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• W1966642608 abstract "Adenine nucleotide translocase-2 (ANT2) catalyzes the exchange of ATP for ADP across the mitochondrial membrane, thus playing an important role in maintaining the cytosolic phosphorylation potential required for cell growth. Expression of ANT2 is activated by growth stimulation of quiescent cells and is down-regulated when cells become growth-arrested. In this study, we address the mechanism of growth arrest repression. Using a combination of transfection, in vivo dimethyl sulfate mapping, and in vitro DNase I mapping experiments, we identified two protein-binding elements (Go-1 and Go-2) that are responsible for growth arrest of ANT2 expression in human diploid fibroblasts. Proteins that bound the Go elements were purified and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as members of the NF1 family of transcription factors. Chromatin immunoprecipitation analysis showed that NF1 was bound to both Go-1 and Go-2 in quiescent human diploid cells in vivo, but not in the same cells stimulated to growth by serum. NF1 binding correlated with the disappearance of ANT2 transcripts in quiescent cells. Furthermore, overexpression of NF1-A, -C, and -X in NIH3T3 cells repressed expression of an ANT2-driven reporter gene construct. Two additional putative repressor elements in the ANT2 promoter, an Sp1 element juxtaposed to the transcription start site and a silencer centered at nucleotide –332, did not appear to contribute to growth arrest repression. Thus, enhanced binding of NF1 is a key step in the growth arrest repression of ANT2 transcription. To our knowledge, this is the first report showing a role for NF1 in growth arrest. Adenine nucleotide translocase-2 (ANT2) catalyzes the exchange of ATP for ADP across the mitochondrial membrane, thus playing an important role in maintaining the cytosolic phosphorylation potential required for cell growth. Expression of ANT2 is activated by growth stimulation of quiescent cells and is down-regulated when cells become growth-arrested. In this study, we address the mechanism of growth arrest repression. Using a combination of transfection, in vivo dimethyl sulfate mapping, and in vitro DNase I mapping experiments, we identified two protein-binding elements (Go-1 and Go-2) that are responsible for growth arrest of ANT2 expression in human diploid fibroblasts. Proteins that bound the Go elements were purified and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as members of the NF1 family of transcription factors. Chromatin immunoprecipitation analysis showed that NF1 was bound to both Go-1 and Go-2 in quiescent human diploid cells in vivo, but not in the same cells stimulated to growth by serum. NF1 binding correlated with the disappearance of ANT2 transcripts in quiescent cells. Furthermore, overexpression of NF1-A, -C, and -X in NIH3T3 cells repressed expression of an ANT2-driven reporter gene construct. Two additional putative repressor elements in the ANT2 promoter, an Sp1 element juxtaposed to the transcription start site and a silencer centered at nucleotide –332, did not appear to contribute to growth arrest repression. Thus, enhanced binding of NF1 is a key step in the growth arrest repression of ANT2 transcription. To our knowledge, this is the first report showing a role for NF1 in growth arrest. The adenine nucleotide translocase (ANT) 1The abbreviations used are: ANT, adenine nucleotide translocase; NF1, nuclear factor-1; Luc, luciferase; nt, nucleotide(s); DMS, dimethyl sulfate; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; GoR, Go repressor; EMSA, electrophoretic mobility shift assay. proteins catalyze the exchange of mitochondria ATP for cytosolic ADP. In doing so, they play an important role in maintaining the cytosolic phosphorylation potential and therefore normal cell growth and function. In addition, the ANTs have been implicated in early events in initiation of mitochondrion-dependent apoptosis (1Marzo I. Brenner C. Zamzami N. Susin S.A. Beutner G. Brdiczka D. Remy R. Xie Z.H. Reed J.C. Kroemer G. J. Exp. Med. 1998; 187: 1261-1271Crossref PubMed Scopus (615) Google Scholar). Three ANT isoforms are encoded in separate genes in mammals (2Neckelmann N. Li K. Wade R.P. Shuster R. Wallace D.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7580-7584Crossref PubMed Scopus (161) Google Scholar, 3Houldsworth J. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 377-381Crossref PubMed Scopus (107) Google Scholar, 4Cozens A.L. Runswick M.J. Walker J.E. J. Mol. Biol. 1989; 206: 261-280Crossref PubMed Scopus (116) Google Scholar, 5Ku D.-H. Kagan J. Chen S.-T. Chang C.-D. Baserga R. Wurzel J. J. Biol. Chem. 1990; 265: 16060-16063Abstract Full Text PDF PubMed Google Scholar) and yeast (6Kolarov J. Kolarova N. Nelson N. J. Biol. Chem. 1990; 265: 12711-12716Abstract Full Text PDF PubMed Google Scholar, 7Lawson J.E. Douglas M.G. J. Biol. Chem. 1988; 263: 14812-14818Abstract Full Text PDF PubMed Google Scholar). Two of these isoforms (ANT1 and ANT2) are differentially expressed in mammalian tissues (8Stepien G. Torroni A. Chung A.B. Hodge J.A. Wallace D.C. J. Biol. Chem. 1992; 267: 14592-14597Abstract Full Text PDF PubMed Google Scholar, 9Dörner A. Pauschinger M. Badorff A. Noutsias M. Giessen S. Schulze K. Bilger J. Rauch U. Schultheiss H.-P. FEBS Lett. 1997; 414: 258-262Crossref PubMed Scopus (106) Google Scholar, 10Dörner A. Olesch M. Giessen S. Pauschinger M. Schultheiss H.-P. Biochim. Biophys. Acta. 1999; 1417: 16-24Crossref PubMed Scopus (45) Google Scholar) and in differentiating cells (8Stepien G. Torroni A. Chung A.B. Hodge J.A. Wallace D.C. J. Biol. Chem. 1992; 267: 14592-14597Abstract Full Text PDF PubMed Google Scholar, 11Battini R. Ferrari S. Kaczmarek L. Calabretta B. Chen S.-T. Baserga R. J. Biol. Chem. 1987; 262: 4355-4359Abstract Full Text PDF PubMed Google Scholar, 12Lunardi J. Attardi G. J. Biol. Chem. 1991; 266: 16534-16540Abstract Full Text PDF PubMed Google Scholar, 13Lunardi J. Hurko O. Engel W.K. Attardi G. J. Biol. Chem. 1992; 267: 15267-15270Abstract Full Text PDF PubMed Google Scholar). ANT2 expression is down-regulated in the latter case (8Stepien G. Torroni A. Chung A.B. Hodge J.A. Wallace D.C. J. Biol. Chem. 1992; 267: 14592-14597Abstract Full Text PDF PubMed Google Scholar, 11Battini R. Ferrari S. Kaczmarek L. Calabretta B. Chen S.-T. Baserga R. J. Biol. Chem. 1987; 262: 4355-4359Abstract Full Text PDF PubMed Google Scholar, 12Lunardi J. Attardi G. J. Biol. Chem. 1991; 266: 16534-16540Abstract Full Text PDF PubMed Google Scholar, 13Lunardi J. Hurko O. Engel W.K. Attardi G. J. Biol. Chem. 1992; 267: 15267-15270Abstract Full Text PDF PubMed Google Scholar). Expression of the ANT2 isoform is also growth-dependent (11Battini R. Ferrari S. Kaczmarek L. Calabretta B. Chen S.-T. Baserga R. J. Biol. Chem. 1987; 262: 4355-4359Abstract Full Text PDF PubMed Google Scholar). Rapid expression of ANT2 mRNA has been demonstrated in a variety of growth-arrested mammalian cell types activated to enter the G1 phase of cell growth (11Battini R. Ferrari S. Kaczmarek L. Calabretta B. Chen S.-T. Baserga R. J. Biol. Chem. 1987; 262: 4355-4359Abstract Full Text PDF PubMed Google Scholar, 14Hirschhorn R.R. Aller P. Yuan Z.A. Gibson C.W. Baserga R. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6004-6008Crossref PubMed Scopus (140) Google Scholar, 15Rittling S.R. Brooks K.M. Cristofalo V.J. Baserga R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3316-3320Crossref PubMed Scopus (203) Google Scholar, 16Kaczmarek L. Calabretta B. Baserga R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5375-5379Crossref PubMed Scopus (96) Google Scholar, 17Kaczmarek L. Calabretta B. Calabretta R.A.B. Biochem. Biophys. Res. Commun. 1985; 133 (B.): 410-416ACrossref PubMed Scopus (14) Google Scholar). ANT2 mRNA expression occurs together with the immediate-early genes required for activation of cell cycle progression and is accounted for solely by the activation of transcription (18Barath P. Luciakova K. Hodny Z. Li R. Nelson B.D. Exp. Cell Res. 1999; 248: 583-588Crossref PubMed Scopus (50) Google Scholar). However, unlike the other immediate-early genes, ANT2 expression is maintained throughout the cell cycle. Expression is down-regulated only as cells become growth-arrested at confluence (18Barath P. Luciakova K. Hodny Z. Li R. Nelson B.D. Exp. Cell Res. 1999; 248: 583-588Crossref PubMed Scopus (50) Google Scholar). The mechanism(s) by which gene expression is repressed in cells entering G0 is poorly understood. Growth arrest-specific genes have been identified in growth-inhibited NIH3T3 cells (19Schneider C. King R.M. Philipson L. Cell. 1988; 54: 787-793Abstract Full Text PDF PubMed Scopus (812) Google Scholar), but these do not participate directly in transcription initiation (20Manfioletti G. Ruaro M.E. Del Sal G. Philipson L. Schneider C. Mol. Cell. Biol. 1990; 10: 2924-2930Crossref PubMed Scopus (221) Google Scholar). Transforming growth factor-β induces growth arrest of many cell types, leading to repression of many individual genes via the Smad proteins (21Shi Y. Bioessays. 2001; 23: 223-232Crossref PubMed Scopus (109) Google Scholar, 22Chang H. Brown C.B. Matzuk M.M. Endocr. Rev. 2002; 23: 787-823Crossref PubMed Scopus (659) Google Scholar). However, microarray analysis of growth-stimulated human primary fibroblasts (23Iyer V.R. Eisen M.B. Ross D.T. Schuler G. Moore T. Lee J.C. Trent J.M. Staudt L.M. Hudson Jr., J. Boguski M.S. Lashkari D. Shalon D. Botstein D. Brown P.O. Science. 1999; 283: 83-87Crossref PubMed Scopus (1730) Google Scholar, 24Zhu H. Cong J.P. Mamtora G. Gingeras T. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14470-14475Crossref PubMed Scopus (411) Google Scholar) revealed up-regulation of relatively few transcription factors, suggesting that modulation of transcription factor expression may not be not a commonly used mechanism for regulating G0-specific gene expression. The mechanism by which ANT2 expression is repressed during growth arrest is not known. We demonstrated previously that removal of a 700-bp upstream region of the human ANT2 promoter prevents growth arrest repression (18Barath P. Luciakova K. Hodny Z. Li R. Nelson B.D. Exp. Cell Res. 1999; 248: 583-588Crossref PubMed Scopus (50) Google Scholar). In the present study, we show that growth arrest of ANT2 is mediated by members of the nuclear factor-1 (NF1) family of transcription factors via two DNA elements (Go-1 and Go-2) in the upstream repressor region. The NF1 family consists of four genes, NF1-A, -B, -C, and -X, and a large number of splice variants that can act either as transcriptional activators or repressors (see Ref. 25Gronostajski R.M. Gene (Amst.). 2000; 249: 31-45Crossref PubMed Scopus (431) Google Scholar for review) depending on the cell context. However, to our knowledge, the repression of ANT2 reported here is the first example of NF1 acting as a growth arrest repressor. Cell Culture—Human primary diploid foreskin fibroblasts were used in passages 7–17. Diploid fibroblasts and NIH3T3 cells were grown as described (18Barath P. Luciakova K. Hodny Z. Li R. Nelson B.D. Exp. Cell Res. 1999; 248: 583-588Crossref PubMed Scopus (50) Google Scholar). For serum starvation, cells were washed twice with phosphate-buffered saline; serum-free medium was added; and incubation was continued for 48 h. Confluent NIH3T3 cells were produced as described (18Barath P. Luciakova K. Hodny Z. Li R. Nelson B.D. Exp. Cell Res. 1999; 248: 583-588Crossref PubMed Scopus (50) Google Scholar). Plasmids—ANT2-Luc reporter plasmids used in stable transfection experiments were prepared using unique restriction enzyme sites (BglII, XbaI, BamHI, and SmaI) in the human ANT2 promoter PstI/PstI fragment (26Li R. Hodny Z. Luciakova K. Barath P. Nelson B.D. J. Biol. Chem. 1996; 271: 18925-18930Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). These restriction fragments were inserted into the HindIII/NheI sites of pGL3-basic (Promega). ANT2 promoter fragments bearing mutations in the C box were prepared as described (26Li R. Hodny Z. Luciakova K. Barath P. Nelson B.D. J. Biol. Chem. 1996; 271: 18925-18930Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) using the Mut-2 sequence. All clones were checked for fragment size and orientation. An oligonucleotide containing the mutated ANT2 Go-2 element (nucleotides (nt) –822 to –794) was prepared by PCR using a mutated 5′-primer (5′-CCA ATT CCT TAA AAG ATC TTT GTC GAA C-3′, where the underlined nucleotides represent mutation of the wild-type GGC sequence) and the GL2 primer (5′-CTT TAT GTT TTT GGC GTC TTC CA-3′) from pGL3-basic. The PstI/PstI ANT2-Luc reporter plasmid (26Li R. Hodny Z. Luciakova K. Barath P. Nelson B.D. J. Biol. Chem. 1996; 271: 18925-18930Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) was used as the template DNA. An oligonucleotide with both the Go-2 (nt –822 to –794) and Go-1 (nt –726 to –701) elements mutated was prepared by PCR amplification of a short fragment using a set of primers in which the core GGC sequence was changed to TAA. The 5′-primer contained the mutated Go-2 element (5′-CCA ATT CCT TAA AAG ATC TTT GTC GAA C-3′), and the 3′-primer contained the mutated Go-1 element (5′-GTG TGC TGT CCT GGA TTA AGT GAA ACC-3′). The amplified fragment was then used as the 5′-primer for another round of amplification using the PstI/PstI ANT2-Luc reporter plasmid (26Li R. Hodny Z. Luciakova K. Barath P. Nelson B.D. J. Biol. Chem. 1996; 271: 18925-18930Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) as the template and the GL2 primer as the 3′-primer. This amplified fragment of ANT2 was used in turn as the template for amplification with the wild-type Go-2 element primer (5′-CCA ATT CCT GGC AAG ATC TTT GTC GAA C-3′) to obtain a combination of mutations. PCR was performed with Vent DNA polymerase (New England Biolabs Inc.) according to the manufacturer's recommendations. All clones were verified by sequencing. Stable transfections were performed as described (18Barath P. Luciakova K. Hodny Z. Li R. Nelson B.D. Exp. Cell Res. 1999; 248: 583-588Crossref PubMed Scopus (50) Google Scholar). Resistant colonies (100–200) for each of the luciferase constructs were pooled and grown in the presence of Geneticin (0.4 mg/ml). Luciferase activity measurements were performed as described (18Barath P. Luciakova K. Hodny Z. Li R. Nelson B.D. Exp. Cell Res. 1999; 248: 583-588Crossref PubMed Scopus (50) Google Scholar). Protein concentration was measured by the Bio-Rad protein assay. In Vitro DNase I Footprinting—Nuclear extracts were prepared from human diploid fibroblasts and NIH3T3 cells by the method of Dignam et al. (27Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar). The DNase I protection assay was performed as described by Promega (28PromegaPromega Protocols and Applications Guide. 2nd ed. Promega, Madison, WI1991Google Scholar). Radioactive probes were prepared by PCR using 5′-32P-labeled chloramphenicol acetyltransferase primer, the M13 primer, and pCAT-ANT2(–917/–654) as the template. In Vivo Dimethyl Sulfate (DMS) Footprinting—DMS footprinting of human diploid cells was performed as described (29Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York1994Google Scholar) with minor modifications. Cells were treated with 0.1% DMS in 2 ml of phosphate-buffered saline for 2 min at room temperature. After washing with phosphate-buffered saline, cells were lysed with 1 ml of lysis solution (50 mm Tris-Cl (pH 8), 300 mm NaCl, 25 mm EDTA, 0.2% SDS, and 200 μg/ml proteinase K). Modified genomic DNA was isolated according to established procedures (29Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York1994Google Scholar). As a control, unmodified genomic DNA isolated from cells was exposed to 0.125% DMS in vitro for 2 min at room temperature. DMS-modified DNA was cleaved with piperidine (29Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York1994Google Scholar) and subjected to ligation-mediated PCR with primers directed against the upper strand. Thus, the radiolabeled bands on the gels represent C residues on the coding strand. Ligation-mediated PCR—DMS-treated DNA (2 μg) was amplified by ligation-mediated PCR using the Vent DNA polymerase as described (29Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York1994Google Scholar). The first cycle of amplification was for 2 min, followed by a gradual increase to 10 min in the last cycle. A total of 21 amplification cycles were performed, after which a third primer, radiolabeled using T4 polynucleotide kinase (MBI Fermentas) and [γ-32P]ATP (Amersham Biosciences), was added, and five more cycles were performed. The final product was analyzed on a 6% sequencing gel in 1× Tris borate/EDTA. The nested 5′-primer sets used were as follows. Primer set –931: primer –982 (5′-ATTCAATCCAAGGGCACTTTACC-3′), primer –947 (5′-AGCCAAATGTCAACGTAGTTC-3′), and primer –931, 5′-AGTTCTTAACCTTCCTAAGCCTC-3′); primer set –703: primer –733 (5′-TTCGTCTGGTTTCACTGGCTCCAGG-3′), primer –718 (5′-TGGCTCCAGGACAGCACACGGTCTA-3′), and primer –703 (5′-ACACGGTCTAGAGTGGGAAGAGTGA-3′); primer set –439: primer –469 (5′-ACAAAACAAGGGGGCCGGCCAG-3′), primer –453 (5′-GGCCAGTAGGATGTAGTTTGCCCATC-3′), and primer –439 (5′-AGTTTGCCCATACGACTTTTTTAAAG-3′); primer set –151: primer –263 (5′-CTCAGAGTCCCAGCGTTCAA-3′), primer –237 (5′-GTGTTCTCTGGACCCGCCCCT-3′), and primer –151 (5′-TCCACGACTCCTCCTCCTGCGAG-3′); linker 11 (5′-GAATTCAGATC-3′); and linker 25 (5′-GCGGTGACCCGGGAGATCTGAATTC-3′). RNA Purification and Northern Analysis—RNA was purified using acidic guanidium thiocyanate/phenol/chloroform extraction (30Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63191) Google Scholar). The samples were separated on 1.2% agarose gels containing 2.2 m formaldehyde, and RNA was transferred to Hybond-N membranes (Amersham Biosciences). Hybridization and membrane washing procedures were performed as described (31Luciakova K. Li R. Nelson B.D. Eur. J. Biochem. 1992; 207: 253-257Crossref PubMed Scopus (42) Google Scholar). Membranes were analyzed on a Fuji BAS-1000 phosphoimager. NF1 Purification—Nuclei were prepared from rat liver (32Blobel G. Potter V.R. Science. 1966; 154: 1662-1665Crossref PubMed Scopus (985) Google Scholar) and HeLa cells (27Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar) as described. Nuclear proteins were extracted into an equal volume of extraction buffer (20 mm HEPES (pH 7.9), 820 mm NaCl, 5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, and 0.5 mm dithiothreitol). One volume of 20 mm HEPES (pH 7.9), 5 mm MgCl2, and 5 mm β-mercaptoethanol was then added, and insoluble particles were removed by ultracentrifugation for1hat 100,000 × g. The cleared supernatant was applied to a heparin-Sepharose XK 16/20 column (Amersham Biosciences) in 20 mm HEPES (pH 7.9), 5 mm MgCl2, 5 mm β-mercaptoethanol, and 200 mm NaCl. Low affinity binding proteins were removed in a 400 mm NaCl wash, and active fractions were subsequently eluted in a 400–800 mm NaCl gradient. Fractions were tested for Go element-binding activity by in vitro DNase I protection assay. Active fractions were desalted on PD-10 columns (Amersham Biosciences) and placed in 20 mm Tris (pH 8.0) and 100 mm NaCl and applied to a ResourceQ column (1 ml; Amersham Biosciences). Proteins were eluted by a 100–300 mm NaCl linear gradient. Active fractions were rebuffered to 20 mm HEPES (pH 7.9), 5 mm MgCl2, 5 mm β-mercaptoethanol, and 70 mm NaCl and applied to a DNA affinity column containing the immobilized Go-2 oligonucleotide (5′-CCA ATT CCT GGC AAG ATC TTT GTC GAA C-3′). The column was prepared as described (33Kadonaga J.T. Methods Enzymol. 1991; 208: 10-23Crossref PubMed Scopus (98) Google Scholar). Proteins were eluted in two salt steps (200 and 500 mm NaCl) in 20 mm HEPES (pH 7.9), 5 mm MgCl2, 5 mm β-mercaptoethanol, 5% glycerol, and 0.1% Nonidet P-40. Protein fractions were stored at –70 °C. SDS-PAGE and Protein Identification by MALDI-TOF/MS—Samples from the DNA affinity column were precipitated for 20 min on ice in 10% trichloroacetic acid, followed by a 15-min centrifugation at 10,000 × g and two washes with ice-cold acetone. Samples were air-dried, dissolved in sample buffer, and separated by 10% SDS-PAGE (34Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). Proteins were visualized by silver staining (35Shevchenko A. Anal Chem. 1996; 68: 850-858Crossref PubMed Scopus (7822) Google Scholar), and bands of interest were cut out. In-gel tryptic digestion and sample preparation were done as described (35Shevchenko A. Anal Chem. 1996; 68: 850-858Crossref PubMed Scopus (7822) Google Scholar). MALDI-TOF analysis was performed in reflector mode using a Voyager-DE STR MALDI-TOF mass spectrometer from Applied Biosystems (Foster City, CA). Internal calibration was done with autodigested trypsin. Data were analyzed using Moverz software (Proteometrics LLC, Winnipeg, Canada), and data base searches were done with Mascot (36Perkins D.N. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6776) Google Scholar). 2Available at www.matrixscience.com. A search of all NCBInr Database entries was performed allowing one missed cleavage for trypsin, carbamidomethylated cysteine residues, and variable modification of oxidized methionine. Peptide tolerance for monoisotopic values was set to 50 ppm. The different forms of NF1 were identified with significant scores. Western Blot Analysis—Trichloroacetic acid-precipitated samples from the DNA affinity column were subjected to 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride membrane. Membranes were incubated with antibodies against the N terminus of human NF1 (Santa Cruz Biotechnology) and developed with alkaline phosphatase-conjugated secondary antibodies as described (29Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York1994Google Scholar). Chromatin Immunoprecipitation—Chromatin immunoprecipitation of NF1 from growth-arrested and growth-induced human diploid cells was performed as described (38Weinmann A.M. Farnham P.J. Methods. 2000; 26: 37-47Crossref Scopus (301) Google Scholar), except that 100 μl of protein A-Sepharose was used instead of Staphylococcus aureus cells. Immunoprecipitation was performed with 2 μl of antiserum 8199 prepared against a central domain of the NF1 C-protein (kindly provided by Dr. Tanese). Amplification of immunoprecipitated DNA fragments (2 μl) was performed using primers –982 and –795 (5′-GTTCGACAAAGATCTTGCCAGGAATTGG-3′) for the Go-2 element and primers –726 (5′-GGTTTCACTGGCTCCAGGACAGCACAC-3′) and –499 (5′-GGGTGAGGCAAGCGAGACAAGGTCATG-3′) for the Go-1 element. PCR was performed for 32 cycles, with 30 s of denaturation at 94 °C, followed by 30 s of annealing at 60 °C and 30 s of extension at 72 °C. The last step included extension for 10 min at 72 °C. Growth Arrest of Human Diploid Cells Down-regulates ANT2 Transcripts—Growth arrest repression of the ANT2 gene (18Barath P. Luciakova K. Hodny Z. Li R. Nelson B.D. Exp. Cell Res. 1999; 248: 583-588Crossref PubMed Scopus (50) Google Scholar) was studied in vivo using human primary foreskin fibroblasts. Exponentially growing cells expressed ANT2 transcripts at high levels (Fig. 1, lanes 1 and 5), but transcripts were barely detectable in growth-arrested cells after 48 h of serum starvation (lane 2). However, ANT2 transcript levels were restored to ∼30% of the control levels after 6 h of serum induction (lane 3) and to 100% after 24 h of induction (lane 4). Mapping of Proteins Bound to the ANT2 Promoter in Growth-arrested Diploid Cells in Vivo—Proteins bound to the ANT2 promoter during growth modulation were mapped in vivo in growing and growth-arrested diploid fibroblasts by DMS modification (Fig. 2; summarized in Fig. 3). Transcription of ANT2 is maintained by two adjacent, synergistically acting Sp1 elements in the proximal promoter (26Li R. Hodny Z. Luciakova K. Barath P. Nelson B.D. J. Biol. Chem. 1996; 271: 18925-18930Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 39Zaid A. Li R. Luciakova K. Barath P. Nery S. Nelson B.D. J. Bioenerg. Biomembr. 1999; 31: 129-135Crossref PubMed Scopus (65) Google Scholar). However, growth arrest repression appears to be located in a distal 700-bp fragment of the promoter (18Barath P. Luciakova K. Hodny Z. Li R. Nelson B.D. Exp. Cell Res. 1999; 248: 583-588Crossref PubMed Scopus (50) Google Scholar). In agreement with these findings, a region of protein contact was detected in vivo within the 700-bp repressor region. In this region, protection from in vivo DMS modification was observed on C residues in growth-arrested cells (Fig. 2A, S lane) that extended over a stretch of ∼60 bp (nt –830 to –767) (Figs. 2A and 3A). More importantly, none of these C residues was protected in vivo in serum-activated (Fig. 2A, I lane) or exponentially growing (E lane) diploid cells Thus, protein binding to the repressor region is detected in diploid fibroblasts in vivo only in the growth-arrested state.Fig. 3Summary of the DMS modifications of the ANT2 promoter region in diploid cells in vivo. A, summary of data presented in Fig. 2A. Nucleotides protected from DMS modification in serum-starved cells are marked with triangles. The boldface underlined regions and asterisks indicate footprinted and hypersensitive sites, respectively, found by in vitro DNase I protection assay (see Figs. 5 and 8, respectively). The NF1 half-sites are boxed. B, summary of data presented in Fig. 2B. The transcription start site is marked by an arrow. The TATA box and the Sp1 A, B, and C elements are boxed. C residues protected under all conditions of growth are marked with triangles. A hypersensitive C residue is marked with an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Protein contact is also detected in vivo within or near the Sp1 activation elements (AB boxes) and the Sp1 repressor element (C box) in the proximal promoter (26Li R. Hodny Z. Luciakova K. Barath P. Nelson B.D. J. Biol. Chem. 1996; 271: 18925-18930Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 39Zaid A. Li R. Luciakova K. Barath P. Nery S. Nelson B.D. J. Bioenerg. Biomembr. 1999; 31: 129-135Crossref PubMed Scopus (65) Google Scholar). Strongly protected nucleotides were found within the A and B boxes under all conditions of growth (i.e. exponentially growing, serum-starved, and serum-induced cells) (Fig. 2B; summarized in Fig. 3B), suggesting that these elements are most likely permanently occupied in vivo. Similarly, C residues within and 5′ of the C box were protected, but less strongly than those in the A and B boxes. In addition, there appears to be at least one C residue between the B and TATA boxes that was protected in all cells, as well as a major hypersensitive site (Figs. 2B and 3B, asterisks). The significance of the latter is not clear. Identification of Growth Arrest DNA Elements in the ANT2 Promoter—To further define the upstream growth arrest repressor in the human ANT2 promoter, stable transfectants were made with 5′-deletion fragments of the promoter (Fig. 4). Since stable transfection of diploid fibroblasts is hampered by their limited life spans, NIH3T3 cells were used. As shown in Fig. 4 (open circles), deletion of a 112-bp fragment between nt –804 and –692 abolished repression of luciferase activity in confluent cells. This is the region in which protein binding was observed in growth-arrested diploid cells in vivo (see above). For convenience, we refer to this extended 112-bp region as the Go repressor (GoR) region. To identify proteins that bind the 112-bp GoR region, an overlapping fragment of the promoter (nt –917 to –654) was mapped in vitro with DNase I using nuclear extracts from human diploid fibroblasts and NIH3T3 cells. A 28-bp region (nt –822 to –794) was strongly protected by nuclear extracts from both cell types in the growth-arrested state (Fig. 5). This footprint, referred to as a Go-2 element, overlaps the 5′-end of the GoR region defined by deletion constructs (Fig. 4) and is part of an extended region that includes a second DNA element, Go-1 (see Figs. 7 and 9 below). DNase I protection of the Go-2 element was interrupted in both cell types by a hypersensitive site (Fig. 5, asterisks), suggesting that the same or a similar protein is bound. In agreement with the in vivo DMS mapping experiments (see above), nuclear extracts from serum-activated human diploid cells did not footprint the –822/–794 Go-2 element (Fig. 5, left panel, compare S and I lanes). Thus, either the amount or the binding ability of the DNA-binding protein is modulated by the growth state of the diploid cells. By contrast, nuclear extracts from serum-activated NIH3T3 cells protected the –822/–794 element from DNase I (Fig. 5, right panel, compare S and I lanes). Although it is not clear why DNA binding is retained in nuclear extracts from" @default.
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• W1966642608 title "Repression of the Human Adenine Nucleotide Translocase-2 Gene in Growth-arrested Human Diploid Cells" @default.
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