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• W2083397383 abstract "We have characterized the 5′-flanking region of the human erythroid-specific 5-amino levulinate synthase (ALAS) gene (the ALAS2 gene) and shown that the first 300 base pairs of promoter sequence gives maximal expression in erythroid cells. Transcription factor binding sites clustered within this promoter sequence include GATA motifs and CACCC boxes, critical regulatory sequences of many erythroid cell-expressed genes. GATA sites at −126/−121 (on the noncoding strand) and −102/−97 were each recognized by GATA-1 proteinin vitro using erythroid cell nuclear extracts. Promoter mutagenesis and transient expression assays in erythroid cells established that both GATA-1 binding sites were functional and exogenously expressed GATA-1 increased promoter activity through these sites in transactivation experiments. A noncanonical TATA sequence at the expected TATA box location (−30/−23) bound GATA-1- or TATA-binding protein (TBP) in vitro. Conversion of this sequence to a canonical TATA box reduced expression in erythroid cells, suggesting a specific role for GATA-1 at this site. However, expression was also markedly reduced when the −30/−23 sequence was converted to a consensus GATA-1 sequence (that did not bind TBP in vitro), suggesting that a functional interaction of both factors with this sequence is important. A sequence comprising two overlapping CACCC boxes at −59/−48 (on the noncoding strand) was demonstrated by mutagenesis to be functionally important. This CACCC sequence bound Sp1, erythroid Krüppel-like factor, and basic Krüppel-like factor in vitro, while in transactivation experiments erythroid Krüppel-like factor activated ALAS2 promoter expression through this sequence. A sequence at −49/−39 with a 9/11 match to the consensus for the erythroid specific factor NF-E2 was not functional. Promoter constructs with 5′-flanking sequence from 293 base pairs to 10.3 kilobase pairs expressed efficiently in COS-1 cells as well as in erythroid cells, indicating that an enhancer sequence located elsewhere or native chromatin structure may be required for the tissue-restricted expression of the gene in vivo. We have characterized the 5′-flanking region of the human erythroid-specific 5-amino levulinate synthase (ALAS) gene (the ALAS2 gene) and shown that the first 300 base pairs of promoter sequence gives maximal expression in erythroid cells. Transcription factor binding sites clustered within this promoter sequence include GATA motifs and CACCC boxes, critical regulatory sequences of many erythroid cell-expressed genes. GATA sites at −126/−121 (on the noncoding strand) and −102/−97 were each recognized by GATA-1 proteinin vitro using erythroid cell nuclear extracts. Promoter mutagenesis and transient expression assays in erythroid cells established that both GATA-1 binding sites were functional and exogenously expressed GATA-1 increased promoter activity through these sites in transactivation experiments. A noncanonical TATA sequence at the expected TATA box location (−30/−23) bound GATA-1- or TATA-binding protein (TBP) in vitro. Conversion of this sequence to a canonical TATA box reduced expression in erythroid cells, suggesting a specific role for GATA-1 at this site. However, expression was also markedly reduced when the −30/−23 sequence was converted to a consensus GATA-1 sequence (that did not bind TBP in vitro), suggesting that a functional interaction of both factors with this sequence is important. A sequence comprising two overlapping CACCC boxes at −59/−48 (on the noncoding strand) was demonstrated by mutagenesis to be functionally important. This CACCC sequence bound Sp1, erythroid Krüppel-like factor, and basic Krüppel-like factor in vitro, while in transactivation experiments erythroid Krüppel-like factor activated ALAS2 promoter expression through this sequence. A sequence at −49/−39 with a 9/11 match to the consensus for the erythroid specific factor NF-E2 was not functional. Promoter constructs with 5′-flanking sequence from 293 base pairs to 10.3 kilobase pairs expressed efficiently in COS-1 cells as well as in erythroid cells, indicating that an enhancer sequence located elsewhere or native chromatin structure may be required for the tissue-restricted expression of the gene in vivo. 5-Aminolevulinate synthase (EC 2.3.1.37) is a nuclear encoded mitochondrial matrix enzyme that catalyzes the formation of 5-aminolevulinate from glycine and succinyl CoA in the heme biosynthetic pathway and is of particular interest, since it is the rate-controlling enzyme (1May B.K. Dogra S.C. Sadlon T.J. Bhasker C.R. Cox T.C. Bottomley S.S. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 1-51Crossref PubMed Scopus (123) Google Scholar, 2Bottomley S.S. May B.K. Cox T.C. Cotter P.D. Bishop D.F. J. Bioenerg. Biomembr. 1995; 27: 161-168Crossref PubMed Scopus (66) Google Scholar, 3Dierks P. Dailey H.A. Biosynthesis of Heme and Chlorophylls. McGraw-Hill Inc., New York1990: 201-233Google Scholar). There are two closely related isozymes of 5-aminolevulinate synthase (ALAS) 1The abbreviations used are: ALAS, 5-aminolevulinate synthase; EKLF, erythroid Krüppel-like factor; BKLF, basic Krüppel-like factor; bp, base pair(s); kb, kilobase(s); MEL, murine erythroleukemia; PBS, phosphate-buffered saline; RSV, Rous sarcoma virus; TBP, TATA-binding protein; GST, glutathione S-transferase; LUC, luciferase. designated ALAS1 and ALAS2, which are encoded by separate genes located on different chromosomes (4Sutherland G.R. Baker E. Callen D.F. Hyland V.J. May B.K. Bawden M.J. Healy H.M. Borthwick I.A. Am. J. Hum. Genet. 1988; 43: 331-335PubMed Google Scholar, 5Cox T.C. Bawden M.J. Abraham N.G. Bottomley S.S. May B.K. Baker E. Chen L.Z. Sutherland G.R. Am. J. Hum. Genet. 1990; 46: 107-111PubMed Google Scholar, 6Bishop D.F. Henderson A.S. Astrin K.H. Genomics. 1990; 7: 207-214Crossref PubMed Scopus (126) Google Scholar). The housekeeping enzyme, ALAS1, is probably expressed in all tissues to provide heme for respiratory cytochromes and other hemoproteins (1May B.K. Dogra S.C. Sadlon T.J. Bhasker C.R. Cox T.C. Bottomley S.S. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 1-51Crossref PubMed Scopus (123) Google Scholar, 7Kappas A. Sassa S. Galbraith R.A. Nordmann Y. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Metabolic and Molecular Basis of Inherited Disease. 7th Ed. McGraw-Hill Inc., New York1995: 2103-2159Google Scholar). The second isozyme, ALAS2, is an erythroid cell-specific enzyme, the synthesis of which is developmentally regulated and is markedly increased during erythropoiesis to meet the demand for heme during hemoglobin production (1May B.K. Dogra S.C. Sadlon T.J. Bhasker C.R. Cox T.C. Bottomley S.S. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 1-51Crossref PubMed Scopus (123) Google Scholar). The genes for ALAS1 and ALAS2 have been isolated from various species (8Maguire D.J. Day A.R. Borthwick I.A. Srivastava G. Wigley P.L. May B.K. Elliot W.H. Nucleic Acids Res. 1986; 14: 1379-1391Crossref PubMed Scopus (36) Google Scholar, 9Yomogida K. Yamamoto M. Yamagami T. Fujita H. Hayashi N. J. Biochem. (Tokyo). 1993; 113: 364-371Crossref PubMed Scopus (20) Google Scholar, 10Schoenhaut D.S. Curtis P.J. Nucleic Acids Res. 1989; 17: 7013-7028Crossref PubMed Scopus (37) Google Scholar, 11Cox T.C. Bawden M.J. Martin A. May B.K. EMBO J. 1991; 10: 1891-1902Crossref PubMed Scopus (308) Google Scholar, 12Lim K.C. Ishihara H. Riddle R.D. Yang Z. Andrews N. Yamamoto M. Engel J.D. Nucleic Acids Res. 1994; 22: 1226-1233Crossref PubMed Scopus (13) Google Scholar) and show a similar exon/intron organization (1May B.K. Dogra S.C. Sadlon T.J. Bhasker C.R. Cox T.C. Bottomley S.S. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 1-51Crossref PubMed Scopus (123) Google Scholar). We have characterized the human ALAS2 gene (11Cox T.C. Bawden M.J. Martin A. May B.K. EMBO J. 1991; 10: 1891-1902Crossref PubMed Scopus (308) Google Scholar) and shown that it consists of 11 exons spanning 22 kb (13Conboy J.G. Cox T.C. Bottomley S.S. Bawden M.J. May B.K. J. Biol. Chem. 1992; 267: 18753-18758Abstract Full Text PDF PubMed Google Scholar) on the X chromosome (5Cox T.C. Bawden M.J. Abraham N.G. Bottomley S.S. May B.K. Baker E. Chen L.Z. Sutherland G.R. Am. J. Hum. Genet. 1990; 46: 107-111PubMed Google Scholar). In the human disorder X-linked sideroblastic anemia, point mutations have been identified in ALAS2 that result in impaired enzyme activity and consequently reduced hemoglobin production (2Bottomley S.S. May B.K. Cox T.C. Cotter P.D. Bishop D.F. J. Bioenerg. Biomembr. 1995; 27: 161-168Crossref PubMed Scopus (66) Google Scholar, 14Cox T.C. Bottomley S.S. Wiley J.S. Bawden M.J. Matthews C.S. May B.K. N. Engl. J. Med. 1994; 330: 675-679Crossref PubMed Scopus (113) Google Scholar). Expression of the ALAS2 gene is regulated at both the transcriptional and post-transcriptional levels. Translation of the ALAS2 mRNA in erythroid cells is controlled by intracellular iron levels through an iron-responsive element located in the 5′-untranslated region to ensure that the production of protoporphyrin is coordinated with iron availability (1May B.K. Dogra S.C. Sadlon T.J. Bhasker C.R. Cox T.C. Bottomley S.S. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 1-51Crossref PubMed Scopus (123) Google Scholar, 11Cox T.C. Bawden M.J. Martin A. May B.K. EMBO J. 1991; 10: 1891-1902Crossref PubMed Scopus (308) Google Scholar, 15Bhasker C.R. Burgiel G. Neupert B. Emery-Goodman A. Kuhn L.C. May B.K. J. Biol. Chem. 1993; 268: 12699-12705Abstract Full Text PDF PubMed Google Scholar). Furthermore, heme may regulate activity of ALAS2 by preventing its import into mitochondria (1May B.K. Dogra S.C. Sadlon T.J. Bhasker C.R. Cox T.C. Bottomley S.S. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 1-51Crossref PubMed Scopus (123) Google Scholar, 16Lathrop J.T. Timko M.P. Science. 1993; 259: 522-525Crossref PubMed Scopus (247) Google Scholar). During erythropoiesis, transcription of the ALAS2 gene is markedly up-regulated (1May B.K. Dogra S.C. Sadlon T.J. Bhasker C.R. Cox T.C. Bottomley S.S. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 1-51Crossref PubMed Scopus (123) Google Scholar) together with an increase in the transcription of genes for the other heme pathway enzymes (17Beaumont C. Deybach J.C. Grandchamp B. Silva V.D. de Verneuil H. Nordmann Y. Exp. Cell Res. 1984; 154: 474-484Crossref PubMed Scopus (31) Google Scholar) and for globin (1May B.K. Dogra S.C. Sadlon T.J. Bhasker C.R. Cox T.C. Bottomley S.S. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 1-51Crossref PubMed Scopus (123) Google Scholar, 3Dierks P. Dailey H.A. Biosynthesis of Heme and Chlorophylls. McGraw-Hill Inc., New York1990: 201-233Google Scholar,18Karlsson S. Nienhuis A.W. Annu. Rev. Biochem. 1985; 54: 1071-1108Crossref PubMed Scopus (112) Google Scholar). Only a small number of erythroid cell-restricted transcription factors have been identified that are involved in erythroid gene transcriptional activation (19Orkin S.H. J. Biol. Chem. 1995; 270: 4955-4958Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), and these include GATA-1 (the prototype of a family of GATA proteins), NF-E2, and the CACCC box-binding protein, EKLF. In the present study, we have identified transcription factors that bind to the ALAS2 promoter to drive its expression and have examined, in detail, the role of GATA and CACCC box-binding proteins in this process. Gel shift assays have been employed to investigate the specificity of protein-DNA interactions in the ALAS2 promoter and the functional contribution of such binding sites evaluated by site-directed mutagenesis and transient expression analysis of ALAS2 promoter/reporter gene constructs. A series of 5′-flanking ALAS2 deletion constructs were generated from subcloned fragments isolated from the human genomic clone, pTC-EA1 (11Cox T.C. Bawden M.J. Martin A. May B.K. EMBO J. 1991; 10: 1891-1902Crossref PubMed Scopus (308) Google Scholar) and ligated into the promoterless firefly luciferase (LUC) reporter gene vector, pGL2-Basic (Promega). The polymerase chain reaction was performed using pTC-EA1 as the template and the following primers: primer 1, 5′-CCCAAGCTTGCACTGAGGACGAACG-3′ at +12/+36 (an introducedHindIII site is underlined), and 5′-GGGTTCTGTAACTACATTGCC-3′, which bound upstream of anAvrII site at −718/−699 and resulted in the amplification of a 730-bp promoter fragment. The amplified product was digested withBglII and HindIII and a 321-bp fragment ligated into the similarly digested pGL2-Basic vector. The resulting construct is designated pALAS−293-LUC and contains ALAS2 promoter sequence from −293 to +28. The amplified product was also digested withSacI and HindIII and a 420-bp fragment ligated into the similarly digested pBluescript KS+ phagemid (pKS+-ALAS). To synthesize plasmids with promoter lengths of −124 and −27, a PvuII site was introduced at these positions by site-directed mutagenesis, and the resulting modified plasmids were digested with SmaI (polylinker) andPvuII and religated to form pALAS−124-LUC and pALAS−27-LUC. The synthesis of the longer promoter constructs was performed in several steps. In separate studies, a HindIII site was introduced at −7/−2 in the ALAS2 promoter by site-directed mutagenesis in a subclone containing −6.0 to +5.0 kb of contiguous human ALAS2 sequence. Subsequent digestion of this subclone withXbaI or KpnI together with HindIII gave promoter lengths of 1.9 and 5.7 kb that were cloned into pGL2-Basic linearized with NheI/HindIII andKpnI/HindIII, respectively. These initial constructs terminated at position −4 and therefore did not contain the native transcription initiation site. To permit strict comparison with the shorter promoter constructs, the sequence from around the native transcription initiation site was then reintroduced into these constructs as follows. An AvrII-HindIII fragment (−700 to −4) was excised from the 1.9 kb promoter construct and replaced with an AvrII-HindIII fragment (−700 to +28) that was amplified by the polymerase chain reaction, resulting in pALAS-1.9kb-LUC. An NcoI-HindIII fragment (−1.0 kb to −4) was removed from the 5.7 kb promoter construct and replaced with the NcoI-HindIII fragment (−1.0 kb to +28) isolated from pALAS-1.9kb-LUC to generate pALAS-5.7kb-LUC. To synthesize the construct containing 10.3 kb of 5′-flanking region (pALAS-10.3kb-LUC), pTC-EA1 was digested with ClaI andXhoI, and a 5.7-kb fragment was cloned into the similarly digested vector pSP72 (Promega). An EcoRV-XhoI fragment isolated from this plasmid was used to replace a 1.1-kbSmaI-XhoI fragment in the construct pALAS-5.7kb-LUC. Constructs with 124 bp of wild type promoter (pALAS−124A-LUC) or a mutation in the −54 bp CACCC site were also synthesized for use in transactivation experiments. A 152-bp fragment (−124 to +28) was generated by the polymerase chain reaction using the plasmids pALAS−293-LUC and pALAS−293mut8-LUC as templates, and two primers: 5′-GGTTTAGATCTTAGCAAGGAAGGGA-3′ at −131/−106 (an introduced BglII site is underlined) and primer 1. Following digestion of the product with BglII and HindIII, the resultant fragment was cloned into the appropriately linearized pGL2-Basic vector. Additional constructs synthesized for use in transactivation experiments included pβ-glob-LUC and p(CAC)4tk-LUC derived from constructs provided by Dr. J. Bieker (20Bieker J.J. Southwood C.M. Mol. Cell. Biol. 1995; 15: 852-860Crossref PubMed Google Scholar). pβ-glob-LUC contained 205 bp of murine β-globin promoter fused to the luciferase reporter gene, and p(CAC)4tk-LUC contained four copies of the murine β-globin CACCC site ligated upstream of the thymidine kinase promoter-luciferase reporter gene fusion. All constructs were verified by restriction mapping and DNA sequence analysis. The human erythroleukemia cell line, K562, was maintained in RPMI 1640 medium containing 10% fetal calf serum. The adherent murine erythroleukemia MEL (F4–12B2) cell line (kindly provided by Dr. G. Bergholz, Hamburg, Germany), COS-1, and CV-1 cells were all maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For electroporation, exponentially growing K562 cells were washed in PBS and 107 cells in 200 μl of PBS containing 10 mm Hepes, pH 7.5, were electroporated with 2 pmol of the reporter construct at 200 V, 960 microfarads using the Bio-Rad Gene Pulser. MEL cells were grown to 80% confluency, harvested by trypsinization, resuspended in media, and washed twice in PBS. MEL cells (107) in 500 μl of cold PBS containing 10 mm Hepes, pH 7.5, were electroporated with 2 pmol of the reporter construct at 300 V, 960 microfarads. COS-1 cells were grown to 80% confluency and harvested by trypsinization. COS-1 cells (5 × 106) were resuspended in 500 μl of cold buffer containing 20 mm Hepes, pH 7.05, 137 mm NaCl, 5 mm KCl, 0.7 mm Na2HPO4, 6 mm dextrose and electroporated with 2 pmol of the reporter construct (300 V, 960 microfarads). All transfections contained 250 μg of sheared salmon sperm DNA (Sigma) as a carrier. As an internal control, K562 and COS-1 cells were co-transfected with 5 μg of the β-galactosidase expression vector, RSV-β-gal, and MEL cells with 10 μg of this vector. Cells were seeded in 60 × 15-mm Petri dishes containing 5 ml of medium and harvested 24 h after transfection. Cell lysates were assayed for luciferase and β-galactosidase activity. Plasmid DNA was prepared by the CsCl/ethidium bromide equilibrium density gradient procedure (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989Google Scholar), quantified by spectrophotometry and analyzed by agarose gel electrophoresis to confirm concentration and supercoiling. All transient transfections were performed in quadruplicate with at least three different plasmid DNA preparations. Transfected cells were harvested, washed once in PBS, and treated with 100 μl of cell culture lysis reagent (Promega) on ice for 10 min. Cells were then snap frozen, thawed on ice, and centrifuged for 5 min to remove cellular debris. Supernatants were assayed to determine total protein concentration (Bio-Rad protein microassay). Subsequent assays (luciferase and β-galactosidase) were performed with 100 μg of cell lysate. Luciferase activity was measured using a luciferase assay system (Promega), and measurements were determined in a Berthold model LB9502 luminometer. β-Galactosidase activity was measured by the procedure of Herbomel et al. (22Herbomel P. Bourachot B. Yaniv M. Cell. 1984; 39: 653-662Abstract Full Text PDF PubMed Scopus (556) Google Scholar) and expressed as (A 420/μg of protein/h) × 100. Luciferase activities were normalized for transfection efficiency using the β-galactosidase activity as an internal control, and the data were expressed as “relative luciferase activity.” All nuclear extracts were prepared by the procedure of Partington et al. (23Partington G.A. Bertwistle D. Nicolas R.H. Kee W.-J. Pizzey J.A. Patient R.K. Dev. Biol. 1997; 181: 144-155Crossref PubMed Scopus (23) Google Scholar) except those used for the detection of CACCC-binding proteins, where the rapid procedure described by Andrews et al. (24Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2214) Google Scholar) was employed. The sequences of sense strand oligonucleotides used in the gel shift experiments are as follows. Binding motifs are underlined: GATA−124, 5′-CTTTGGGTTTTATCTCTAGCAAGG-3′; GATA−100, 5′-AAGGGACTGAGATACCTTTGGGGC-3′; β-globin GATA-cons (25Wall L. deBoer E. Grosveld F. Genes Dev. 1988; 2: 1089-1100Crossref PubMed Scopus (242) Google Scholar), 5′-TTGGCTCCCTTATCATGTCCCTG-3′; GATA−27, 5′-GAGGAGAAGGGATAAATGCCAGGT-3′; GATA−27G, 5′-TCAGAGGAGACATGATAAGTGCCAGGTCCT-3′; TATA 5′-GAGGAGAAGGTATAAATGCCAGGT-3′; βglobin CACCC (26Miller L.J. Bieker J.J. Mol. Cell. Biol. 1993; 13: 2776-2786Crossref PubMed Scopus (657) Google Scholar), 5′-AGCTAGCCACACCCTGAAGCT-3′; CACCC−54, 5′-CAGAAGGCAGGGTGGGTGGGGCTGAGTC-3′; nonspecific competitor (NRF-1 site in the rat somatic cytochrome cpromoter (27Evans M.J. Scarpulla R.C. Genes Dev. 1990; 4: 1023-1034Crossref PubMed Scopus (335) Google Scholar)), 5′-GCTAGCCCGCATGCGCGCGCACCTTG-3′. Single-stranded oligonucleotides were gel-purified, and 100 ng of each labeled with [γ32P]ATP using T4polynucleotide kinase. A 3-fold molar excess of the unlabeled complementary oligonucleotide was annealed to the32P-labeled oligonucleotide in 100 mm NaCl by incubation at 100 °C for 2 min and 70 °C for 10 min and then allowing the samples to slowly cool to room temperature. The labeled annealed oligonucleotides were precipitated and washed to remove unincorporated radioactivity and resuspended in 100 μl of water. Unlabeled oligonucleotides were also annealed for use in competition assays. Binding reactions used in the detection of GATA-binding proteins contained 5 μg of nuclear protein, 2 μg of poly(dI-dC) in 15 μl of 25 mm Hepes, pH 7.9, containing 60 mm KCl, 7.5% glycerol, 0.1 mm EDTA, 5 mm MgCl2, 0.75 mm dithiothreitol, and 2 mm spermidine and incubated on ice for 10 min. Radiolabeled probe (1 ng) was added to the reaction and incubated on ice for a further 30 min. In supershift assays, 2 μl of the GATA-1-specific monoclonal antibody, N-6 (28Ito E. Toki T. Ishihara H. Ohtani H. Gu L. Yokoyama M. Engel J.D. Yamamoto M. Nature. 1993; 362: 466-468Crossref PubMed Scopus (258) Google Scholar) (provided by Dr. G. Partington), was incubated in the binding reaction prior to the addition of probe. Retarded nuclear protein complexes were resolved on a 6% nondenaturing polyacrylamide gel in 0.25 × Tris borate-EDTA buffer at 180 V for 2.5 h at 4 °C. The gels were dried and exposed to Kodak X-Omat AR film. For the detection of protein binding to the −27 GATA site, the binding reaction protocol described by Fong and Emerson (29Fong T.C. Emerson B.M. Genes Dev. 1992; 6: 521-532Crossref PubMed Scopus (69) Google Scholar) was used. Purified recombinant human TBP was obtained from Promega, and poly(dI-dC) was omitted from the binding reaction. In experiments designed to determine the binding affinity constants (K d ) of GATA-1 and TBP, binding reactions and electrophoresis conditions were as described above with a constant amount of radiolabeled oligonucleotide probes and serial dilutions of TBP or a purified GST-GATA-1 zinc finger fusion protein (GST-GATA-1(f)) (30Crossley M. Merika M. Orkin S.H. Mol. Cell. Biol. 1995; 15: 2448-2456Crossref PubMed Scopus (158) Google Scholar) prepared as described previously (31Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). Binding reactions used in the detection of CACCC-binding proteins and the supershift assays using polyclonal antibodies to BKLF, EKLF, and Sp1 were performed as described by Crossley et al. (32Crossley M. Whitelaw E. Perkins A. Williams G. Fujiwara Y. Orkin S.H. Mol. Cell. Biol. 1996; 16: 1695-1705Crossref PubMed Scopus (209) Google Scholar). The polyclonal antibodies to BKLF and EKLF were generously provided by Dr. M. Crossley. The Sp1 polyclonal antibody, Sp1 (PEP 2) (Santa Cruz Biotechnology, Inc.), was a gift from Dr. M. F. Shannon. Gel shift competition assays were performed with unlabeled competitor oligonucleotides included in the binding reactions. Site-directed mutagenesis was performed using the Bio-Rad Muta-Gene M13 in vitromutagenesis kit according to the manufacturer's instructions. The plasmid pKS+-ALAS, containing ALAS2 promoter sequence (−392 to +28), was transformed into the Escherichia coliCJ236 strain, and following superinfection with the helper phage M13KO7, single-stranded DNA was purified and used as a template in the mutagenesis reaction. In the final step, aBglII-HindIII fragment harboring the mutation was excised from pBluescript KS+ and subcloned into theBglII/HindIII-digested pGL2-Basic vector. To synthesize promoter lengths of −124 and −27 and to inactivate the GATA sites, PvuII sites were introduced at −124, −100, and −27. The −27 GATA site was also converted to a canonical TATA box and to a consensus GATA-1 site. The CACCC and NF-E2 sites were also inactivated by conversion to a PvuII site. Mutant clones were confirmed by DNA sequence analysis. The primers used in these reactions are as follows with the mutations underlined: −124 GATA, 5′-ACTTTGGGTTTCAGCTGTTAGCAAGGAA-3′; −100 GATA, 5′-GGAAGGGACTGGCAGCTGTTTGGGGCCA-3′; −27 GATA, 5′-AGAGGAGAAGGCAGCTGTGCCAGGTCCT-3′; GATA−27G, 5′-TCAGAGGAGACATGATAAGTGCCAGGTCCT-3′; TATA, 5′-AGAGGAGAAGGTATAAATGCCAGG-3′; −54 CACCC, 5′-CAGAAGGCAGGCAGCTGGGGGCTGAGTC-3′; −44 NF-E2, 5′-GTGGGTGGGGCAGCTGCAGAGGAGAAG-3′. Transactivation experiments in COS-1 cells were performed with 2 pmol of the reporter construct and 5 μg of the murine GATA-1 cDNA expression clone, pXM/GF-1 (provided by Dr. S. H. Orkin) or 10 μg of each of the cDNA expression clones, pMT2/RINFE and pMT2/p18w-1, for NF-E2 (provided by Dr. N. Andrews). For transactivation experiments in K562 cells, 2 pmol of the reporter construct and 7.5 μg of the EKLF cDNA expression clone, pSG5/EKLF (26Miller L.J. Bieker J.J. Mol. Cell. Biol. 1993; 13: 2776-2786Crossref PubMed Scopus (657) Google Scholar) (provided by Dr. J. Bieker), were employed. The vectors pGL2-Basic and ptk-LUC containing the thymidine kinase promoter were included as controls. Cells were harvested 24 h after transfection and 100 μg of total protein assayed for luciferase activity. The -fold transactivations were determined following subtraction of the background activity obtained with the appropriate progenitor vectors. We previously reported the isolation of genomic clones for human ALAS2 (11Cox T.C. Bawden M.J. Martin A. May B.K. EMBO J. 1991; 10: 1891-1902Crossref PubMed Scopus (308) Google Scholar), and a partial restriction map of the first 10.3 kb of 5′-flanking sequence of the gene is shown in Fig. 1 A. To determine regions that contribute to expression, constructs generated with different 5′ lengths (−10.3 kb to −27 bp) and with a common 3′ end (+28) were fused to the firefly luciferase reporter gene (Fig.1 B). These constructs were transiently transfected into K562, MEL (F4–12B2), or COS-1 cells, the latter as a nonerythroid control, and luciferase activity was determined in cell lysates. The activity of the longest construct (pALAS-10.3kb-LUC) in each cell line was assigned a value of 100 (Fig. 1 B). The promoter expressed strongly in both erythroid cell lines, and maximal activity was seen with 293 bp of promoter (pALAS−293-LUC). A low level of activity was obtained with the −27 bp promoter construct (pALAS−27-LUC). Expression of the constructs was also observed in COS-1 cells and followed a similar pattern to that in erythroid cells except that 1.9 kb of promoter (pALAS-1.9kb-LUC) gave maximal expression (Fig. 1 B). In this study, we have investigated the basis for the strong transcriptional activity of the first 300 bp of promoter. Sequence analysis of this region revealed a clustering of potential binding sites in the first 140 bp, including those for the erythroid-specific transcription factors GATA-1 and NF-E2 as well as CACCC (19Orkin S.H. J. Biol. Chem. 1995; 270: 4955-4958Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) and CCAAT (33Chodosh L. Baldwin A.S. Carthew R.W. Sharp P.A. Cell. 1988; 53: 11-24Abstract Full Text PDF PubMed Scopus (435) Google Scholar) box proteins and the Ets family of proteins (34Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (811) Google Scholar) (Fig.1 C). Three putative GATA-1 binding sites were identified at −126/−121 (on the noncoding strand), −102/−97, and −30/−23 (Fig.1 C). The sites centered at −124 and −100 were first investigated. The −124 GATA site (5′-AGATAA-3′) conforms to the consensus for GATA-1 (35Merika M. Orkin S.H. Mol. Cell. Biol. 1993; 13: 3999-4010Crossref PubMed Scopus (566) Google Scholar, 36Ko L.J. Engel J.D. Mol. Cell. Biol. 1993; 13: 4011-4022Crossref PubMed Scopus (511) Google Scholar) while the −100 site (5′-AGATAC-3′) deviates by one nucleotide. Binding of nuclear proteins to these sites was determined using GATA−124 and GATA−100 probes in gel shift assays with nuclear extracts from K562, MEL, or COS-1 cells and also from COS-1 cells transfected with the murine GATA-1 cDNA expression vector, pXM/GF-1. A β-globin GATA-1 consensus sequence (GATA-cons) was employed as a control probe (25Wall L. deBoer E. Grosveld F. Genes Dev. 1988; 2: 1089-1100Crossref PubMed Scopus (242) Google Scholar). A major protein complex was obtained with the GATA−124 probe (Fig.2 A, lanes 2 and3) and GATA-cons probe (lanes 12 and13) using nuclear extracts from K562 and MEL cells. A complex with the same mobility was detected with the GATA−100 probe, although the intensity was reduced (lanes 7 and8). Similar results were also observed with all three probes using nuclear extracts from COS-1 cells expressing recombinant GATA-1 (lanes 5, 10, and 15) but were not detected with nuclear extracts from mock-transfected COS-1 cells (lanes 4 and 9), although a minor band was observed with the GATA-cons probe (lane 14). To confirm whether the protein complex that bound to the −124 and −100 sites in the erythroid cell extracts was indeed GATA-1, gel supershift assays were undertaken with the GATA-1 monoclonal antibody, N-6 (28Ito E. Toki T. Ishihara H. Ohtani H. Gu L. Yokoyama M. Engel J.D. Yamamoto M. Nature. 1993; 362: 466-468Crossref PubMed Scopus (258) Google Scholar), and nuclear extracts from either MEL cells or COS-1 cells expressing recombinant GATA-1. The antibody substantially supershifted the major band obtained with the GATA−124 probe, the GATA-cons probe (Fig. 2 B), and the GATA−100 probe (data not shown). Competition experiments using the GATA-cons probe and nuclear extracts from" @default.
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• W2083397383 date "1997-10-01" @default.
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• W2083397383 title "Transcriptional Regulation of the Human Erythroid 5-Aminolevulinate Synthase Gene" @default.
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