Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

ZNF143 Mediates Basal and Tissue-specific Expression of Human Transaldolase

2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês

10.1074/jbc.m307039200

ISSN

1083-351X

Autores

Craig E. Grossman, Yueming Qian, Katalin Bánki, András Perl,

Tópico(s)

Methemoglobinemia and Tumor Lysis Syndrome

Resumo

Transaldolase regulates redox-dependent apoptosis through controlling NADPH and ribose 5-phosphate production via the pentose phosphate pathway. The minimal promoter sufficient to drive chloramphenicol acetyltransferase reporter gene activity was mapped to nucleotides –49 to –1 relative to the transcription start site of the human transaldolase gene. DNase I footprinting with nuclear extracts of transaldolase-expressing cell lines unveiled protection of nucleotides –29 to –16. Electrophoretic mobility shift assays identified a single dominant DNA-protein complex that was abolished by consensus sequence for transcription factor ZNF143/76 or mutation of the ZNF76/143 motif within the transaldolase promoter. Mutation of an AP-2α recognition sequence, partially overlapping the ZNF143 motif, increased TAL-H promoter activity in HeLa cells, without significant impact on HepG2 cells, which do not express AP-2α. Cooperativity of ZNF143 with AP-2α was supported by supershift analysis of HeLa cells where AP-2 may act as cell type-specific repressor of TAL promoter activity. However, overexpression of full-length ZNF143, ZNF76, or dominant-negative DNA-binding domain of ZNF143 enhanced, maintained, or abolished transaldolase promoter activity, respectively, in HepG2 and HeLa cells, suggesting that ZNF143 initiates transcription from the transaldolase core promoter. ZNF143 overexpression also increased transaldolase enzyme activity. ZNF143 and transaldolase expression correlated in 21 different human tissues and were coordinately upregulated 14- and 34-fold, respectively, in lactating mammary glands compared with nonlactating ones. Chromatin immunoprecipitation studies confirm that ZNF143/73 associates with the transaldolase promoter in vivo. Thus, ZNF143 plays a key role in basal and tissue-specific expression of transaldolase and regulation of the metabolic network controlling cell survival and differentiation. Transaldolase regulates redox-dependent apoptosis through controlling NADPH and ribose 5-phosphate production via the pentose phosphate pathway. The minimal promoter sufficient to drive chloramphenicol acetyltransferase reporter gene activity was mapped to nucleotides –49 to –1 relative to the transcription start site of the human transaldolase gene. DNase I footprinting with nuclear extracts of transaldolase-expressing cell lines unveiled protection of nucleotides –29 to –16. Electrophoretic mobility shift assays identified a single dominant DNA-protein complex that was abolished by consensus sequence for transcription factor ZNF143/76 or mutation of the ZNF76/143 motif within the transaldolase promoter. Mutation of an AP-2α recognition sequence, partially overlapping the ZNF143 motif, increased TAL-H promoter activity in HeLa cells, without significant impact on HepG2 cells, which do not express AP-2α. Cooperativity of ZNF143 with AP-2α was supported by supershift analysis of HeLa cells where AP-2 may act as cell type-specific repressor of TAL promoter activity. However, overexpression of full-length ZNF143, ZNF76, or dominant-negative DNA-binding domain of ZNF143 enhanced, maintained, or abolished transaldolase promoter activity, respectively, in HepG2 and HeLa cells, suggesting that ZNF143 initiates transcription from the transaldolase core promoter. ZNF143 overexpression also increased transaldolase enzyme activity. ZNF143 and transaldolase expression correlated in 21 different human tissues and were coordinately upregulated 14- and 34-fold, respectively, in lactating mammary glands compared with nonlactating ones. Chromatin immunoprecipitation studies confirm that ZNF143/73 associates with the transaldolase promoter in vivo. Thus, ZNF143 plays a key role in basal and tissue-specific expression of transaldolase and regulation of the metabolic network controlling cell survival and differentiation. Metabolism of glucose through the pentose phosphate pathway (PPP) 1The abbreviations used are: PPP, pentose phosphate pathway; NE, nuclear extracts; Abs, antibodies; PMSF, phenylmethylsulfonyl fluoride; ChIP, chromatin immunoprecipitation; EMSAs, electrophoretic mobility shift assays; G6PD, glucose-6-phosphate dehydrogenase; ROS, reactive oxygen species; TAL, transaldolase; np, nucleotide positions; snRNA, small nuclear RNA; CAT, chloramphenicol acetyltransferase; H-TAL, human TAL; M-TAL, murine TAL; Staf, selenocysteine tRNA gene transcription activating factor; GPx, glutathione peroxidase. 1The abbreviations used are: PPP, pentose phosphate pathway; NE, nuclear extracts; Abs, antibodies; PMSF, phenylmethylsulfonyl fluoride; ChIP, chromatin immunoprecipitation; EMSAs, electrophoretic mobility shift assays; G6PD, glucose-6-phosphate dehydrogenase; ROS, reactive oxygen species; TAL, transaldolase; np, nucleotide positions; snRNA, small nuclear RNA; CAT, chloramphenicol acetyltransferase; H-TAL, human TAL; M-TAL, murine TAL; Staf, selenocysteine tRNA gene transcription activating factor; GPx, glutathione peroxidase. fulfills two unique functions: formation of ribose 5-phosphate for the synthesis of nucleotides, RNA and DNA; and generation of NADPH as a reducing equivalent for biosynthetic reactions. Medical importance of PPP was first appreciated when deficiencies of certain enzymes of the pathway, most often deficiency of glucose-6-phosphate dehydrogenase (G6PD), were found to be associated with hemolytic anemia (1Cooper R.A. Bunn H.F. Wilson J.D. Braunwald E. Isselbacher K.J. Petersdorf R.G. Martin J.B. Fauci A.S. Root R.K. Harrison's Principles of Internal Medicine. McGraw-Hill Inc., New York1991: 1531-1543Google Scholar). PPP is important in host defense mechanisms in all tissues against oxidative stress (2Mayes P.A. Murray R.K. Granner D.K. Mayes P.A. Rodwell V.W. Harper's Biochemistry. Appleton & Lange, East Norwalk, CT1993: 201-211Google Scholar), in embryogenesis/morphogenesis (3Stark K.L. Harris C. Juchau M.R. Biochem. Pharmacol. 1989; 38: 2685-2692Crossref PubMed Scopus (23) Google Scholar), neurulation (4Baquer N.Z. Hothersall J.S. McLean P. Greenbaum A.L. Dev. Med. Child Neurol. 1977; 19: 81-104Crossref PubMed Scopus (51) Google Scholar), myelination (5Jacobson S. J. Comp. Neurol. 1963; 121: 5-29Crossref PubMed Scopus (259) Google Scholar), inflammation (6Kelso T.B. Shear C.R. Max S.R. Am. J. Physiol. 1989; 257: E885-E894PubMed Google Scholar, 7Spolarics Z. Bagby G.J. Lang C.H. Spitzer J.J. Biochem. J. 1991; 278: 515-519Crossref PubMed Scopus (21) Google Scholar, 8Held K.D. Tuttle S.W. Biaglow J.E. Radiat. Res. 1993; 134: 383-389Crossref PubMed Scopus (18) Google Scholar, 9Costa Rosa L.F.B.P. De Almeida A.F. Safi D.A. Curi R. Physiol. Behav. 1993; 53: 651-656Crossref PubMed Scopus (13) Google Scholar), lymphocyte activation (10Pilz R.B. Willis R.C. Boss G.R. J. Biol. Chem. 1984; 259: 2927-2935Abstract Full Text PDF PubMed Google Scholar, 11Boss G.R. J. Biol. Chem. 1984; 259: 2936-2941Abstract Full Text PDF PubMed Google Scholar), phagocytosis (7Spolarics Z. Bagby G.J. Lang C.H. Spitzer J.J. Biochem. J. 1991; 278: 515-519Crossref PubMed Scopus (21) Google Scholar, 9Costa Rosa L.F.B.P. De Almeida A.F. Safi D.A. Curi R. Physiol. Behav. 1993; 53: 651-656Crossref PubMed Scopus (13) Google Scholar), cardiac arrhythmias (12Bhatnagar A. Srivastava S.K. Szabo G. Circ. Res. 1990; 67: 535-549Crossref PubMed Scopus (92) Google Scholar), radiation resistance of malignant tumors (13Tuttle S. Varnes M.E. Mitchell J.B. Biaglow J.E. Int. J. Radiat. Oncol. Biol. Phys. 1992; 22: 671-675Abstract Full Text PDF PubMed Scopus (70) Google Scholar), and toxicity of high glucose concentrations in diabetes mellitus (14Asahina T. Kashiwagi A. Nishio Y. Ikebuchi M. Harada N. Tanaka Y. Saeki Y. Kikkawa R. Shigeta Y. Diabetes. 1995; 44: 520-526Crossref PubMed Scopus (105) Google Scholar, 15Kashiwagi A. Asahina T. Nishio Y. Ikebuchi M. Tanaka Y. Kikkawa R. Diabetes. 1996; 45: 84-86Crossref PubMed Google Scholar, 16Greene D.A. Stevens M.J. Obrosova I. Feldman E.L. Eur. J. Pharmacol. 1999; 375: 217-223Crossref PubMed Scopus (205) Google Scholar). Many of these processes are associated with apoptosis, a fundamental form of programmed cell death (17Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6179) Google Scholar). Apoptosis is indispensable for normal development and maintenance of homeostasis within multicellular organisms (18Wyllie A.H. Kerr J.F.R. Currie A.E. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6712) Google Scholar). Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic existence, but evidence is now accumulating that controlled levels of ROS modulate cellular function and are necessary for signal transduction pathways, including those mediating programmed cell death (19Perl A. Gergely Jr., P. Puskas F. Banki K. Antiox. Redox Signal. 2002; 4: 427-443Crossref PubMed Scopus (61) Google Scholar). A normal reducing atmosphere, required for cellular integrity is provided by reduced GSH that protects cells from damage by ROS (2Mayes P.A. Murray R.K. Granner D.K. Mayes P.A. Rodwell V.W. Harper's Biochemistry. Appleton & Lange, East Norwalk, CT1993: 201-211Google Scholar). Regeneration of GSH from its oxidized form, GSSG, is dependent on NADPH produced by the PPP (2Mayes P.A. Murray R.K. Granner D.K. Mayes P.A. Rodwell V.W. Harper's Biochemistry. Appleton & Lange, East Norwalk, CT1993: 201-211Google Scholar). Understanding how the PPP is regulated has been complicated by the fact that the pathway is composed of two separate phases, oxidative and nonoxidative. Reactions in the oxidative phase are irreversible, whereas all reactions of the nonoxidative phase are fully reversible. Nevertheless, the two phases are functionally connected. The nonoxidative phase can convert ribose 5-phosphate into glucose 6-phosphate for the oxidative phase, and thus, indirectly, it can also contribute to generation of NADPH (20Horecker B.L. J. Biol. Chem. 2002; 277: 47965-47971Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The rate-limiting enzymes for the two phases are different. The oxidative phase is primarily dependent on G6PD (21Wood T. The Pentose Phosphate Pathway. Academic Press, New York1985Google Scholar). Although the control of the nonoxidative branch is less well established, transaldolase (TAL) has been proposed as its rate-limiting enzyme (21Wood T. The Pentose Phosphate Pathway. Academic Press, New York1985Google Scholar, 22Heinrich P.C. Morris H.P. Weber G. Cancer Res. 1976; 36: 3189-3197PubMed Google Scholar). TAL catalyzes the transfer of a 3-carbon fragment, corresponding to dihydroxyacetone, to d-glyceraldehyde 3-phosphate, d-erythrose 4-phosphate, and a variety of other acceptor aldehydes, including nonphosphorylated trioses and tetroses. Enzymatic activity of TAL is regulated in a tissue-specific (21Wood T. The Pentose Phosphate Pathway. Academic Press, New York1985Google Scholar, 22Heinrich P.C. Morris H.P. Weber G. Cancer Res. 1976; 36: 3189-3197PubMed Google Scholar, 23Novello F. McLean P. Biochem. J. 1968; 107: 775-791Crossref PubMed Google Scholar, 24Severin S.E. Stepanova N.G. Adv. Enzyme Regul. 1980; 19: 235-255Crossref PubMed Scopus (13) Google Scholar, 25James H.M. Williams S.G. Bais R. Rofe A.M. Edwards J.B. Conyers R.A. Int. J. Vitam. Nutr. Res. 1985; 28 (suppl.): 29-46Google Scholar, 26Kauffman F.C. Harkonen M.H. J. Neurochem. 1977; 28: 745-750Crossref PubMed Scopus (18) Google Scholar, 27Hothersall J.S. Baquer N.Z. McLean P. Enzyme (Basel). 1982; 27: 259-267Crossref PubMed Scopus (13) Google Scholar, 28Banki K. Colombo E. Sia F. Halladay D. Mattson D.H. Tatum A.H. Massa P.T. Phillips P.E. Perl A. J. Exp. Med. 1994; 180: 1649-1663Crossref PubMed Scopus (122) Google Scholar) and developmentally specific manner (4Baquer N.Z. Hothersall J.S. McLean P. Greenbaum A.L. Dev. Med. Child Neurol. 1977; 19: 81-104Crossref PubMed Scopus (51) Google Scholar, 29Thom E. Mohlman T. Quick W.P. Camara B. Neuhaus H.E. Planta. 1998; 204: 226-233Crossref Scopus (38) Google Scholar). In the brain, TAL is expressed selectively in oligodendrocytes at high levels (28Banki K. Colombo E. Sia F. Halladay D. Mattson D.H. Tatum A.H. Massa P.T. Phillips P.E. Perl A. J. Exp. Med. 1994; 180: 1649-1663Crossref PubMed Scopus (122) Google Scholar). This is particularly interesting because myelin sheaths are formed by oligodendrocytes, and lesions in the most common demyelinating disease of the central nervous system, multiple sclerosis, are characterized by a progressive loss of oligodendrocytes and demyelination (30Martin R. McFarland H.F. McFarlin D.E. Annu. Rev. Immunol. 1992; 10: 153-187Crossref PubMed Scopus (945) Google Scholar). High levels of TAL expression in oligodendrocytes may be related to synthesis of lipids as major constituents of myelin and exquisite susceptibility of these cells to damage by reactive oxygen species, nitric oxide, and tumor necrosis factor α released by activated macrophages and astrocytes (31Merrill J.E. Ignarro L.J. Sherman M.P. Melinek J. Lane T.E. J. Immunol. 1993; 151: 2132-2141PubMed Google Scholar). Data from this laboratory have provided evidence that TAL can regulate susceptibility to apoptosis through control of the balance between the two branches of the PPP and its overall output as measured by NADPH and GSH production (32Banki K. Hutter E. Colombo E. Gonchoroff N.J. Perl A. J. Biol. Chem. 1996; 271: 32994-33001Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Overexpression of TAL in Jurkat and H9 human T cell lines results in a decrease in G6PD and 6-phosphogluconate dehydrogenase activities and NADPH and GSH levels and renders the cell highly susceptible to apoptosis induced by serum deprivation, H2O2, nitric oxide, tumor necrosis factor α, Fas antibody, or infection by human immunodeficiency virus, type 1 (32Banki K. Hutter E. Colombo E. Gonchoroff N.J. Perl A. J. Biol. Chem. 1996; 271: 32994-33001Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 33Banki K. Hutter E. Gonchoroff N.J. Perl A. J. Biol. Chem. 1998; 273: 11944-11953Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 34Banki K. Hutter E. Gonchoroff N.J. Perl A. J. Immunol. 1999; 162: 1466-1479PubMed Google Scholar). In addition, reduced levels of TAL results in increased G6PD and 6-phosphogluconate dehydrogenase activities and increased GSH levels with inhibition of apoptosis. Disruption of the mitochondrial transmembrane potential (Δψm), the point of no return in the effector phase of programmed cell death (35Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 25-37Crossref PubMed Scopus (589) Google Scholar, 36Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar), is subject to regulation by an oxidation-reduction equilibrium of ROS, pyridine nucleotides (NAD/NADH and NADP/NADPH), and GSH levels (37Constantini P. Chernyak B.V. Petronilli V. Bernardi P. J. Biol. Chem. 1996; 271: 6746-6751Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). The extent of Fas-induced mitochondrial ROI production, changes in Δψm, caspase activation, and cell death are regulated by TAL expression levels (34Banki K. Hutter E. Gonchoroff N.J. Perl A. J. Immunol. 1999; 162: 1466-1479PubMed Google Scholar). The impact of TAL on apoptotic signaling (32Banki K. Hutter E. Colombo E. Gonchoroff N.J. Perl A. J. Biol. Chem. 1996; 271: 32994-33001Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) may be related to an overwhelming influence of TAL-catalyzed dihydroxyacetone transfer reactions on the balance between the two branches of PPP (32Banki K. Hutter E. Colombo E. Gonchoroff N.J. Perl A. J. Biol. Chem. 1996; 271: 32994-33001Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) that controls intracellular NADPH levels and neutralization of ROS (38Ni T.-C. Savageau M.A. J. Biol. Chem. 1996; 271: 7927-7941Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). TAL-dependent changes in NADP/NADPH levels may regulate expression of G6PD through oxidative stress-response elements located in the G6PD promoter (39Ursini M.V. Lettieri T. Braddock M. Martini G. Virology. 1993; 196: 338-343Crossref PubMed Scopus (20) Google Scholar). Therefore, regulation of TAL expression may play a pivotal role in tissue- and cell type-specific metabolic signaling (19Perl A. Gergely Jr., P. Puskas F. Banki K. Antiox. Redox Signal. 2002; 4: 427-443Crossref PubMed Scopus (61) Google Scholar, 38Ni T.-C. Savageau M.A. J. Biol. Chem. 1996; 271: 7927-7941Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). TAL-H is a single copy gene located on the short arm of human chromosome 11 at p15.4–15.5 (40Banki K. Eddy R.L. Shows T.B. Halladay D.L. Bullrich F. Croce C.M. Jurecic V. Baldini A. Perl A. Genomics. 1997; 45: 233-238Crossref PubMed Scopus (15) Google Scholar). The eight exons encoding a 337-amino acid protein of 37.6 kDa (41Banki K. Halladay D. Perl A. J. Biol. Chem. 1994; 269: 2847-2851Abstract Full Text PDF PubMed Google Scholar) are contained within a 17.5-kb genomic locus (TALDO1) (42Perl A. Colombo E. Samoilova E. Butler M.C. Banki K. J. Biol. Chem. 2000; 275: 7261-7272Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). In the present study, we localized the core promoter of TAL-H to nucleotide positions (np) –49 to –1 that harbors a DNA element that is recognized and functionally activated by transcription factor ZNF143, a human homolog of Xenopus selenocysteine tRNA gene transcription activating factor (Staf) (43Myslinski E. Krol A. Carbon P. J. Biol. Chem. 1998; 273: 21998-22006Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 44Rincon J.C. Engler S.K. Hargrove B.W. Kunkel G.R. Nucleic Acids Res. 1998; 26: 4846-4852Crossref PubMed Scopus (27) Google Scholar). This is a seven-zinc finger transcription factor capable of enhancing transcription of mRNA, tRNASec, snRNA, and snRNA-type genes via RNA polymerase II or III (43Myslinski E. Krol A. Carbon P. J. Biol. Chem. 1998; 273: 21998-22006Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 44Rincon J.C. Engler S.K. Hargrove B.W. Kunkel G.R. Nucleic Acids Res. 1998; 26: 4846-4852Crossref PubMed Scopus (27) Google Scholar, 45Schuster C. Myslinski E. Krol A. Carbon P. EMBO J. 1995; 14: 3777-3787Crossref PubMed Scopus (69) Google Scholar, 46Schaub M. Myslinski E. Schuster C. Krol A. Carbon P. EMBO J. 1997; 16: 173-181Crossref PubMed Scopus (89) Google Scholar, 47Schuster C. Krol A. Carbon P. Mol. Cell. Biol. 1998; 18: 2650-2658Crossref PubMed Scopus (33) Google Scholar). ZNF143 is encoded by a single copy genomic locus within a 10-megabase distance from the TAL-H locus at band p15.4 of human chromosome 11 (48Tommerup N. Vissing H. Genomics. 1995; 27: 259-264Crossref PubMed Scopus (123) Google Scholar). Site-directed mutagenesis of the ZNF143/76 motif abolished transcriptional activity of the core TAL-H promoter. Expression of TAL-H correlated with that of ZNF143 in 21 different human tissues and both were up-regulated in lactating mouse mammary glands as compared with nonlactating controls. Overexpression of ZNF143 enhanced TAL-H promoter activity, and a dominant-negative form of ZNF143 blocked transcription from the TAL-H promoter, indicating that ZNF143 may play a key role in regulating tissue-specific expression of TAL-H. Cell Culture—HepG2 human hepatoma cells (ATCC, Manassas, VA) were cultured in minimum essential medium with Earle's salts, 10% fetal bovine serum, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate, 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. HeLa human cervix carcinoma cells (from ATCC) and MO3.13 human oligodendroglioma cells (kindly provided by Dr. Neil Cashman, Montreal Neurological Institute, Montreal, Canada) were grown in Dulbecco's modified Eagle's medium with 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10 μg/ml amphotericin B. HOG human oligodendroglioma cells (from Dr. Glyn Dawson, University of Chicago) were maintained in Dulbecco`s modified Eagle's medium with 2 mm l-glutamine, 0.1 mm nonessential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10 μg/ml amphotericin B. Jurkat human T cell leukemia cells (49Puskas F. Gergely Jr., P. Banki K. Perl A. FASEB J. 2000; 14: 1352-1361Crossref PubMed Google Scholar) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, and antibiotics. All cell lines were maintained in a humidified atmosphere with 5% CO2 at 37 °C. Cell culture products were purchased from Cellgro (Mediatech Inc., Herndon, VA). Plasmids and Oligonucleotides—Mutagenesis of TAL-H promoter constructs were generated by PCR using the QuickChange Site-directed Mutagenesis kit as suggested by the manufacturer (Stratagene, La Jolla, CA). 25 ng of template was incubated with 125 ng of sense and antisense primers, dNTPs, and subjected to 18 PCR cycles with Pfu turbo DNA polymerase with denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, and extension at 68 °C for 10 min. DpnI was used to digest the parental supercoiled double-stranded methylated DNA for 1 h at 37 °C. Transformations were performed in Escherichia coli XL-1 Blue cells using DpnI-treated DNA. Plasmids were prepared using Qiagen Plasmid Maxi Kit Columns (Qiagen, Valencia, CA). Introduction of mutations into the TAL-H promoter constructs were verified by DNA sequencing. AP-2 and Sp1 consensus oligonucleotides were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). TARE-6 oligonucleotide, derived from full-length TARE-6 (42Perl A. Colombo E. Samoilova E. Butler M.C. Banki K. J. Biol. Chem. 2000; 275: 7261-7272Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), was used as a nonspecific competitor in electrophoretic mobility shift assays (EMSAs). ZNF143/76 (50Schaub M. Krol A. Carbon P. J. Biol. Chem. 1999; 274: 24241-24249Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and SOX5 (51Denny P. Swift S. Connor F. Ashworth A. EMBO J. 1992; 11: 3705-3712Crossref PubMed Scopus (235) Google Scholar) consensus sequences were generated based on published reports. All oligonucleotides used in EMSAs and for generating TAL-H promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs were synthesized by Ransom Hill Bioscience (Ramona, CA). Transient Transfections and Reporter Gene Assays—HepG2 cells were transfected with 1.6 μg of pBLCAT3-based (52Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar) TAL-H promoter reporter constructs at 80% confluency in 9.5-cm2 wells using 20 μl of PLUS reagent and 4.8 μl of LipofectAMINE reagent (Invitrogen). 80% confluent HeLa cells in 3.8-cm2 wells were transfected with 0.8 μg of pBLCAT3-based TAL-H promoter vectors using 10 μl of PLUS reagent and 2.4 μl of LipofectAMINE reagent. Each cell line was cotransfected with pRSVβ-gal (β-galactosidase reporter gene driven by the Rous sarcoma virus promoter (53Gorman C.M. Merlino G.T. Willingham M.C. Pastan I. Howard B.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6777-6781Crossref PubMed Scopus (879) Google Scholar)) using 1.6 μg for HepG2 and 0.8 μg for HeLa cells, respectively, in order to normalize transfection efficiency. In each transfection, the promoterless pBLCAT3 vector was used as a negative control. For overexpression of recombinant transcription factors ZNF76 and ZNF143, the corresponding expression vectors or pCAGGS empty vector (54Kubota H. Yokota S. Yanagi H. Yura T. J. Biol. Chem. 2000; 275: 28641-28648Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) was cotransfected with the TAL-H promoter CAT reporter plasmids and internal control pRSVβ-gal using 1 μg of each plasmid, 20 μl of PLUS reagent, and 8 μl of LipofectAMINE reagent. After 4½ h of exposure to the DNA-LipofectAMINE complex in serum- and antibiotic-free media, the DNA complex was removed, and cells were further cultured for 36 h in complete growth media. Cells were harvested in 150 μl of 250 mm Tris, pH 7.8, and solubilized by 3 rounds of freezing and thawing. For β-galactosidase assay, 30 μl of lysate was incubated with 270 μl of reaction mixture (1 mm MgCl2, 50 mm β-mercaptoethanol, 3 mm o-nitrophenyl β-d-galactopyranoside, and 0.1 m NaPi, pH 7.5) at 37 °C and terminated with the addition of 500 μl of 1 m Na2CO3 once the reaction turned yellow. Absorbance values were measured at 420 nm and employed to adjust the quantity of cell lysates to be used in the CAT assay for normalization of transfection efficiencies. Prior to CAT assay, lysates were heated to 65 °C for 10 min to deactivate acetylases. CAT assays were performed at 37 °C in a 50-μl reaction mixture with normalized volumes of cell lysates in 250 mm Tris, pH 7.8, 0.4 mm acetyl coenzyme A, and 0.025 μCi of [14C]chloramphenicol. Acetylated chloramphenicol was extracted with ethyl acetate and dried in a vacuum centrifuge. Pellets were resuspended in ethyl acetate, spotted on a silica gel TLC plate (Analtech, Newark, DE), and resolved in an equilibrated chromatography tank containing 19:1 chloroform/methanol until the solvent front approached the top of the TLC plate. A 445 SI PhosphorImager with ImageQuant software (Amersham Biosciences) was used to determine the ratio of acetylated to unacetylated [14C]chloramphenicol. All assays were conducted within the range of linearity of CAT activities with respect to incubation time, based on the β-galactosidase assay. Each transfection experiment was repeated at least four times. Preparation of Nuclear Extracts—Nuclear extracts (NE) were prepared according to a protocol described previously (55Lee K.A. Bindereif A. Green M.R. Gene Anal. Tech. 1988; 5: 22-31Crossref PubMed Scopus (394) Google Scholar) at 4 °C. Briefly, cells in log phase were harvested, washed with cold phosphate-buffered saline, and resuspended in 1× packed cell volume of ice-cold hypotonic buffer A (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm dithiothreitol, and 10 mm leupeptin). Following a 15-min incubation on ice, the suspension was rapidly flushed eight times through a 25-gauge syringe to lyse cells. Homogenates were centrifuged at 16,000 × g, and pelleted nuclei were resuspended in 2/3× original packed cell volume of ice-cold buffer C (20 mm HEPES, pH 7.9, 20% glycerol, 550 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm PMSF, 1 mm dithiothreitol, and 10 mm leupeptin). After constant stirring for 30 min, samples were centrifuged for 5 min at 16,000 × g, and the supernatant containing nuclear proteins was aliquoted and stored at –80 °C. Protein concentrations were determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad). DNase I Footprinting—DNase I footprinting was carried out with modifications of an established protocol (56Brenowitz M. Senear D.F. Shea M.A. Ackers G.K. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8462-8466Crossref PubMed Scopus (146) Google Scholar, 57Brenowitz M. Senear D.F. Shea M.A. Ackers G.K. Methods Enzymol. 1986; 130: 132-181Crossref PubMed Scopus (367) Google Scholar). Briefly, a doublestranded gel-purified TAL-H promoter DNA fragment (np –153 to +52) from a BamHI-TaqI digestion of clone 1994 (Fig. 3) was end-labeled by incorporating 3000 Ci/mmol [α-32P]dCTP (ICN Biomedicals, Aurora, OH) into the 3′-end of the sense strand using Sequenase version 2.0 DNA polymerase (U. S. Biochemical Corp.) and purified through a Sephadex G-25 spin column (Shelton Scientific, Shelton, CT). Binding reactions were conducted in 50 μl of EMSA binding buffer (see below) without bovine serum albumin incubating 0.035 pmol (65,000–80,000 cpm) of TAL-H promoter probe with 40 μg of NE for 10 min at room temperature. Then the reaction was supplemented with 50 μl of 5 mm CaCl2, 10 mm MgCl2 and allowed to equilibrate for an additional minute at room temperature. Subsequently, the mixture was treated with 1.5 units of DNase I (Promega, Madison, WI) for 1 min at room temperature and terminated with the addition of 90 μl of 30 mm EDTA. Nuclear proteins were digested with 25 μg of proteinase K for 10 min at room temperature. After phenol/chloroform extraction, the aqueous phase was ethanol-precipitated overnight with 30 μg of GlycoBlue (Ambion, Inc., Austin, TX). The DNA pellet was resuspended in 5 μl of 1:2 diluted loading dye (U. S. Biochemical Corp. Stop Solution: 95% formamide, 20 mm EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol FF), denatured at 95 °C for 5 min, and separated on a 6% polyacrylamide, 8 m urea sequencing gel run at 65 watts in 1× Tris borate/EDTA (TBE) buffer. The gel was fixed in 10% methanol, 10% acetic acid for 30 min, dried under vacuum at 80 °C for 1 h, and subjected to autoradiography at –80 °C with an intensifying screen. Control reaction consisted of 0.035 pmol of probe incubated with 0.75 units of DNase I in the absence of nuclear extract. Identification of the location of the protected regions within the TAL-H promoter was determined by alignment with a 35S-labeled sequencing ladder run alongside the footprinting reactions. Electrophoretic Mobility Shift Assay—Double-stranded synthetic oligonucleotides (Fig. 4) were generated by heating primer pairs to 95 °C for 5 min in 100 mm NaCl, 10 mm Tris, pH 8, 1 mm EDTA, and slowly cooling to room temperature. These double-stranded oligonucleotides were 5′-end-labeled with 4,500 Ci/mmol [γ-32P]ATP (ICN Biomedicals) and T4 polynucleotide kinase (New England Biolabs, Beverly, MA) for 1 h at 37 °C and purified on Sephadex G-25 spin columns for use as EMSA probes. 10 μg of NE was incubated with 20 fmol (45,000–60,000 cpm) of double-strand

Referência(s)