Milan, Italy). The Sp1 mammalian and Drosophila expression constructs pAct-Sp1, pPacUSp3, pRc/CMV/Sp3, and pCGN-Sp1 and their relevant empty parent vectors that were used as negative controls were all provided by Dr. Paul Gardner (Brudnick Neuropsychiatric Research Institute, Worcester, MA) and were used with the kind permission of Drs. Edward Seto (University of South Florida, FL), Thomas Shenk (Princeton University, NJ), and Guntram Suske (Philipps-University, Marburg, Germany). The LKLF mammalian expression construct pBKCMVwtLKLF was a generous gift from Dr. Jerry Lingrel (University of Cincinnati College of Medicine, OH) and the KLF8 and BKLF expression constructs pMT2.BKLF, pMT2.KLF8, and pPac.BKLF were kindly provided by Dr. Merlin Crossley (University of Sydney, New South Wales, Australia). Transient transfections and subsequent luciferase reporter assays in actively proliferating cells were performed as described previously (10Maclean K.N. Janosik M. Kraus E. Kozich V. Allen R.H. Raab B.K. Kraus J.P. J. Cell. Physiol. 2002; 192: 81-92Crossref PubMed Scopus (54) Google Scholar). Analysis of CBS Expression—CBS enzyme activity was determined by a previously described radioisotope assay using [14C]serine as the labeled substrate (15Kraus J.P. Methods Enzymol. 1987; 143: 388-394Crossref PubMed Scopus (74) Google Scholar). One unit of activity is defined as the amount of CBS that catalyzes the formation of 1 μmol of cystathionine in 1 h at 37 °C. SDS Western blots were performed after running 10- to 75-μg samples of total protein on 9% polyacrylamide gels. Transfer, primary, and secondary antibody incubation conditions and subsequent detection using ECL chemiluminescent reagents were performed as described previously (16Janosik M. Oliveriusova J. Janosikova B. Sokolova J. Kraus E. Kraus J.P. Kozich V. Am. J. Hum. Genet. 2001; 68: 1506-1513Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Nuclear Extracts and EMSA—Nuclear extracts were prepared from actively proliferating HepG2 cells (60% confluent) as described previously (17Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Electrophoretic mobility shift assays (EMSAs) were performed using a lightshift chemiluminescent EMSA kit according to the manufacturer's standard protocol. The minimally active –1b promoter region was generated for EMSA by synthesizing oligonucleotides representing the proximal 32-bp probe of sequence. The oligonucleotide 5′-GCCAATCACCGCCCGCCTGCTCCCCTCGCCGT-3′ and its reverse complement, 5′-ACGGCGAGGGGAGCAGGCGGGCGGTGATTGGC-3′, were labeled using a biotin 3′-end labeling kit according to the manufacturer's instructions (Pierce, Rockford, IL). Standard EMSA reactions contained 20 μg of cell extract and 60 fmol of biotin end-labeled DNA in a 20-μl volume binding reaction in the presence of 2.5% glycerol, 5 mm MgCl2, 50 ng/μl of poly(dI·dC), and 0.05% Nonidet P-40. EMSA reactions were incubated at room temperature for 30 min, terminated by adding 2 μl of 10× loading buffer (0.2% (w/v) bromphenol blue and 0.2% xylene cyanol containing 10% (v/v) glycerol), and then separated on 6% polyacrylamide gels pre-run at 100 V for 30 min in 0.5× TAE buffer (0.02 m Tris-acetate, 0.001 m EDTA) and subsequently run at 150 V for 4 h after sample loading. Reaction products were then transferred to a Biodyne B membrane (Pierce) by capillary transfer and fixed by UV cross-linking. The biotin-labeled reaction products were then visualized by incubation with streptavidin horseradish peroxidase conjugate and subsequent incubation with ECL chemiluminescent reagents. DNase I Footprinting—The SphI-HindIII DNA fragment bearing 210 bp of –1b proximal sequence was excised from pCBS474 as an SphI-HindIII fragment into the high copy number vector pUC19. This region was then excised as a BamHI-HindIII fragment, gel-purified, 5′-end-labeled with 32P on either the top (at the BamH1 site) or bottom strand (at the HindIII site), and used as a probe in DNase I footprinting as described previously (18Wade D.P. Lindahl G.E. Lawn R.M. J. Biol. Chem. 1994; 269: 19757-19765Abstract Full Text PDF PubMed Google Scholar). Approximately 4 × 104 cpm of labeled probe was incubated with either 50 or 75 μg of crude nuclear extract derived from actively proliferating (∼60% confluence) HepG2 cells for 10 min at room temperature in DNase I digestion buffer (100 mm NaCl, 50 mm Tris-HCl (pH 8.0), 3 mm MgCl2, 0.15 mm spermine, and 0.5 mm spermidine). The reaction mixture was then subjected to digestion with DNase I (20 μg/ml) for 90 s at room temperature. Reaction products were run on a 6% polyacrylamide/8 m urea sequencing gel with control probe that had been incubated with DNase I in the absence of nuclear extract and a guanine sequencing ladder derived from the fragment for sequence positioning purposes. After electrophoresis, the gel was dried and visualized by autoradiography. Defining the CBS –1b Basal Promoter—In our first study of the CBS –1b promoter we examined the 388-bp region between the end of exon –1a and the start of exon –1b for promoter activity (6Kraus J.P. Oliveriusova J. Sokolova J. Kraus E. Vlcek C. de Franchis R. Maclean K.N. Bao L. Bukovska G. Patterson D. Paces V. Ansorge W. Kozich V. Genomics. 1998; 52: 312-324Crossref PubMed Scopus (102) Google Scholar). Subsequent reports regarding the CBS basal promoter activity have been essentially confined to this 481-bp region (12Ge Y. Konrad M.A. Matherly L.H. Taub J.W. Biochem. J. 2001; 357: 97-105Crossref PubMed Scopus (65) Google Scholar, 13Ge Y. Matherly L.H. Taub J.W. J. Biol. Chem. 2001; 276: 43570-43579Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 19Ge Y. Jensen T.L. Matherly L.H. Taub J.W. Blood. 2003; 101: 1551-1557Crossref PubMed Scopus (42) Google Scholar). However, it is not unusual for sequence elements several kbp upstream of the transcriptional start sites to contain sequence elements that significantly modulate basal activity or confer tissue-specific expression. To fully define the CBS –1b basal promoter and to search for both enhancer and repressor sequence elements, we examined a series of CBS –1b promoter reporter constructs with up to 5276 kbp of 5′-flanking sequence in promoter assays after transient transfection into HepG2 cells. We used our previously reported 479-bp CBS –1b luciferase reporter construct pCBS47 (–4055 to –3576) as a reference for comparative purposes. The results of these experiments (Fig. 1) indicated that the presence of an additional 4787 kbp of flanking sequence did not lead to any significant change in promoter activity relative to the original pCBS47 CBS –1b base construct. Deletion of an additional 259 bp from the pCBS47 construct did not adversely affect promoter activity. Further deletion of the CBS –1b promoter region was accompanied by significant reductions in the resultant promoter activity allowing us to define the 210 bp proximal to exon –1b as the basal promoter region. The observation that deletion of the region between the SphI and ApaI sites is accompanied by an approximate 50% reduction in promoter activity indicates that the 84-bp region between –3877 and –3792 contains sequence elements that contribute significantly to basal expression of the CBS –1b promoter. As Little As 32 bp of Proximal CBS –1b Promoter Sequence Is Capable of Driving Transcription in HepG2 Cells—To define the minimal portion of the CBS –1b promoter sequence capable of driving transcription, we generated a series of progressive 5′ deletion derivatives of pCBS47 and assayed them for promoter activity after transient transfection into HepG2 cells (Fig. 1). We were able to delete down from 82 to 32 bp (–3699 to –3576) before losing all detectable promoter activity. This definition of the minimal promoter differs significantly (4-fold smaller) from the previously published version, and, consequently, we examined the function of this region in detail. The pGL-enhancer vector used to make our pCBS472 expression construct contains an SV40 enhancer sequence located downstream of luc+ and the poly (A) signal. To assess the possible contribution of this downstream enhancer element to the sensitivity of our promoter assays, the –1b minimal promoter region was subcloned into pGL-basic, which lacks this SV40 enhancer sequence but is otherwise identical. When this 32-bp minimal promoter region was examined in the absence of the enhancer sequence in HepG2 cells it was found to have essentially identical promoter activity as was observed previously. Analysis of the minimal promoter sequence (Fig. 2A) shows one candidate Sp1 binding site that overlaps with a binding site for the zinc-finger transcription factor early growth-response protein (Egr1), one CCAAT box, and putative binding sites for Ap2, MZF1, IK2, and Gfi1. Ap2 is unlikely to contribute to the promoter activity of this construct, because it is not expressed in HepG2 cells (20Zeng Y.X. Somasundaram K. el-Deiry W.S. Nat. Genet. 1997; 15: 78-82Crossref PubMed Scopus (260) Google Scholar) and has previously been shown to down-regulate CBS when heterologously expressed in these cells (10Maclean K.N. Janosik M. Kraus E. Kozich V. Allen R.H. Raab B.K. Kraus J.P. J. Cell. Physiol. 2002; 192: 81-92Crossref PubMed Scopus (54) Google Scholar). The tissue distribution of Gfi1, IK2, and MZF1 (21Karsunky H. Zeng H. Schmidt T. Zevnik B. Kluge R. Schmid K.W. Duhrsen U. Moroy T. Nat. Genet. 2002; 30: 295-300Crossref PubMed Scopus (257) Google Scholar) (22Georgopoulos K. Winandy S. Avitahl N. Annu. Rev. Immunol. 1997; 15: 155-176Crossref PubMed Scopus (213) Google Scholar, 23Hromas R. Boswell S. Shen R.N. Burgess G. Davidson A. Cornetta K. Sutton J. Robertson K. Leukemia. 1996; 10: 1049-1050PubMed Google Scholar, 24Gaboli M. Kotsi P.A. Gurrieri C. Cattoretti G. Ronchetti S. Cordon-Cardo C. Broxmeyer H.E. Hromas R. Pandolfi P.P. Genes Dev. 2001; 15: 1625-1630Crossref PubMed Scopus (113) Google Scholar) effectively precludes any role for these transcription factors in the regulation of CBS in tissues such as liver, kidney, pancreas, and brain, which are the main sites of transsulfuration in humans (8Mudd S.H. Finkelstein J.D. Irreverre F. Laster L. J. Biol. Chem. 1965; 240: 4382-4392Abstract Full Text PDF PubMed Google Scholar). Consequently, the contribution of these transcription factors to CBS –1b promoter activity was not investigated. To assess the relative importance of the CCAAT box and the Sp1 site, we generated derivatives of the pCBS472 construct where these binding sites had been mutated. The effect of these mutations was examined by measuring their relative promoter activities in HepG2 cells (Fig. 2B). Mutagenesis of the CAAT box (pCBS472-1) did not affect the level of basal promoter activity in HepG2 cells indicating that this sequence does not play a crucial role in directing transcription from the –1b promoter in these cells. Mutagenesis of the Sp1/Egr1 site in the pCBS472-2 construct lead to the complete abolition of promoter activity indicating that the promoter activity of the CBS –1b minimal promoter sequence is dependent upon the Sp1-Egr1 binding site. DNA Footprinting Reveals Key Areas of the CBS –1b Minimal Promoter Region—DNase I footprinting analysis was performed using double-stranded fragments corresponding to nucleotides –3877 to –3786. This SphI-HindIII restriction fragment from pCBS474 contains 210 bp of –1b proximal sequence and was used as a probe in DNase I footprinting using a HepG2 cell extract generated as described under "Experimental Procedures" (Fig. 3, A and B). Scoring and interpretation of protected regions was performed blind by two independent observers with no prior knowledge of the CBS promoter or the results of our deletion and mutagenesis study described above. Three distinct regions designated Fp1 (–3808 to –3787 on the top strand and –3810 to –3783 on the bottom strand), Fp2 (–3715 to –3681 on the bottom strand), and Fp3 (–3664 to –3639 on the bottom strand) were found to be either partially or totally protected from DNase I digestion. Examination of the sequence contained within the protected Fp1 region revealed two adjacent Sp1 binding sites separated by a MZF1 site. Interestingly, these binding sites are present at the 3′ end of the 85-bp region that was shown in the previous section to contribute significantly to basal transcription indicating that these Sp1 sites are of considerable importance for maximal promoter activity. The vast majority of the Fp1 region was also found to be protected in a DNase I footprinting analysis of the –1b promoter region performed previously where it was described as region D (13Ge Y. Matherly L.H. Taub J.W. J. Biol. Chem. 2001; 276: 43570-43579Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The second protected region (Fp2) was not previously reported to be protected (13Ge Y. Matherly L.H. Taub J.W. J. Biol. Chem. 2001; 276: 43570-43579Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and contains the Sp1/Egr1 overlapping binding site that was shown to be essential for the minimal promoter function in the previous section and serves as an independent confirmation of the functional importance of this binding site. This protection is relatively weak, but it is part of the nature of the DNase I footprinting methods that the relative binding affinities of different sites afford different levels of protection from the DNase I treatment. The lower degree of protection of this site is presumably due to a relatively reduced level of Sp1 binding that is entirely consistent with the level of promoter activity that this site was shown to confer in the deletion analysis described above. Interestingly, the inverted CCAAT box-NF-Y binding site 40 bp upstream of the –1b minimal promoter sequence that has previously been identified as playing a key role in regulating the CBS –1b promoter (12Ge Y. Konrad M.A. Matherly L.H. Taub J.W. Biochem. J. 2001; 357: 97-105Crossref PubMed Scopus (65) Google Scholar, 13Ge Y. Matherly L.H. Taub J.W. J. Biol. Chem. 2001; 276: 43570-43579Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) was not protected. We have independently found that NF-Y can weakly transactivate the CBS –1b promoter (but not –1a) in SL2 cells. 2K. N. Maclean, unpublished results. However, this site is close to a DNase I-hyper-sensitive site, which is typically indicative of conformational changes induced by protein binding and often occurs at sites where only a small fraction of the probe molecules are bound by protein. This finding suggests that NF-Y may bind to this sequence but is not present at a sufficiently high concentrat