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J. Biol. Chem., Vol. 277, Issue 19, 16585-16591, May 10, 2002

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Role of Sp1 and Sp3 in the Nutrient-regulated Expression of the Human Asparagine Synthetase Gene^*

Van Leung-Pineda and Michael S. Kilberg

From the Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610

Received for publication, November 15, 2001, and in revised form, February 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The human asparagine synthetase (AS) gene responds to depletion of mammalian cells for either amino acids or carbohydrates. Five specific cis-elements have been implicated: three GC boxes (GC-I, GC-II and GC-III) and two nutrient-sensing response elements (NSRE-1, -2). This study shows that all three GC boxes are required to maintain basal transcription and to obtain maximal induction of the AS gene by amino acid limitation. However, there is not complete redundancy among the three GC boxes, and there is a hierarchy of importance with regard to transcription (GC-III > GC-II > GC-I). In vitro, two GC boxes formed protein-DNA complexes (GC-II and GC-III) with Sp1 and Sp3. Although transcription of the AS gene is elevated by nutrient limitation, the absolute amount of these protein-DNA complexes and the total pools of Sp1 and Sp3 did not increase. A small, but detectable portion of Sp1 was modified by phosphorylation following amino acid deprivation. In vivo, expression of Sp1 and Sp3 in Drosophila SL2 cells increased AS promoter activity. Sp1 expression increased basal transcription but did not cause a further increase when SL2 cells were amino acid-deprived. Sp3 expression enhanced both the basal and the starvation-induced transcription.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Metabolite control of gene transcription in mammalian cells is an important factor in regulating protein expression in response to nutrient changes in the environment. An example of a gene that is highly regulated by the nutritional status of the cell is asparagine synthetase (AS),¹ which encodes the enzyme that produces asparagine from glutamine, ATP, and aspartic acid (1). AS expression is induced following depletion of the extracellular medium for amino acids (2-5), which activates the amino acid response (AAR) pathway (reviewed in Refs. 6 and 7), or for glucose (4, 5, 8), which activates the ER stress response pathway, also known in yeast as the unfolded protein response pathway (9-11). Typically, these two independent pathways enhance transcription of their respective target genes through completely different cis-acting elements (7, 12). However, AS is the first gene identified that is activated by the AAR and the ER stress response pathway through the same cis-elements, termed nutrient-sensing response elements-1,2 (NSRE-1, NSRE-2). The first analysis of amino acid-dependent control of the human AS gene was triggered by the observation that a mutation in AS led to a block in G₁ phase of the cell cycle (13). Those studies led Guerrini et al. (14) to identify an amino acid responsive sequence in the proximal promoter, which corresponds to the NSRE-1 site shown by Barbosa-Tessman et al. (4, 5) to mediate both amino acid and glucose effects. Guerrini et al. (14) also noted two GC boxes and postulated, based on sequence only, that they may be binding sites for the transcription factor Sp1. Those authors showed that when the GC boxes were deleted, the rate for basal transcription was reduced.

More recently, in vivo footprinting analysis by Barbosa- Tessmann et al. (5) identified six putative protein-binding sites in the AS proximal promoter region, five of which are thought to be important for metabolite control. Three of these sites are GC-rich sequences, referred to as GC-I (nt 148 to 139), GC-II (nt 128 to 119), and GC-III (nt 107 to 97). The other two sites, NSRE-1 and NSRE-2, are responsible for increasing transcription following amino acid or glucose limitation (4, 5). Promoter deletion analysis by Barbosa-Tessmann et al. (5) showed that after mutating either of the NSRE both basal and induced transcription were significantly reduced. Those authors also showed that if all three GC boxes were deleted, both basal and nutrient-regulated transcription were reduced significantly (5). Deleting both GC-I and GC-II, while retaining GC-III, reduced basal transcription, but the induction following amino acid or glucose deprivation still occurred. However, when GC-III was mutated in the presence of functional copies of both GC-I and GC-II, basal and regulated transcriptional roles were unaffected, suggesting possible redundancy between the three GC boxes.

Given the GC-rich sequence of sites I-III, occupation by one of the Sp family of proteins is possible, if not probable. Sp1 was the first protein identified (15) in a family of proteins that recognizes a similar binding sequence and is involved in regulating the transcription of many genes (reviewed in Refs. 16, 17). In recent years, other members of the family have been identified. Sp2 has been reported to bind a GT-rich box instead of a GC-rich sequence, whereas Sp3 and Sp4 are known to bind with similar affinity to a sequence that can also be recognized by Sp1.

This report shows that each of the three GC boxes within the human AS proximal promoter are required to maintain the highest basal transcription rates and that they act as modulators to allow maximal activation via the NSRE sites following nutrient deprivation. There is a hierarchy in the relative contribution of the GC boxes (GC-III > GC-II > GC-I), indicating that their relative importance is not completely redundant. As a minimum, at least one functional GC box is needed for maximal induction of AS transcription by amino acid deprivation. Two of the GC boxes were able to form protein-DNA complexes in vitro with either Sp1 or Sp3. In amino acid-limited cells, the absolute amounts of Sp1 and Sp3 were unchanged, but a portion of the Sp1 protein was phosphorylated. In vivo, when Sp1 was expressed in an Sp-negative background, it increased the basal transcriptional activity of the AS promoter but did not increase the transcriptional rate following amino acid deprivation. In contrast, Sp3 was capable of enhancing both the basal and the starvation-induced activation of the AS promoter.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture-- Human HepG2 hepatoma cells and Drosophila SL2 cells were obtained from American Type Culture Collection (Manassas, VA). HepG2 cells were maintained in minimal essential medium (MEM), pH 7.4, and supplemented with 25 mM NaHCO₃, 2 mM glutamine, 10 µg/ml streptomycin sulfate, 100 µg/ml penicillin G, 28.4 µg/ml gentamicin, 0.023 µg/ml N-butyl-p-hydroxybenzoate, 0.2% (w/v) bovine serum albumin, and 10% (v/v) fetal bovine serum. The cells were grown at 37 °C in a 5% CO₂, 95% air incubator. Drosophila SL2 cells were maintained in Schneider's insect medium supplemented with 100 mg/liter streptomycin sulfate, 62.8 mg/liter penicillin G, and 10% (v/v) fetal bovine serum. SL2 cells were grown at 25 °C without CO₂.

Mutagenesis and Transient Transfection-- The reporter plasmid utilized to assay the transcriptional activity of the AS promoter by Northern analysis contained nucleotides 173 to +51 of the human AS promoter linked to the human growth hormone (GH) gene (AS^173/+51/GH) (4, 5). Reporter constructs defective in one or more of the GC boxes were created by mutating the GC-rich sites within the AS^173/+51/GH plasmid using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's directions. The mutations for each site are shown in Table I.

For HepG2 cells, a batch transfection protocol was performed (4). The cells were seeded on 100-mm dishes (6.6 × 10⁶ cells) 24 h before transfection. Transfection was performed with Superfect reagent (Qiagen, Valencia, CA) at a ratio of 60 µl of Superfect to 10 µg of DNA. For each transfection, 10 µg of the AS^173/+51/GH reporter plasmid was used along with 10 µg of the co-transfection control plasmid, which was the pcDNA3.1 vector containing lacZ driven by the cytomegalovirus promoter. Transfection was performed as described previously (4), and after 24 h transfected cells from each 100-mm dish were divided by passage into multiple 60-mm dishes and cultured for another 24 h before treatment. The cells were then transferred to either fresh complete MEM or histidine-free MEM for 18 h. Both media were supplemented with 10% dialyzed fetal bovine serum. Using this batch transfection protocol, cells exposed to the two different media conditions arose from the same initial transfection. At least three independent transfections were performed for each experiment.

Drosophila SL2 cells were transfected following a protocol similar to the one used by Whetstine and Matherly (18). The SL2 cells were seeded (9.2 × 10⁵) on 60-mm dishes and transfected using FuGENE 6 reagent according to the manufacturer's instructions (Roche Molecular Diagnostics). A firefly luciferase reporter construct was made by inserting the AS promoter region 173/+51 into the HindIII site of the pGL3 plasmid (AS^173/+51/Luc). Expression plasmids for Sp1 (pPacSp1), full-length Sp3 (pPacUSp3), and truncated Sp3 (pPacSp3) isoforms were obtained thanks to the generosity of Dr. Guntram Suske (Marburg, Germany). The control pPac0 plasmid, having no cDNA insert, was constructed by removing the Sp1 cDNA from the pPacSp1 plasmid using XhoI. The expression of each transcription factor is driven by the presence of the Drosophila actin promoter (19). The cells were incubated with 2 µg of the AS/luciferase reporter plasmid and 400 ng of the appropriate Sp expression vector. Approximately 30 h after transfection, the cells in each 60-mm dish were divided into two wells of a 6-well dish and then incubated for 18 h in either fresh Schneider's medium alone or Schneider's medium containing 1 unit/ml of asparaginase to deplete both asparagine and glutamine from the medium. Previous studies have shown that the induction of AS gene transcription is similar regardless of whether one uses histidine-deficient medium or asparaginase treatment of the medium (20). Cells were then lysed and assayed for luciferase activity (Promega, Madison, WI), which was normalized to protein concentrations in the cell lysates. At least three independent transfections were performed for each experiment.

RNA Isolation and Northern Analysis-- Total cellular RNA was isolated from HepG2 cells using the Qiagen RNeasy Kit (Qiagen, Valencia, CA). For Northern analysis, 15 µg of RNA was size-fractionated, capillary-transferred to a Hybond-N (Amersham Biosciences) nylon membrane using 10× SSC covalently cross-linked to the membrane and then hybridized with a ³²P-labeled cDNA (GH or LacZ) as described previously (8). The blots were exposed to Biomax MR (Kodak, Rochester, NY) film and analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The results were quantified using Quantity One software (Bio-Rad, Hercules, CA).

Nuclear Protein Extraction-- HepG2 cells were scraped from 100-mm dishes (four dishes per condition) using ice-cold phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄·7H₂O, 1.4 mM KH₂PO₄) and centrifuged for 5 min at 240 × g. All remaining steps were performed at 4 °C. The cells were resuspended in 1 ml of lysis buffer (20 mM Hepes, pH 7.6, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, and 0.1% (v/v) Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 tablet/10 ml of buffer of complete mini-protease inhibitor mixture (Roche Molecular Biochemicals)) and incubated for 5 min. The lysate was transferred to a 1.5-ml microcentrifuge tube and centrifuged at 510 × g for 5 min. After discarding the supernatant, 1 ml of nuclear extraction buffer (20 mM Hepes, pH 7.6, 20% glycerol, 400 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 tablet/10 ml of buffer of protease inhibitors) was added to the pellet, and the samples were incubated with slow rotation for 1-2 h. After extraction, the samples were centrifuged for 10 min at 12,900 × g, and then the supernatant was dialyzed overnight in a Spectra/Por 2 dialysis membrane with a molecular mass cutoff of 12,000-14,000 Daltons (Spectrum Laboratories, Rancho Dominguez, CA). The dialysis buffer was composed of 20 mM Hepes, pH 7.8, 20% glycerol, 100 mM KCl, 10 mM MgCl₂, 0.2 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride, adjusted to pH 7.8. Protein concentration was determined by using a modified Lowry assay (21).

Electrophoresis Mobility Shift Assay-- Double-stranded oligonucleotides were radiolabeled by extension of overlapping ends with Klenow fragment in the presence of either [-³²P]dATP or [-³²P]dCTP. For each binding reaction, 5 µg of protein was incubated with 40 mM Tris-base, 200 mM NaCl, 2 mM dithiothreitol, 10% glycerol, 0.05% Nonidet P-40, 1 µg of poly(dI-dC) (Amersham Biosciences), and 0.05 mM EDTA for 20 min on ice. The radiolabeled probe was added at a concentration of 0.008 pmol/reaction (~10,000 cpm), and where indicated unlabeled competitor oligonucleotides were added at a 200-fold excess. The reaction mixture, 18 µl of final volume, was incubated at room temperature for 20 min. For those samples to be treated with antibody, 2 µg of anti-Sp3, anti-Sp4, (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-Sp1 (Upstate Biotechnology, Lake Placid, NY) was added, and the samples were incubated for an additional 20 min at room temperature. The reactions were subjected to electrophoresis in a non-denaturing gel that contained 15% acrylamide on the bottom 4 cm and 6% acrylamide for the remaining 12 cm. After electrophoresis for 2.5 h at 200 V, the gel was dried and exposed to Biomax MR (Kodak) film.

Immunoblotting-- Nuclear extracts (30 µg/sample) were separated on a 7.5% SDS/PAGE and electrotransferred to a Protran nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was stained with Fast Green stain to check for equal loading and then incubated with 5% blocking solution (5% (w/v) Carnation non-fat dry milk, 30 mM Tris-base, pH 7.5, 0.1% (v/v) Tween 10, and 200 mM NaCl) for 2 h at room temperature on a rotator. Immunoblotting was performed using rabbit polyclonal antibodies against Sp1 (Upstate Biotechnology, Lake Placid, NY) or Sp3 (Santa Cruz Biotechnology, Santa Cruz, CA) at a concentration of 0.2 µg/ml in 1% dry milk blocking solution for 2 h at 4 °C. The blots were washed 5 × 5 min in 1% blocking solution on a shaker and then incubated with peroxidase-conjugated goat anti-rabbit secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD) at a 1:20000 dilution for 45 min at room temperature. The blots were then washed for 5 × 5 min in 1% dry milk blocking solution and 2 × 5 min in Tris-buffered saline/Tween (30 mM Tris-base, 0.1% Tween 20, and 200 mM NaCl) pH 7.5. The bound secondary antibody was detected by using an enhanced chemiluminescence kit (Amersham Biosciences) and exposed to Biomax MR film (Kodak).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Human AS Promoter Sequence-- The human AS promoter region has been partially characterized in previous reports (4, 5). Within the initial 173 nt upstream of the transcription start site (Fig. 1), there are three GC-rich sequences (GC-I, GC-II, GC-III) and two cis-elements (NSRE-1 and NSRE-2) that make up the nutrient sensing response unit. These sequences are responsible for increased transcription following activation of the gene by glucose (ER stress response) or amino acid (AAR) deprivation.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Summary of cis-elements previously identified in the proximal promoter of the human AS gene. Six putative transcription factor binding sites were identified by in vivo footprinting (5), and the five shown are associated with nutrient-regulated transcription of the human AS gene. Three GC-rich sequences showed constitutive binding regardless of nutrient status (GC-I, nt 148 to 139; GC-II, nt 128 to 119; GC-III, nt 107 to 97). Single nucleotide mutagenesis defined the boundaries for NSRE-1, nt 68 to 60 and NSRE-2, nt 48 to 43) (5). (C. Chen and M. S. Kilberg, unpublished results.) These two elements function as a regulatory unit to mediate activation of the gene in response to glucose (ER stress response) or amino acid deprivation (amino acid response). Nucleotides included in the gray boxes result in loss of basal and nutrient-regulated transcription if mutated.

A GC Box Is Necessary for Maximal Transcription of the AS Gene-- To determine the importance of each of the GC boxes I-III in the transcriptional activity of the human AS promoter, each site was mutated in the context of the AS^173/+51/GH reporter construct (Table I). The expression driven by the wild-type promoter was compared with each mutant by transient transfection of HepG2 cells and subsequent Northern analysis (Fig. 2). To serve as negative control for amino acid deprivation, the mouse metallothionein-I promoter was used and, as expected, did not respond to medium lacking histidine. The wild-type 173/+51 fragment of the AS gene promoter resulted in basal transcription that was increased about 3-fold following histidine deprivation. Mutation of GC-I did not affect basal transcription significantly, but did cause a 46% decrease in the stimulated rate of transcription (Fig. 2). This result indicates that GC-I functions primarily as a component of the stimulatory mechanism of amino acid limitation rather than establishing the basal rate. Mutation of either GC-II or GC-III significantly inhibited basal transcription by about 60 and 75% of the wild-type rate, respectively, and the amino acid deprivation-induced transcription in both cases was inhibited by about 40% of the level observed for the wild-type sequence (Fig. 2). Given that, on a percentage basis, the decreases in the basal values were greater than those for the stimulated transcription, the calculated fold-induction was actually higher for the two mutants compared with the wild-type sequence. However, the absolute value of the reduction in the MEM medium, when considered as the actual transcription rate, was less than the decrease in the absolute value of the transcription rate following amino acid limitation. For example, mutating the GC-III sequence caused the basal rate to drop from 1.0 to 0.3 units of GH mRNA, a difference of 0.7 units, whereas the starvation-activated rate dropped from 3.0 to 1.7, a difference of 1.3 units, or nearly twice that of the decrease in basal units (Fig. 2). Therefore, the data indicate that these GC sequences make an important contribution to both basal and activated transcription.

View this table: [in this window] [in a new window]   Table I Mutagenesis of the GC boxes within the human AS promoter Underlined bases represent substitutions for each mutant. These mutations were tested for their ability to drive transcription by transient transfection of the wild-type and mutant sequences within the 173/+51 AS promoter background (see Figs. 2 and 3). The wild-type or mutated sequences were also used in oligonucleotides to test for competition in electrophoresis mobility shift analyses (Fig. 4).

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Site-directed mutagenesis of individual GC boxes within the AS promoter. A, mutations within each of the three GC boxes were introduced in the context of the AS173/+51/GH reporter (see Table I). GH reporter driven by the mouse metallothionein-I promoter was used as a negative control for amino acid depletion. HepG2 cells were transfected as described under "Materials and Methods" and then incubated in complete MEM or MEM lacking histidine (His) for 18 h. RNA was isolated and subjected to Northern analysis to measure GH (reporter) and LacZ (transfection normalization) mRNA. B, the graph represents the GH/LacZ mRNA ratio from PhosphorImager data. The value for the wild-type sequence expressed in cells maintained in complete MEM medium was set to 1.0, and the other values were normalized to it. The data are presented as the mean ± S.D. from at least three independent transfection experiments. The asterisk (*) represents p  0.05 compared with wild-type.

The GC Boxes Do Not Have Complete Redundancy-- To determine whether the GC boxes were redundant, two (or all three) of the sites were mutated simultaneously, leaving only one of the sites functional (Fig. 3). If all three GC boxes were mutated simultaneously, an 80% reduction in basal transcription and a 45% reduction in the regulated transcription was observed. It is clear that unlike the NSRE-1 or NSRE-2 sites that are absolutely required for regulated transcription (4, 5), the total lack of a GC box reduces but does not preclude induction of AS promoter-driven transcription. If only GC-I was left intact, basal transcription was inhibited by 87% relative to the wild-type sequence, and the transcription rate after histidine deprivation was decreased by 68%. In the presence of a functional GC-II alone, basal and stimulated transcription declined by 73 and 22%, respectively. Leaving an intact GC-III sequence by itself supported a basal rate that was reduced by about 40% relative to the native promoter sequence, and the induced transcription rate was actually higher than the wild-type (Fig. 3).

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Site-directed mutagenesis of multiple GC boxes within the human AS promoter. A, mutations were introduced within two or three of the GC boxes in the context of the AS173/+51/GH reporter (see Table I). HepG2 cells were transfected as described under "Materials and Methods" and then incubated in complete MEM or MEM lacking histidine (His) for 18 h. RNA was isolated and subjected to Northern analysis to measure GH (reporter) and LacZ (transfection normalization) mRNA. B, the graph represents the GH/LacZ mRNA ratio from PhosphorImager data. The value for the wild-type sequence expressed in cells maintained in complete MEM medium was set to 1.0, and the other values were normalized to it. The data are presented as the mean ± S.D. from at least three independent transfection experiments. One asterisk (*) represents p  0.05, two asterisks (**) represent p  0.005 compared with wild-type.

Sp1 and Sp3 Bind to the GC Boxes in Vitro-- It was hypothesized that GC boxes I-III might be binding sites for members of the Sp family because of their GC-rich sequences. Therefore, protein-DNA complex formation in vitro was examined by electrophoresis mobility shift assays performed with each individual GC box plus some flanking sequence on each end. Using a radiolabeled oligonucleotide that contained GC-I, (5'-CCCTTCCGCCGCCCCACTTAGTC-3') no protein-DNA complex formation was detected in vitro (data not shown). In contrast, a GC-II-specific probe (5'-ACTTAGTCCTGCTCCGCCCCGGACACC-3') revealed three primary complexes (Fig. 4A, lane 2). Unlabeled specific competitor (200X) inhibited formation of all three complexes (Fig. 4A, lane 5), but these complexes were neither competed away by an excess of unlabeled nonspecific DNA sequence (Fig. 4A, lane 3) nor by an oligonucleotide that contained the mutated GC-II sequence shown in Table I (Fig. 4A, lane 4). To determine whether the observed GC-II complexes contained Sp proteins, antibodies against Sp1, Sp3, and Sp4 were incubated with the complexes. Sp1 antibody caused a supershift of the slowest migrating complex (complex-1) (Fig. 4A, lane 6), whereas the middle (complex-2) and lower (complex-3) complexes were supershifted by Sp3 antibody (Fig. 4A, lane 7). Sp4 antibody did not supershift any of the complexes (Fig. 4A, lane 8) as expected because Sp4 expression is restricted largely to the brain and certain epithelia (reviewed in Ref. 17). Thus, Sp4 antibody serves as a good negative control.

View larger version (63K): [in this window] [in a new window]   Fig. 4.   Sp1 and Sp3 protein-DNA complexes with AS promoter GC-II and GC-III sequences. An oligonucleotide that contained either the GC-II (A) or GC-III (B) sequence as the core binding site (see "Results" for complete sequence) was incubated with nuclear extracts from HepG2 cells. Prior to preparation of the nuclear extracts, the cells had been incubated for 18 h in either complete MEM or MEM lacking histidine (His). The three major protein-DNA complexes (1-3) formed are indicated by the arrows. For supershifts, antibody for the specific protein designated was added to the binding reaction and incubated for an additional 20 min as described under "Materials and Methods." Each experiment was repeated with at least three different batches of nuclear extract.

Although the absolute amount of protein-DNA complex formation was slightly reduced by histidine deprivation in the particular set of samples shown in Fig. 4A (compare lanes 2 and 9), analysis of many different nuclear extract preparations typically showed little or no difference. However, the supershift patterns observed for Sp1 and Sp3 antibodies were faithfully reproduced in every experiment so that the data shown are representative of their recognition of those sequences. The observed pattern of complexes and the corresponding supershifts are indicative of Sp1 and Sp3 binding. Confirmation of the Sp protein composition of these complexes has been summarized by Suske (17). Complex-2 contains the full-length Sp3 isoform, whereas complex-3 (often a doublet) contains one or both of the N-terminally truncated Sp3 isoforms.

Three major complexes were also detected using the GC-III oligonucleotide (5'-CCCGCGGCCCCCGCCCCTGTGC-3') as radiolabeled probe (Fig. 4B). Once again, Sp1 antibody supershifted the slowest migrating complex (complex-1), whereas the Sp3 antibody supershifted the two lower complexes (complex-2 and complex-3). Sp4 served as a negative control to demonstrate that GC-III did not bind all Sp proteins. Furthermore, when used as a radiolabeled probe, a GC-III-containing oligonucleotide, with the mutations shown in Table I, did not produce any specific protein-DNA complexes (data not shown).

Nuclear Sp1 and Sp3 Protein Content Is Not Increased by Amino Acid Limitation-- To determine whether cells subjected to histidine deprivation regulated the absolute amount of nuclear Sp1 and Sp3 protein, nuclear extracts were examined by immunoblotting (Fig. 5). Consistent with the electrophoresis mobility shift assay data, the level of Sp3 (Fig. 5A) and Sp1 (Fig. 5B) proteins in nuclear extracts from either control or histidine-deprived cells was similar. When whole cell extracts were also analyzed for Sp1 and Sp3, histidine limitation did not alter protein levels (data not shown). The pattern of Sp3 isoforms was the same as that reported by others (22, 23), a primary band representing the full-length protein and the two truncated isoforms at about 60-70 kDa. These isoforms likely arise from different translation initiation sites (17). Although there was no change in the nuclear protein content, the Sp1 immunoblots did reveal a band of slower migration that was increased in those cells that were deprived of histidine (Fig. 5C). Sp1 is known to be modified posttranslationally by phosphorylation (24), so it was possible that a portion of the Sp1 was phosphorylated following amino acid deprivation. Treatment of nuclear extract samples with calf intestinal phosphatase resulted in elimination of the slower migrating Sp1 species in the samples from histidine-deprived cells (Fig. 5C).

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Sp1 and Sp3 protein content and Sp1 phosphorylation in response to amino acid deprivation. Nuclear extracts from HepG2 cells incubated in complete MEM medium or MEM lacking histidine (His) for 18 h were analyzed by immunoblotting for Sp3 (A) or Sp1 (B) protein content. In addition, nuclear extracts were treated with calf intestinal phosphatase (CIP) to analyze the phosphorylation state of Sp1 protein (C). The blots shown are representative of results obtained with at least two independently prepared nuclear extracts.

Sp1 and Sp3 Activate Transcription Driven by the AS Promoter-- To determine the role of Sp1 and Sp3 on the transcriptional activity of the AS promoter in vivo, transient expression experiments were performed in Drosophila SL2 cells that lack Sp proteins (25). Using this Sp-negative background, a luciferase reporter plasmid driven by the AS promoter sequence 173/+51 was co-transfected along with empty vector (pPac0), Sp1 expression plasmid (pPacSp1), or one of two different Sp3 expression plasmids (pPacUSp3, pPacSp3). The pPacUSp3 plasmid encodes the full-length Sp3, whereas the pPacSp3 encodes a short isoform of Sp3 that lacks one of the two activation domains (17). In the absence of an available insect cell culture medium lacking individual amino acids, transfected SL2 cells were incubated in Schneider's complete media without (control) or with the enzyme asparaginase (1 unit/ml) for 18 h. Asparaginase will cause complete depletion of medium asparagine and glutamine within minutes of addition, inducing amino acid limitation conditions (20). Induction of AS expression is the same regardless of whether histidine is removed from the MEM culture medium or asparaginase is added to complete medium (26).

Cells transfected with the vector only (pPac0) expressed a low level of luciferase activity that was not increased by amino acid limitation (Fig. 6). SL2 cells co-transfected with pPacSp1 had more than a 4-fold increase in basal (control) transcriptional activity relative to cells transfected with empty vector (pPac0). However, even in the presence of Sp1 expression, amino acid depletion did not increase transcription further (Fig. 6). In contrast, the full-length form of Sp3 also had an activating effect on the basal transcription driven by the AS promoter (3.5-fold relative to vector only), but when the cells were histidine-deprived, this basal rate of transcription was doubled. The truncated isoform of Sp3, lacking one of the trans-activation domains, did not activate the AS promoter regardless of nutrient status and thus served as a negative control. To investigate the possibility that the increased transcription following amino acid depletion of Sp3-transfected cells was simply due to an asparaginase-induced increase in Sp3 expression, two independent approaches were used. First, immunoblotting for Sp3 protein in HepG2 cells incubated with or without asparaginase showed no difference in Sp3 protein content. Second, SL2 cells were transfected with Sp1 and Sp3 vectors and an AS promoter construct containing a mutant (i.e. non-functional) NSRE-1 site, and in the absence of starvation-induced transcription, Sp3 did not further enhance the activity observed in the presence of ASNase. Collectively, these control studies indicate that regulatory mechanisms other than absolute Sp3 expression level are responsible for the functional difference between Sp1 and Sp3 shown in Fig. 6.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   AS promoter activity following expression of Sp1 or Sp3 protein in Drosophila SL2 cells. Drosophila SL2 cells were co-transfected with 2 µg of luciferase reporter plasmid (AS173/+51/Luc) and 400 ng of transcription factor expression plasmid (pPac0, pPacSp1, pPacUSp3, or pPacSp3). The pPacUSp3 vector contains the full-length isoform of Sp3, whereas the pPacSp3 encodes a truncated isoform of Sp3, which lacks one of the trans-activation domains. Luciferase activity was normalized to the protein concentration of each cell lysate. The data from three independent transfections are presented as the means ± S.D.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mammalian AS is a model for gene regulation by nutrient availability. This report demonstrates that binding of Sp1 and Sp3 proteins plays an important role in both the basal and the metabolite-regulated transcription of the human AS gene. In vivo footprinting had documented constitutive protein binding at three GC-rich sequences within the 5' upstream region of the human AS gene (5). Although in vivo footprinting is a powerful tool to show binding site occupancy in the intact cell, it does not identify the protein bound. Based on site-directed mutagenesis and transient transfection assays, the present data show that at least one of these three GC-boxes is necessary to maintain high rates of transcription, and all three are required to ensure maximal transcription from the AS promoter. Mutagenesis of these elements individually or in pairs indicated that there is a hierarchy based on their ability to maintain the basal transcription rate as well as to support the induction of transcription by amino acid limitation. GC-III (nt 107 to 97), the one most proximal to the two known nutrient responsive elements NSRE-1 and NSRE-2, is the most effective of all three, followed by GC-II (nt 128 to 119), and then GC-I (nt 148 to 139). When all three GC-boxes were mutated simultaneously, the basal transcription rate was only about 20% of that observed for the wild-type sequence, and although transcriptional activation following amino acid deprivation was still detectable, the absolute rate was nearly half that in the presence of all three GC boxes. When one (Fig. 2) or two (Fig. 3) of the GC boxes were mutated, both basal and nutrient-regulated transcription was affected. In fact, there are two ways to consider the data. First, if the fold-induction relative to the MEM-incubated cells is considered for each mutant, the value is actually greater in some of the mutants than it is for the wild-type sequence, demonstrating that the GC boxes are not absolutely necessary for a nutrient-regulated response to occur. On the other hand, in terms of absolute transcription rates, the numerical value for the reduction of basal transcription was always less than the decrease observed for the stimulated condition, so one would argue that the GC boxes make a positive contribution to the actual transcription rates achieved following amino acid limitation. The question that could be debated is whether the quantitative decrease in the absolute rates of starvation-induced transcription is more important than the difference between the basal and stimulated rates (i.e. the fold-induction). In either case, the mutagenesis results demonstrate that the GC-rich elements serve to establish the degree of responsiveness to nutrient deprivation by modulating the level of regulated transcription mediated by the NSRE-1 and NSRE-2 sequences.

The present data also extend previous in vivo footprinting results (5) by documenting that two of the GC boxes, GC-II and GC-III, form protein-DNA complexes in vitro. This pattern of three primary protein-DNA complexes is consistent with that seen by other investigators using GC-rich sequences as a probe, and the Sp1 (complex-1 and Sp3 (complex-2 and complex-3) composition of these complexes has been documented (17). Footprinting of the AS promoter region corresponding to GC-I suggested constitutive protein binding in vivo (5). The reason that GC-I failed to form a complex in vitro is not clear, but it is possible that the protein that binds at the GC-I site might require occupancy at other sites in the promoter as a prerequisite. Sp1 association with complex-1 and Sp3 with complex-2 and complex-3 was the same regardless of whether the radiolabeled probe contained the GC-II or GC-III sequence. These results demonstrate that these two elements are qualitatively similar in their binding specificity, but the mutagenesis data document that they are not identical functionally. The inability of each antibody to supershift the same complex indicates that Sp1 and Sp3 are not components of the same complex. Furthermore, the results for the nuclear extracts from control and histidine-deprived cells were the same with regard to the migration rate of the complexes, the absolute amount of each complex, and the Sp1/Sp3 composition of each complex. These in vitro results are consistent with the in vivo footprinting data indicating that protein binding at these sites was not altered by amino acid availability (5).

The in vivo expression studies illustrate that Sp1 and Sp3 are not functionally equivalent with regard to the ability to modulate nutrient control of AS gene transcription. In vivo studies using Sp-deficient Drosophila SL2 cells demonstrated that expression of either Sp1 or the full-length Sp3 were capable of enhancing basal AS transcription. The electrophoresis mobility shift assay data indicated that both proteins can bind to either GC-II or GC-III, but that they are not simultaneously bound to the same complex at either site. Together these results suggest that the SL2 cell core transcription machinery is capable of recognizing two different complexes, either one containing Sp1 or one containing Sp3, to initiate basal transcription from the AS promoter. However, given that only Sp3, not Sp1, could enhance the starvation-induced transcription in response to amino acid limitation, it appears that the two proteins are not functionally equivalent in their ability to interact (directly or indirectly) with the NSRE-1/NSRE-2 binding proteins. Also, given that the absolute nuclear content of Sp1 and Sp3 proteins in HepG2 cells did not change following histidine deprivation, a change in the ratio of Sp1/Sp3 does not appear to be responsible for regulation of the AS gene, as reported for other genes (27). However, the observation that a small, but detectable portion of Sp1 is phosphorylated by amino acid limitation suggests that this posttranslational modification might have a role in Sp1 function under these conditions.

Collectively, the results presented in this report document the importance of a series of three GC-rich cis-elements in modulating the transcription driven by the human AS promoter, both the basal and the nutrient-regulated activity. The results also represent the first demonstration that: 1) both Sp1 and Sp3 can bind to these two AS promoter sites, 2) Sp1 and Sp3 do not appear to be part of a common complex, 3) the amount of Sp1 or Sp3 bound does not change in control versus amino acid-deprived cells, and 4) in vivo, Sp1 and Sp3 are not functionally equivalent in their ability to regulate nutrient control of the human AS gene. Future studies should unravel the relationship and possible interactions between Sp1/Sp3 binding and the trans-factors that bind at the nutrient sensing response cis-elements within the AS promoter.

	FOOTNOTES

^* This work was supported by NIDDK, Grant DK-52064 (to M. S. K.) from the NIH.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Florida College of Medicine, Box 100245, Gainesville, FL 32610-0245. Tel.: 352-392-2711; Fax: 352-392-6511; E-mail: mkilberg@ufl.edu.

Published, JBC Papers in Press, February 26, 2002, DOI 10.1074/jbc.M110972200

	ABBREVIATIONS

The abbreviations used are: AS, asparagine synthetase; AAR, amino acid response; ER, endoplasmic reticulum; NSRE, nutrient sensing response element; nt, nucleotide(s); MEM, minimal essential medium; GH, growth hormone.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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