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• W1597493603 abstract "Transcription from the human asparagine synthetase (AS) gene is increased in response to either amino acid (amino acid response) or glucose (unfolded protein response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the AS promoter, which are referred to as nutrient-sensing response element (NSRE)-1 and -2, both of which are absolutely necessary for gene activation. The NSRE-1 sequence was used to identify the corresponding transcription factor by yeast one-hybrid screening. Based on those results, electrophoretic mobility shift assays for individual CCAAT/enhancer-binding protein-β (C/EBP) family members were performed to test for supershifting of complexes by specific antibodies. The results indicated that of all the family members, C/EBPβ bound to the NSRE-1 sequence to the greatest extent and that the absolute amount of this complex was increased when extracts from amino acid- or glucose-deprived cells were tested. Using electrophoretic mobility shift assays, mutation of the NSRE-1 sequence completely prevented formation of the C/EBPβ-containing complexes. In contrast, mutation of the NSRE-2 sequence did not block C/EBPβ binding. Overexpression in HepG2 hepatoma cells of the activating isoform of C/EBPβ increased AS promoter-driven transcription, whereas the inhibitory dominant-negative isoform of C/EBPβ blocked enhanced transcription following amino acid or glucose deprivation. Collectively, the results provide both in vitro and in vivo evidence for a role of C/EBPβ in the transcriptional activation of the AS gene in response to nutrient deprivation. Transcription from the human asparagine synthetase (AS) gene is increased in response to either amino acid (amino acid response) or glucose (unfolded protein response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the AS promoter, which are referred to as nutrient-sensing response element (NSRE)-1 and -2, both of which are absolutely necessary for gene activation. The NSRE-1 sequence was used to identify the corresponding transcription factor by yeast one-hybrid screening. Based on those results, electrophoretic mobility shift assays for individual CCAAT/enhancer-binding protein-β (C/EBP) family members were performed to test for supershifting of complexes by specific antibodies. The results indicated that of all the family members, C/EBPβ bound to the NSRE-1 sequence to the greatest extent and that the absolute amount of this complex was increased when extracts from amino acid- or glucose-deprived cells were tested. Using electrophoretic mobility shift assays, mutation of the NSRE-1 sequence completely prevented formation of the C/EBPβ-containing complexes. In contrast, mutation of the NSRE-2 sequence did not block C/EBPβ binding. Overexpression in HepG2 hepatoma cells of the activating isoform of C/EBPβ increased AS promoter-driven transcription, whereas the inhibitory dominant-negative isoform of C/EBPβ blocked enhanced transcription following amino acid or glucose deprivation. Collectively, the results provide both in vitro and in vivo evidence for a role of C/EBPβ in the transcriptional activation of the AS gene in response to nutrient deprivation. asparagine synthetase nucleotide(s) amino acid response element electrophoretic mobility shift assay unfolded protein response nutrient-sensing response element CCAAT/enhancer-binding protein C/EBP homologyprotein amino acid response liver-enriched activating protein liver-enriched inhibitory protein minimal essential medium activating transcription factor general control non-depressible Amino acid availability is clearly an important factor in general nutrition, particularly during development (1Morgane P.J. Austinlafrance R. Bronzino J. Tonkiss J. Diazcintra S. Cintra L. Kemper T. Galler J.R. Neurosci. Biobehav. Rev. 1993; 17: 91-128Crossref PubMed Scopus (530) Google Scholar), as well as in the progression of a wide range of diseases, including diabetes (2Hoffer L.J. Can. J. Physiol. Pharmacol. 1993; 71: 633-638Crossref PubMed Scopus (9) Google Scholar), kwashiorkor (3Roediger W.E.W. J. Pediatr. Gastroenterol. Nutr. 1995; 21: 130-136Crossref PubMed Scopus (42) Google Scholar), and hepatic encephalopathy (4Mizock B.A. Nutrition. 1999; 15: 220-228Crossref PubMed Scopus (34) Google Scholar), and in cancer chemotherapy. With regard to the latter, asparagine starvation, induced by administration of the enzyme l-asparaginase, is included in all accepted protocols for the treatment of childhood acute lymphoblastic leukemia (5Amylon M.D. Shuster J. Pullen J. Berard C. Link M.P. Wharam M. Katz J., Yu, A. Laver J. Ravindranath Y. Kurtzberg J. Desai S. Camitta B. Murphy S.B. Leukemia (Baltimore). 1999; 13: 335-342Crossref PubMed Scopus (272) Google Scholar). However, the impact of amino acid availability on the control of fundamental cellular processes is just beginning to be investigated extensively (reviewed in Refs. 6Jousse C. Bruhat A. Fafournoux P. Curr. Opin. Clin. Nutr. Metab. Care. 1999; 2: 297-301Crossref PubMed Scopus (20) Google Scholar and 7Fafournoux P. Bruhat A. Jousse C. Biochem. J. 2000; 351: 1-12Crossref PubMed Scopus (225) Google Scholar). A number of laboratories have identified specific mRNAs, proteins, and cellular activities that are induced following amino acid deprivation (reviewed in Refs. 7Fafournoux P. Bruhat A. Jousse C. Biochem. J. 2000; 351: 1-12Crossref PubMed Scopus (225) Google Scholar and 8Laine R.O. Hutson R.G. Kilberg M.S. Prog. Nucleic Acid Res. Mol. Biol. 1996; 53: 219-248Crossref PubMed Scopus (29) Google Scholar). An example is asparagine synthetase (AS),1 which catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine. Nearly 2 decades ago, Arfin et al. (9Arfin S.M. Simpson D.R. Chiang C.S. Andrulis I.L. Hatfield G.W. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2367-2369Crossref PubMed Scopus (32) Google Scholar, 10Andrulis I.L. Hatfield G.W. Arfin S.M. J. Biol. Chem. 1979; 254: 10629-10633Abstract Full Text PDF PubMed Google Scholar) showed that asparagine starvation of Chinese hamster ovary cells decreases the level of asparaginyl-tRNAAsn, and subsequently, AS enzymatic activity increases; refeeding these cells asparagine rapidly reverses this response. Induction of the AS activity also occurs in temperature-sensitive Chinese hamster ovary cell mutants for asparaginyl-, leucyl-, methionyl-, and lysyl-tRNA synthetases (10Andrulis I.L. Hatfield G.W. Arfin S.M. J. Biol. Chem. 1979; 254: 10629-10633Abstract Full Text PDF PubMed Google Scholar). Consistent with these data, starvation of HeLa or HepG2 hepatoma cells for any one of a number of individual amino acids causes the accumulation of AS mRNA, documenting that this nutrient-sensing mechanism broadly detects amino acid availability (11Gong S.S. Guerrini L. Basilico C. Mol. Cell. Biol. 1991; 11: 6059-6066Crossref PubMed Scopus (80) Google Scholar, 12Hutson R.G. Kilberg M.S. Biochem. J. 1994; 303: 745-750Crossref Scopus (64) Google Scholar, 13Jousse C. Bruhat A. Ferrara M. Fafournoux P. J. Nutr. 2000; 130: 1555-1560Crossref PubMed Scopus (48) Google Scholar). Guerrini et al. (14Guerrini L. Gong S.S. Mangasarian K. Basilico C. Mol. Cell. Biol. 1993; 13: 3202-3212Crossref PubMed Scopus (87) Google Scholar) were the first to document the presence of a genomic element, within the human AS promoter, that mediates amino acid-dependent regulation of transcription. Using deletion analysis and scanning mutagenesis in conjunction with transient transfection, these authors identified a region from nt −70 to −62 that functions as an amino acid response element (AARE). Electrophoretic mobility shift assays (EMSAs) using oligonucleotides containing this sequence documented the formation of specific protein-DNA complexes in vitro (14Guerrini L. Gong S.S. Mangasarian K. Basilico C. Mol. Cell. Biol. 1993; 13: 3202-3212Crossref PubMed Scopus (87) Google Scholar). The sequence within this region does not correspond precisely to a consensus sequence for any known transcription factors. Subsequently, Barbosa-Tessmann et al. (15Barbosa-Tessmann I.P. Pineda V.L. Nick H.S. Schuster S.M. Kilberg M.S. Biochem. J. 1999; 339: 151-158Crossref PubMed Scopus (38) Google Scholar, 16Barbosa-Tessmann I.P. Chen C. Zhong C. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 1999; 274: 31139-31144Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 17Barbosa-Tessmann I.P. Chen C. Zhong C. Siu F. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 2000; 275: 26976-26985Abstract Full Text Full Text PDF PubMed Google Scholar) demonstrated that the human AS gene is also induced by glucose deprivation and that this process is mediated by the unfolded protein response (UPR) pathway. Through the use of in vivo footprinting and single-nucleotide mutagenesis, it was demonstrated that the promoter sequence 5′-TGATGAAAC-3′ (nt −68 to −60), the region first identified by Guerrini et al. (14Guerrini L. Gong S.S. Mangasarian K. Basilico C. Mol. Cell. Biol. 1993; 13: 3202-3212Crossref PubMed Scopus (87) Google Scholar), is also responsible for induction of the AS gene following activation of the UPR pathway (17Barbosa-Tessmann I.P. Chen C. Zhong C. Siu F. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 2000; 275: 26976-26985Abstract Full Text Full Text PDF PubMed Google Scholar). UPR pathway activation demonstrates that this sequence serves in a broader capacity than simply as an AARE; and, to reflect this broader substrate-detecting capability, this sequence has been labeled nutrient-sensing response element (NSRE)-1. A second element (5′-GTTACA-3′, nt −48 to −43), 11 nucleotides downstream, is also absolutely required for activation of the gene by the nutrient-sensing response pathway (17Barbosa-Tessmann I.P. Chen C. Zhong C. Siu F. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 2000; 275: 26976-26985Abstract Full Text Full Text PDF PubMed Google Scholar) and is referred to as NSRE-2. The term nutrient-sensing response unit has been coined to describe the collective function of these sequences. The C/EBP homology protein (CHOP) is a member of the C/EBP family (18Ron D. Habener J.F. Gene. Dev. 1992; 6: 439-453Crossref PubMed Scopus (968) Google Scholar), and Bruhat et al. (19Bruhat A. Jousse C. Wang X.-Z. Ron D. Ferrara M. Fafournoux P. J. Biol. Chem. 1997; 272: 17588-17593Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,20Bruhat A. Jousse C. Carraro V. Reimold A.M. Ferrara M. Fafournoux P. Mol. Cell. Biol. 2000; 20: 7192-7204Crossref PubMed Scopus (165) Google Scholar) have identified, within the promoter of the humanCHOP gene, a sequence (5′-TGATGCAAT-3′, nt −302, order to −310) that functions as an AARE and differs by only two nucleotides from the NSRE-1 sequence in the AS promoter. Although transcription from the CHOP gene is also induced by the UPR pathway, an NSRE-2 sequence is not present; and, unlike the AS gene, the CHOP promoter has a separate sequence (5′-CCAATCAGAAAGTGGCACG-3′, nt −75 to −93) that mediates the activation by the UPR pathway (21Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Mol. Cell. Biol. 2000; 20: 6755-6767Crossref PubMed Scopus (770) Google Scholar). Therefore, the mechanisms for activation of these two genes by the amino acid response (AAR) and UPR pathways are distinct. Consistent with this proposal, Jousse et al. (13Jousse C. Bruhat A. Ferrara M. Fafournoux P. J. Nutr. 2000; 130: 1555-1560Crossref PubMed Scopus (48) Google Scholar) have documented that transcriptional control of a reporter gene driven by the AS and CHOP promoters responds differently when cells are deprived of selected individual amino acids. This study was designed to identify transcription proteins capable of binding to NSRE-1 of the AS promoter and to establish their role with regard to induction of the gene by the AAR and UPR pathways. Using the NSRE-1 sequence as bait, yeast one-hybrid screening indicated that members of the C/EBP family bind this element. Among the members of the C/EBP family, antibodies specific for C/EBPβ caused the greatest amount of a supershifted complex, and the abundance of this complex was increased when extracts from amino acid- or glucose-deprived cells were used. Mutation of individual nucleotides within the NSRE-1 sequence known to be critical for regulated transcription (16Barbosa-Tessmann I.P. Chen C. Zhong C. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 1999; 274: 31139-31144Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 17Barbosa-Tessmann I.P. Chen C. Zhong C. Siu F. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 2000; 275: 26976-26985Abstract Full Text Full Text PDF PubMed Google Scholar) caused loss of C/EBPβ binding. Expression of the activating isoform (liver-enriched activating protein (LAP)) of C/EBPβ in HepG2 cells caused increased transcription from anAS promoter/luciferase reporter construct, whereas expression of the dominant-negative C/EBPβ isoform (liver-enriched inhibitory protein (LIP)) blocked transcriptional induction following activation of either the AAR or UPR pathway. Collectively, the results demonstrate that C/EBPβ binds in vitro to NSRE-1 within the human AS promoter and is able to regulate AS promoter-driven transcription in vivo. Human HepG2 hepatoma cells were cultured in minimal essential medium (MEM), pH 7.4, supplemented with 25 mm NaHCO3, 4 mm glutamine, 10 μg/ml streptomycin sulfate, 100 μg/ml penicillin G, 28.4 μg/ml gentamycin, 0.023 μg/mlN-butyl-p-hydroxybenzoate, 0.2% (w/v) bovine serum albumin, and 10% (v/v) fetal bovine serum. Cells were maintained at 37 °C in a 5% CO2 and 95% air incubator. HepG2 cells were cotransfected with a reporter plasmid composed of firefly luciferase (pGL3 vector, Promega, Madison, WI) driven by humanAS promoter sequence −115 to +1 and, where indicated, the pSCT vector containing either the activating (LAP) or dominant-negative (LIP) isoform of rat C/EBPβ, originally described by Descombes and Schibler (22Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (855) Google Scholar) and kindly provided by Dr. Harry S. Nick (University of Florida). Transfection efficiency was monitored by cotransfection with the Renilla luciferase reporter (phRL-SV40) driven by the SV40 promoter (Promega). The cells were transfected to 50–70% confluence in 24-well plates using the batch transfection technique described by Barbosa-Tessmann et al. (16Barbosa-Tessmann I.P. Chen C. Zhong C. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 1999; 274: 31139-31144Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). After transfection and a subsequent 24-h incubation in MEM, cells from each well were divided into multiple wells of a new 24-well plate and cultured for another 24 h prior to incubation for 12 h in complete MEM, histidine-free MEM, or glucose-free MEM, each supplemented with 10% dialyzed fetal bovine serum (15Barbosa-Tessmann I.P. Pineda V.L. Nick H.S. Schuster S.M. Kilberg M.S. Biochem. J. 1999; 339: 151-158Crossref PubMed Scopus (38) Google Scholar, 16Barbosa-Tessmann I.P. Chen C. Zhong C. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 1999; 274: 31139-31144Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In this way, cells incubated in the three different nutrient media arose from the same transfection. The firefly and Renilla luciferase activities were assayed by the dual-luciferase reporter system (Promega) according to the manufacturer’s directions. The data are expressed as the means ± S.D. of three to four assays, and each experiment was repeated with multiple batches of cells. Total cellular RNA was isolated using an RNeasy minikit (QIAGEN Inc., Valencia, CA) according to the procedure described by the supplier.32P-Radiolabeled cDNA probe synthesis and Northern analysis were performed as described by Aslanian et al. (23Aslanian A.M. Fletcher B.S. Kilberg M.S. Biochem. J. 2001; 357: 321-328Crossref PubMed Scopus (133) Google Scholar). The probe for C/EBPβ was the rat LIP isoform cDNA, which detected both transfected isoforms, as well as endogenous human C/EBPβ. A yeast one-hybrid screening system for the identification of DNA-binding proteins was purchased from CLONTECH (Palo Alto, CA). Three tandem copies of the NSRE-1 sequence (5′-GCAGGCATGATGAAACTTC-3′) were cloned in front of the minimal promoter within the two reporter vectors pHisi-1 and pLacZi and then used to produce the corresponding stably expressing yeast strains. These two cell lines permit a dual selection using HIS3 complementation for cell growth and β-galactosidase activity as a secondary screen for elimination of false positives. Following selection for stable transformants, the yeast strains were transformed with a human pancreatic cDNA library fused to the yeast Gal4 activation domain. Initial positives were isolated by growth on histidine-free medium containing 45 mm 3-amino-1,2,4-triazole to reduce the background expression of HIS3 in the reporter strain. Colonies that grew in the absence of histidine were used to isolate specific clones, and then each of these clones was tested for activation of the pLacZi reporter gene by back-transformation of the appropriate yeast strain, followed by a β-galactosidase filter lift assay. All procedures were performed according to the manufacturer’s recommendations. Nuclear extracts were prepared from HepG2 cells incubated for 16 h in complete MEM (control) or in MEM lacking glucose or histidine. Nuclear extracts were prepared from four 150-mm dishes of cells, and all steps were performed at 4 °C. The cells were rinsed with ice-cold phosphate-buffered saline (10 mm sodium phosphate and 0.9% sodium chloride, pH 7.5), collected by scraping in 5 ml of phosphate-buffered saline/dish, and then pelleted by centrifugation at 500 ×g for 10 min. After resuspension in phosphate-buffered saline, the cells were collected by centrifugation at 1160 ×g for 5 min. To the pellet were added 600 μl of TM-2 buffer (10 mm Tris-HCl, 2 mm magnesium chloride, and 0.5 mm phenylmethylsulfonyl fluoride, pH 7.4) and 4 μl of a protease inhibitor mixture stock solution (2 μg each of aprotinin, leupeptin, pepstatin, N-tosylphenylalanine chloromethyl ketone, and N-tosyllysine chloromethyl ketone); Triton X-100 was added to a final concentration of 0.5%; and the mixture was incubated for an additional 5 min. The cells were lysed by gently passing through a 22-gauge needle three times and then centrifuged for 10 min at 130 × g. The supernatant was carefully removed and discarded, and then 300 μl of TM-2 buffer was added to the lower layer (nuclei) and gently mixed. The mixture was centrifuged for 10 min at 130 × g, and the upper supernatant layer was discarded as before. To the lower layer containing the nuclei were added 800 μl of nuclear extraction buffer (20 mm HEPES, 20% (v/v) glycerol, 400 mmsodium chloride, 1.5 mm magnesium chloride, 1 mm dithiothreitol, 0.2 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride, pH 7.6) and 4 μl of the protease inhibitor mixture. The mixture was gently mixed by constant rotation for 1 h at 4 °C, and the supernatant was collected after centrifugation at 18,000 × g for 10 min. This mixture was dialyzed against dialysis buffer (20 mm HEPES, 20% (v/v) glycerol, 100 mm potassium chloride, 10 mm magnesium chloride, 1 mm dithiothreitol, 0.2 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride, pH 7.8) overnight at 4 °C, quickly frozen in liquid nitrogen, and then stored at −70 °C until used. Double-stranded oligonucleotide probes were32P-radiolabeled by extension of overlapping ends with Klenow fragment in the presence of [α-32P]dCTP (24Kerrigan L.A. Kadonaga J.T. Ausubel F.M. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1994Google Scholar). An aliquot of nuclear extract (5 μg of protein) was preincubated for 20 min at 4 °C in a total volume of 20 μl containing 40 mm Tris, pH 7.5, 200 mm NaCl, 2 mmdithioerythritol, 10% glycerol, 0.05% Nonidet P-40, 2 μg of poly(dI-dC), and 0.05 mm EDTA. Then 0.004 pmol of32P-radiolabeled probe (∼10,000 dpm) and, where indicated, unlabeled competitor oligonucleotides were added, and the incubation was continued for 20 min at room temperature. If antibody binding was to be tested, 4 μg (2 μl) of the indicated polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added, and an additional incubation for 20 min at room temperature was included. After electrophoresis for 2–3 h at 200V on a 5% polyacrylamide gel with a 15% cushion on the bottom 2 cm, the gel was dried, and the results were visualized by autoradiography. All experiments were repeated with at least two independently prepared nuclear extracts. To identify the transcription proteins that bound the NSRE-1 sequence, yeast one-hybrid screening was performed using three copies of the AS promoter NSRE-1 sequence placed in front of two independent reporter systems (HIS3 and LacZ) and a human pancreatic cDNA library fused to the Gal4 activation domain. A total of 2.51 × 106 colonies were screened for growth on histidine-free plates, and seven colonies were judged to be potential positives based on growth rate. DNA from each of the seven positives was isolated and used to perform a back-transformation. The positive colonies were then subjected to a β-galactosidase filter lift assay as a secondary screen. Three clones contained β-galactosidase activity that was much greater than the background level. These three positives were used to perform a back-transformation and confirmed that complementation of histidineindependent growth occurred. When these clones were sequenced, they were shown to have identity to the C/EBP family of transcription factors. EMSA was performed to determine which of the C/EBP family members were present in human HepG2 hepatoma cells and which had affinity for the NSRE-1 sequence. The radiolabeled oligonucleotide probe (oligonucleotide-1) corresponded to nt −79 to −53 within the human AS proximal promoter and therefore contained the NSRE-1 sequence (nt −68 to −60) (Fig.1). Incubation of the NSRE-1 probe with nuclear extract from cells cultured in complete MEM resulted in the formation of three primary complexes (denoted A–C in Fig.2), two of which were detected as faint broad bands, which are often difficult to detect in extracts from cells maintained in complete medium. Inclusion of an excess of the unlabeled NSRE-1 oligonucleotide completely eliminated formation of these three complexes, but also revealed the presence of a nonspecific complex as well (Fig. 2, lane 2). Formation of complexes A and C was blocked by the presence of an excess of an unlabeled oligonucleotide containing a C/EBP consensus sequence, but complex B remained (Fig. 2,lane 3). When extracts from cells cultured in the absence of histidine for 16 h were assayed, the amount of each of the specific complexes increased significantly, but the content of complex A was enhanced to the greatest extent (Fig. 2, compare lanes 4 and 10). To test for the binding of individual C/EBP family members, antibodies against C/EBPα, C/EBPβ, C/EBPδ, C/EBPε, and CHOP were screened for the ability to supershift these complexes. Antibodies specific for C/EBPα, C/EBPδ, C/EBPε, and CHOP each caused formation of one or more supershifted complexes (denoted by asterisks in Fig. 2), but the absolute amount of these complexes was relatively small in both fed and histidine-deprived cells. In contrast, the anti-C/EBPβ antibody caused the formation of two shifted complexes that were substantially increased when nuclear extracts from histidine-limited cells were tested (Fig. 2, comparelanes 6 and 12). It appeared that the primary complex shifted by the anti-C/EBPβ antibody was complex C (Fig. 2, compare lanes 4 and 6 or lanes 10 and 12). To show that the C/EBPβ-induced supershift was not cell-specific, experiments were performed using nuclear extracts from human embryonic kidney 293 cells, and similar results were obtained (data not shown).Figure 2EMSAs with the human AS promoter NSRE-1 sequence. Nuclear extracts prepared from HepG2 cells maintained for 16 h in complete MEM (MEM) or histidine-free MEM (−His) were incubated with32P-radiolabeled oligonucleotide-1 (see Fig. 1). Lane 1 illustrates the effect of including an unrelated sequence as an unlabeled, nonspecific (NS) competitor at a 100-fold excess.Lane 2 represents a 100-fold excess of unlabeled oligonucleotide-1 (Sp), and lane 3 illustrates the effect of a 100-fold excess of the unlabeled C/EBP consensus sequence (C/E; 5′-TGCAGATTGCGCAATCTGCA-3′). Where indicated, the incubation protocol included no antibody (control (Con)) or an antibody specific for one of the C/EBP family members, as shown above the lanes. CH, CHOP. Arrows A–C denote specific complexes that were increased in amount when extracts from histidine-deprived cells were tested, and the asterisks to the left of a lane indicate supershifted complexes. The autoradiographic film shown is representative of at least two separate experiments using independently prepared nuclear extracts.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Addition of increasing amounts of unlabeled NSRE-1 oligonucleotide from 5- to 200-fold demonstrated a concentration-specific competition for the formation of C/EBPβ complexes A–C (Fig.3, lanes 3–7). Using a 5–200-fold excess of the C/EBP-binding consensus sequence illustrated that complex C was blocked most effectively by the C/EBP sequence, followed by complex B. Complex A formation was the least affected, and a significant amount of the complex remained in the presence of a 200-fold excess of the C/EBP sequence (Fig. 3, lanes 8–12). Including the anti-C/EBPβ antibody illustrated that a 100-fold excess of either the NSRE-1 or C/EBP consensus sequence as an unlabeled competitor prevented formation of the supershifted complex (Fig. 3,lanes 13–15). Bruhat et al. (20Bruhat A. Jousse C. Carraro V. Reimold A.M. Ferrara M. Fafournoux P. Mol. Cell. Biol. 2000; 20: 7192-7204Crossref PubMed Scopus (165) Google Scholar) have identified ATF-2 as a protein required for amino acid starvation-dependent induction of the CHOP gene. As mentioned above, the element within the CHOP promoter that is responsible for this regulation is similar to the NSRE-1 sequence in the AS promoter, but differs by two nucleotides. To determine whether ATF-2 bound to the AS NSRE-1 sequence in vitro, EMSAs were performed in conjunction with supershift assays using an antibody specific for ATF-2 (Figs. 4 and5). Although the protein-DNA complexes that are induced following histidine (Fig. 4) or glucose (Fig. 5) deprivation were clearly present, no shifted complexes were detectable after incubation with the anti-ATF-2 antibody with nuclear extracts from cells maintained in MEM (Fig. 4, compare lanes 3 and 4), histidine-deprived cells (Fig. 4, compare lanes 8 and 9), or glucose-deprived cells (Fig. 5, lane 6). Within the AS NSRE-1 sequence, there is a half-site (5′-ATGA-3′) for the c-Jun transcription factor, which can dimerize with c-Fos to form the activator protein-1 complex and can also associate with C/EBPβ (25Hsu W. Kerppola T.K. Chen P.L. Curran T. Chen-Kiang S. Mol. Cell. Biol. 1994; 14: 268-276Crossref PubMed Scopus (181) Google Scholar). To investigate the possible binding of c-Fos or c-Jun to the NSRE-1 sequence, EMSAs were performed with or without antibodies specific for these two transcription proteins. No evidence could be obtained to suggest that either c-Fos or c-Jun was bound to the NSRE-1 sequence in either fed cells (Fig. 4, lanes 5–7) or histidine-deprived cells (lanes 10–12). These results are consistent with data published by Guerrini et al. (14Guerrini L. Gong S.S. Mangasarian K. Basilico C. Mol. Cell. Biol. 1993; 13: 3202-3212Crossref PubMed Scopus (87) Google Scholar), who also concluded that this regulatory sequence within the AS promoter does not bind activator protein-1 complexes.Figure 5Formation of C/EBP β-containing complexes at the NSRE-1 sequence following activation of the UPR pathway by glucose deprivation. Nuclear extracts prepared from HepG2 cells maintained for 16 h in MEM lacking glucose (−Glc) were incubated with 32P-radiolabeled oligonucleotide-1 (see Fig. 1) to monitor formation of protein-DNA complexes. Lane 1 shows the complexes that remained if a 100-fold excess of an unlabeled, nonspecific (NS) sequence was added, and lane 2 illustrates the competition of the specific complexes by a 100-fold excess of unlabeled oligonucleotide-1 (Sp) itself. As indicated above lanes 3–12, the additional remaining incubations included no antibody (control (Con)) or an antibody specific for C/EBPβ (β), CHOP (CH), or ATF-2. Arrows A–C denote complexes that were increased in amount when extracts from glucose-deprived cells were tested. The data shown are representative of several experiments using independently prepared nuclear extracts.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The NSRE-1 sequence within the AS promoter differs functionally from the closely related AARE sequence within theCHOP gene because it is also required for activation of the gene by the UPR pathway (17Barbosa-Tessmann I.P. Chen C. Zhong C. Siu F. Schuster S.M. Nick H.S. Kilberg M.S. J. Biol. Chem. 2000; 275: 26976-26985Abstract Full Text Full Text PDF PubMed Google Scholar). To determine whether the abundance of the C/EBPβ-containing NSRE-1 co" @default.
• W1597493603 created "2016-06-24" @default.
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• W1597493603 creator A5056480265 @default.
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• W1597493603 date "2001-12-01" @default.
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• W1597493603 title "CCAAT/Enhancer-binding Protein-β Is a Mediator of the Nutrient-sensing Response Pathway That Activates the Human Asparagine Synthetase Gene" @default.
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