INTRODUCTION

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The control of gene expression in multicellular organisms differs in many aspects from that operating in single-cell organisms and involves complex interactions of hormonal, neuronal, and nutritional factors. Although not as widely appreciated as the other factors, nutritional and metabolic signals play an important role in controlling gene expression in mammalian cells. It has been shown that major (carbohydrates, fatty acids, and sterols) or minor (minerals and vitamins) dietary constituents participate in the regulation of gene expression in response to nutritional changes (16, 20, 54, 60). Much less is known about the role of amino acids in the control of gene expression. The relative concentrations of amino acids are altered in response to various forms of stresses, such as sepsis, fevers, thermal burns, or malnutrition. For example, alteration of the amino acid concentration has been reported when there is a deficiency of any one of the essential amino acids, a dietary imbalance of amino acids, or an insufficient intake of protein (3).

The molecular mechanisms involved in the control of gene expression in response to amino acid availability have been extensively studied in yeast (24). In addition to specific controls of genes involved in the synthesis of individual amino acids, the yeast employs a general control process whereby multiple genes in nine different biosynthetic pathways are regulated by starvation of the cell for a single amino acid (25, 26). In mammalian cells, a few examples of enzymes, transporters, and mRNAs that are regulated by amino acid availability have been reported (32, 35). At the molecular level, the current understanding of amino acid-dependent control of gene expression is limited. It was reported that up-regulation of asparagine synthetase mRNA in response to amino acid limitation involves both transcriptional and posttranscriptional components (18). These authors defined a region of the asparagine synthetase promoter involved in transcriptional regulation in response to amino acid starvation but did not demonstrate transfer of amino acid responsiveness to a heterologous promoter.

Among the amino acid-regulated genes in mammalian cells, CHOP (also called GADD153) expression exhibits the greatest induction level (40). CHOP encodes a small nuclear protein that regulates certain aspects of the cell response to stress (62, 65). The induction of CHOP expression by stress agents is generally linked to the activation of an endoplasmic reticulum stress (ER stress), manifested as the accumulation of malfolded proteins in the endoplasmic reticulum (unfolded protein response) (61, 65). CHOP protein belongs to the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors that have been implicated in the regulation of processes relevant to energy metabolism (43), cellular proliferation, and differentiation and expression of cell type-specific genes (7, 9, 56). By forming heterodimers with the members of the C/EBP family, CHOP protein can influence gene expression both as a dominant negative regulator of C/EBP binding to one class of DNA targets and by directing CHOP-C/EBP heterodimers to other sequences (5, 6, 14, 51, 55, 62).

It has been shown previously that leucine limitation in human cell lines leads to induction of CHOP mRNA and protein in a dose-dependent manner (8). In addition, the regulation of CHOP mRNA expression by leucine has both transcriptional and posttranscriptional components (8). The signaling pathway that links amino acid deprivation to CHOP expression is not known. However, it has been demonstrated that amino acid starvation regulates the expression of CHOP through a specific pathway that is distinct from the ER stress signaling cascade (31). In the present study, we report the characterization of cis- and trans-acting elements involved in the transcriptional activation of CHOP by amino acid deprivation. By deletion and mutation analysis of the CHOP promoter region, we characterize an amino acid response element (AARE). In addition, we show that the AARE binds activating transcription factor 2 in vitro and that this transcription factor is essential for the transcriptional activation of CHOP by leucine starvation.

	MATERIALS AND METHODS

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Cell culture and treatment conditions. Cells were cultured at 37°C in Dulbecco's modified Eagle's medium F12 (DMEM F12) (Sigma) containing 10% (mouse embryonic fibroblasts [MEF], HeLa, and HepG2) or 20% (Caco-2) fetal bovine serum. When indicated, DMEM F12 lacking leucine was used. For other amino acid starvation experiments, MEM medium (Life Technologies, Inc.) was used. In all experiments involving amino acid starvation, 10% dialyzed calf serum was used. MEF deficient in C/EBP were a gift of David Ron (65). MEF deficient in ATF-2 were produced from decapitated, eviscerated day 14.5 ATF-2^0/0 embryos (39) using a 3T3 protocol until cells passed through crisis, typically by passage 18 (53).

DNA transfection and luciferase assay. Cells were plated in 12-well dishes and transfected by the calcium phosphate coprecipitation method as described previously (8). Two micrograms of luciferase plasmid was transfected into the cells along with 0.1 µg of pCMV-Gal, a plasmid carrying the bacterial -galactosidase gene fused to the human cytomegalovirus immediate-early enhancer-promoter region, as an internal control. In the experiments using cDNA expression plasmids, a mixture containing 1 µg of luciferase plasmid, 1 µg of cDNA expression plasmid, and 0.05 µg of pCMV-Gal was transfected in the cells. The total amount of plasmid DNA was adjusted to 2 µg by the addition of the control plasmid lacking the cDNA to be expressed. Cells were then exposed to the precipitate for 16 h, washed twice in phosphate-buffered saline (PBS), and then incubated with DMEM F12 containing 10% calf serum. Twenty-four hours after transfection, cells were starved for 16 h. After starvation, cells were harvested in 150 µl of lysis buffer (Promega) and centrifuged at 13,000 × g for 2 min. Twenty microliters of the supernatant was assayed for luciferase activity (PRODEMAT, Anduze, France). -Galactosidase activity was measured as described previously (23). Relative luciferase activity was given as the ratio of relative light units to relative -galactosidase units. All values are the means calculated from the results of at least three independent experiments.

RNA isolation and Northern blot analysis. Total RNA was prepared as previously described (12). Northern blots were performed according to the procedure of Sambrook et al. (48). The membranes were UV cross-linked, and then prehybridization was carried out for 2 h at 55°C in 50% formamide-6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-5× Denhardt's reagent-0.5% sodium dodecyl sulfate (SDS)-0.1 mg of sonicated salmon sperm DNA/ml-10 µg of yeast tRNA/ml. The human CHOP cDNA (BH1), generously provided by N. J. Holbrook (46), was used as a probe. BH1 plasmid was linearized by PstI, and ³²P-riboprobes were synthesized (48) using T7 RNA polymerase (Promega). Hybridization was carried out for 16 h at 55°C. The membranes were washed for 15 min at 55°C successively in 2× SSC containing 0.1% SDS, 0.5× SSC containing 0.1% SDS, and 0.1× SSC containing 0.1% SDS. Labeled bands were detected by autoradiography. Autoradiogram signals were visualized by using a PhosphorImager and IMAGEQUANT software (Molecular Dynamics). To control for variation in either the amount of RNA in different samples or loading errors, all blots were rehybridized with a DNA probe corresponding to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Relative CHOP mRNA was determined as the ratio of CHOP mRNA to GAPDH mRNA.

Antibodies. Anti-C/EBP serum was generously provided by U. Schiebler. Anti-ATF-4 serum was a gift from D. Ron. Anti-ATF-1 (catalog number sc-241X), anti-ATF-2 (catalog number sc-6233X), anti-ATF-3 (catalog number sc-188X), and anti-c-Jun (catalog number sc-1694X) were purchased from Santa Cruz Biotechnologies.

Nuclear extract preparation. Nuclear extracts were prepared from HeLa cells plated in 150-mm-diameter dishes. Cells were washed twice with ice-cold PBS, harvested with a rubber policeman, and centrifuged at 4°C. Cell pellets were resuspended in 10 volumes of ice-cold lysis buffer (20 mM HEPES [pH 7.9], 10 mM KCl, 3 mM MgCl₂, 0.5 mM EDTA, 1 mM dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 1 µg of leupeptin/ml), incubated on ice for 5 min, and centrifuged 5 min at 2,000 × g. Nuclear pellets were resuspended in 2 volumes of ice-cold extract buffer (20 mM HEPES [pH 7.9], 450 mM KCl, 3 mM MgCl₂, 0.5 mM EDTA, 25% glycerol, 1 mM DTT, 1 mM PMSF, 1 µg of leupeptin/ml, 0.5 mM spermidine, 0.15 mM spermine) and incubated on ice for 30 min. After centrifugation for 10 min at 14,000 × g, the supernatant was dialyzed for two 2-h periods against 100 volumes of dialysis buffer (20 mM HEPES [pH 7.9], 50 mM KCl, 0.2 mM EDTA, 10% glycerol, 1 mM DTT, 0.5 mM PMSF), frozen, and stored at 80°C.

Oligonucleotides. Oligonucleotides were from Eurogentec. When double-stranded oligonucleotides were required, equal numbers of moles of complementary strands were heated to 90°C for 1 min and annealed by slow cooling to room temperature.

Gel mobility shift assays. Probes for the gel mobility shift assay were obtained by labeling synthetic double-stranded oligonucleotides with T4 polynucleotide kinase (Eurogentec). HeLa nuclear extracts (5 µg) were preincubated for 10 min on ice in the presence of 3 µg of poly(dI-dC) in a reaction mixture containing 25 mM HEPES (pH 7.9), 60 mM KCl, 2.5 mM MgCl₂, 0.1 mM EDTA, 0.75 mM DTT, 1 mM PMSF, and 5% glycerol. Fifty femtomoles (i.e., 100,000 cpm) of end-labeled oligonucleotides was then added and incubated for a further 10 min. Competition for specific complex formation was performed by preincubation of the extract with a 25-, 50-, or 100-fold molar excess of the unlabeled competitors. To test the effect of specific antibodies, 1 µl of antiserum (see above) was added to the incubation mixture on ice 2 h prior to the addition of the labeled probe. The samples were loaded onto a prerun, 16-cm-long, 1.5-mm-thick 4% acrylamide-bisacrylamide (29:1) gel, prepared in 1× TGE (25 mM Tris base, 190 mM glycine, 1 mM EDTA [pH 8.5]). Electrophoresis was carried out at 240 V in 1× TGE for 2 h at 4°C. Radioactive bands were visualized by using a PhosphorImager and IMAGEQUANT software (Molecular Dynamics). Each mobility shift experiment was repeated three times to confirm the reproducibility of the results.

Plasmid constructions. All constructs containing deletions in the CHOP promoter were generated by PCR from cloned genomic DNA, using Pfu polymerase (Stratagene), primers, and antisense primers containing appropriate restriction sites at their 5' end. Amplified fragments were then cloned into the pGL3-basic reporter construct (Promega) using the corresponding restriction sites. For 5' CHOP promoter deletions (pCHOP-LUC series [see Fig. 1]), XhoI-ended primers and HindIII-ended antisense primers were used. For internal CHOP promoter deletions (pCHOP-LUC series [see Fig. 2]), fragments of the 5' part of the CHOP promoter were generated by using SstI-ended primers and MluI-ended antisense primers and fragments of the 3' part by using XhoI-ended primers and HindIII-ended antisense primers.

	RESULTS

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Transcriptional activation of CHOP by leucine starvation requires sequences located upstream from the start site. To understand the regulation of gene expression by amino acids at a molecular level, we have studied the regulation of CHOP expression in response to leucine limitation because (i) leucine is an essential amino acid that is poorly utilized by HeLa cells during a 16-h incubation period (data not shown), and (ii) leucine, which is transported by system L, is rapidly equilibrated through the cell membrane (34, 42). It has been shown previously that regulation of CHOP transcription by leucine starvation is mediated through the promoter sequence situated between nucleotide position 954 and +91 (8). To identify amino acid-responsive elements within the CHOP promoter, a series of deletions in this region was created by PCR and fused to the coding region of the luciferase (LUC) reporter gene (Fig. 1). These constructs were transiently transfected into HeLa cells, and the response to leucine was determined by LUC assay, in starved and nonstarved conditions. Deletion to 649 had no significant effect on the activation of the CHOP promoter by leucine starvation (row 2). By contrast, deletion from 280 decreased the amino acid inducibility, suggesting that the region between 649 and 280 contains cis-positive elements involved in the transcriptional activation of CHOP by leucine starvation (row 3). Further deletions of the promoter from 221 to 40 resulted in a similar fold reduction in amino acid responsiveness and a progressive reduction of basal LUC activities (rows 4 to 6).

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Effect of leucine limitation on 5' deletions of the CHOP promoter. HeLa cells were transiently transfected with LUC constructs containing progressive 5' deletions of the CHOP promoter, as described in Materials and Methods. Twenty-four hours after transfection, cells were incubated for 16 h in DMEM F12 (420 µM leucine) or in DMEM F12 lacking leucine (0 µM leucine) and then were harvested for preparation of cell extracts and determination of LUC activity. Relative LUC activities were determined as described in Materials and Methods. The relative fold induction, defined as the ratio of the relative LUC activity of leucine-starved cells to unstarved cells, is indicated in parentheses to the right of the bars.

Localization of an upstream positive element involved in activation of CHOP transcription by leucine starvation. To localize the positive elements involved in activation of CHOP transcription by leucine starvation, HeLa cells were transfected with a series of constructs containing internal deletions of the CHOP promoter (Fig. 2A). These deletions can be divided into two groups according to their level of amino acid inducibility. The first group includes three deletions that produced high levels of amino acid inducibility (rows 2, 4, and 5), while the second group of deletions led to reduced levels of induction (rows 3, 6, 7, and 8). These findings demonstrate that a 33-bp CHOP promoter region from 318 to 286 contains a cis-positive element that is essential for amino acid regulation. Close inspection of this 33-bp promoter sequence reveals the presence of a site that is similar to both the C/EBP consensus and the ATF/CRE-like sequence (referred to as a C/EBP-ATF composite site [15, 63]) and the presence of a putative SP-1 binding site (Fig. 2B). To determine the importance of each site in the leucine responsiveness of the CHOP promoter, we mutated these sites and assayed the LUC activity of the constructs. Figure 2C shows that mutation of the C/EBP-ATF site resulted in a decrease of amino acid responsiveness (row 11), while mutations in the SP-1 site did not affect the activation of the CHOP promoter by leucine starvation (row 10). These results show that the C/EBP-ATF composite site is essential for transcriptional activation of CHOP in response to leucine starvation.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Identification of an upstream cis-positive element involved in the activation of CHOP transcription by leucine starvation. HeLa cells were transiently transfected with LUC constructs containing internal 5' deletions (A) or 5' mutations (C) of the CHOP promoter as described in Materials and Methods. The experiments were carried out as described in the legend to Fig. 1. The relative fold induction, defined as the ratio of the relative LUC activity of leucine-starved cells to unstarved cells, is indicated in parentheses to the right of the bars. (B) Nucleotide sequence from 318 to 286 relative to the start site of CHOP transcription. The positions of the SP-1 and composite C/EBP-ATF sites are indicated. The open circles represent the positions of substitution mutations in either the SP-1 site or the C/EBP-ATF site.

The cis-positive element in the CHOP promoter is an AARE. To determine whether the above-described CHOP cis-positive element could, by itself, render a heterologous promoter amino acid responsive, one or two copies of a 19-bp segment of the CHOP promoter from 313 to 295 containing the positive element were cloned 5' of the minimal herpes simplex virus promoter for thymidine kinase (TK). As shown in Fig. 3A, a single copy of the CHOP-positive sequence was able to regulate the basal promoter in response to leucine (row 2). Furthermore, the leucine starvation-induced activity was enhanced synergistically by the presence of two copies of the positive element in the promoter (row 3).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   The cis-positive element is an AARE. (A) HeLa cells were transfected with LUC constructs containing a single or two copies of a 19-bp segment of the CHOP promoter including the positive element (313 to 295) inserted 5' to the TK promoter. The 19-bp segment of the CHOP promoter is indicated by a black diamond. Twenty-four hours after transfection, cells were incubated for 16 h in DMEM F12 (420 µM leucine) or in DMEM F12 lacking leucine (0 µM leucine) and then were harvested for preparation of cell extracts and determination of LUC activity. (B) HeLa, HepG2, and Caco-2 cells were transfected with LUC constructs containing two copies of the 19-bp segment of the CHOP promoter including the positive element (313 to 295) inserted 5' to the TK promoter. Twenty-four hours after transfection, cells were incubated for 16 h in MEM control medium (MEM) or in MEM lacking one amino acid (MEM-AA) and then were harvested for preparation of cell extracts and determination of LUC activity. Relative LUC activities were determined as described in Materials and Methods. (A) The relative fold induction, defined as the ratio of the relative LUC activity of leucine-starved cells to unstarved cells, is indicated in parentheses to the right of the bars.

The minimum AARE core sequence is 5'-ATTGCATCA-3'. To pinpoint the exact nucleotides of the CHOP AARE that are involved in amino acid regulation, we scanned the 313 to 295 promoter region by site-directed mutagenesis. Each mutation consisted of a 3-bp substitution in a context of a single copy of the CHOP AARE sequence inserted upstream of the TK promoter (Fig. 4A). Mutations mt3 and mt8 did not affect the activation of the TK promoter construct by leucine starvation, and mutation mt7 caused a slight decrease in amino acid responsiveness. By contrast, mutations mt4, mt5, and mt6 resulted in a complete loss of the amino acid inducibility, suggesting that nine nucleotides are essential for conferring amino acid sensitivity. Furthermore, mutation mt9 demonstrates that the minimum core sequence able to render a promoter amino acid responsive is 5'-ATTGCATCA-3'.

View larger version (51K): [in this window] [in a new window]   FIG. 4.   Identification of a CHOP AARE core sequence. (A) HeLa cells were transfected with LUC constructs containing a single copy of native or mutant CHOP AARE (313 to 295) inserted 5' to the TK promoter. The open circles represent the positions of substitution mutations in the 19-bp CHOP AARE sequence. The minimum AARE core sequence is shown in bold letters. Twenty-four hours after transfection, cells were incubated for 16 h in DMEM F12 (420 µM leucine) or in DMEM F12 lacking leucine (0 µM leucine) and then were harvested for preparation of cell extracts and determination of LUC activity. Relative LUC activities were determined as described in Materials and Methods. The relative fold induction, defined as the ratio of the relative LUC activity of leucine-starved cells to unstarved cells, is indicated in parentheses. Each data point represents the mean of at least three independent experiments performed in triplicate. (B) Gel mobility shift assays of nuclear extracts from HeLa cells incubated for 16 h in DMEM F12 (420 µM leucine) or in DMEM F12 lacking leucine (0 µM leucine) in the presence of the 19-bp CHOP AARE probe. The AARE probe carried sequences 313 to 295 (wild type). The 19-bp CHOP AARE and different mutated AARE oligonucleotides (mt3 to 8) were used as competitors at a 25- or 50-fold molar excess relative to the probe. The specific DNA-protein complex is indicated by an arrow.

CHOP AARE binds members of C/EBP and ATF protein families in vitro. Sequence comparison of the CHOP AARE with database sequences did not reveal any perfect homology with known transcription factor binding sites. However, the minimum CHOP AARE core sequence 5'-ATTGCATCA-3' showed some homology with binding sites of the C/EBP and ATF/CREB protein families and was referred to as a C/EBP-ATF composite site (15, 63) (Fig. 5A). To investigate whether DNA binding sites for C/EBP or ATF transcription factors could mediate amino acid inducibility, two copies of several candidate sites were cloned immediately upstream of the TK promoter (see Fig. 5A for sequences). The adenovirus E4-ATF binding site (E4 ATF) (27, 36) (Fig. 5B, row 3), a consensus C/EBP binding site (C/EBP) (50) (row 4), the cyclic AMP response element of the fibronectin gene (FN CRE) (45) (row 5), and a chimeric C/EBP-CRE site (C/EBP-CRE) (50) (row 6) could not render a basal promoter amino acid responsive. A low level of inducible promoter activity was observed with the distal AP-1 binding site in the c-jun gene promoter (jun2TRE) (58) (row 7), but five copies of this binding site did not produce higher amino acid responsiveness (data not shown). These findings demonstrate that previously identified binding sites for ATF or C/EBP are not able to mediate amino acid inducibility.

View larger version (34K): [in this window] [in a new window]   FIG. 5.   Comparison of CHOP AARE with C/EBP and ATF/CRE binding sites. (A) Sequence comparison of the CHOP AARE (313 to 295) with the ATF binding site in the adenovirus E4 promoter (E4 ATF), a consensus C/EBP binding site (C/EBP), the ATF/CRE box of the fibronectin gene promoter (FN CRE), a chimeric C/EBP-CRE binding site (C/EBP-CRE), and the distal AP-1 binding site in the c-jun gene promoter (jun2-TRE). The position of the minimum AARE core sequence is boxed. Identical nucleotides are shaded in grey. (B) HeLa cells were transfected with LUC constructs containing two copies of the above-described sequences inserted 5' to the TK promoter. Twenty-four hours after transfection, cells were incubated for 16 h in DMEM F12 (420 µM leucine) or in DMEM F12 lacking leucine (0 µM leucine) and then were harvested for preparation of cell extracts and determination of LUC activity. Relative LUC activities were determined as described in Materials and Methods. The relative fold induction, defined as the ratio of the relative LUC activity of leucine-starved cells to unstarved cells, is indicated in parentheses to the right of the bars. Each data point represents the mean of at least three independent experiments performed in triplicate. (C) Gel mobility shift assays of nuclear extracts from HeLa cells incubated for 16 h in DMEM F12 in the presence of the 19-bp CHOP AARE probe. The CHOP AARE carried sequences 313 to 295. Competing oligonucleotides (see panel A for sequence) were added at a 25-, 50-, and 100-fold molar excess relative to the probe. The specific DNA-protein complex is indicated by an arrow.

View larger version (40K): [in this window] [in a new window]   FIG. 6.   Presence of ATF-2 and C/EBP in the DNA-protein complex binding to the CHOP AARE. Supershift assays using specific antibodies to different proteins and nuclear extracts from HeLa cells incubated for 16 h in DMEM F12 (A) or DMEM F12 lacking leucine (B). HeLa nuclear extracts were first incubated with 1 µl of rabbit nonimmune serum or specific antiserum, and then the preincubation mixture was incubated with the 19-bp CHOP AARE probe as described in Materials and Methods. The 19-bp CHOP AARE carried sequences 313 to 295.

ATF-2 has a critical role in the transcriptional activation of CHOP by amino acids. To assess the respective roles of ATF-2 and C/EBP in the transcriptional activation of CHOP by leucine, we first measured the effect of leucine starvation on CHOP mRNA expression in MEF deficient in ATF-2 or C/EBP (65) and in the corresponding wild-type cells. As shown in Fig. 7, CHOP exhibited a normal response to leucine starvation and to an agent (tunicamycin) that induces ER stress (31) in C/EBP^+/+ (lanes 1 to 3) and ATF-2^+/+ cells (lanes 7 to 9). Lack of C/EBP did not affect the induction of CHOP mRNA by leucine starvation and tunicamycin (lanes 4 to 6). By contrast, lack of ATF-2 resulted in a complete loss of the CHOP mRNA amino acid inducibility (lanes 10 and 11) but did not affect the induction of mRNA level by tunicamycin (lane 12). The result concerning the effect of the ATF-2 genotype on CHOP induction was strengthened by a kinetic analysis of the CHOP mRNA level in ATF-2^+/+ and ATF-2^/ cells exposed to medium lacking leucine (Fig. 8A). CHOP mRNA was only detectable in ATF-2^+/+ cells 4 h after starvation, and a maximum level was reached after 16 h. Moreover, as shown in Fig. 8B, the defect in endogenous CHOP induction in the ATF-2^/ cells extended to the response to deprivation of some other amino acids (lysine, arginine, and phenylalanine). Deprivation of these amino acids had been shown above to increase LUC activity from the CHOP AARE LUC reporter construct (see Fig. 3B). Taken together, these results provide evidence that ATF-2 is essential in the specific amino acid pathway that leads to the induction of CHOP mRNA.

View larger version (30K): [in this window] [in a new window]   FIG. 7.   CHOP mRNA level is induced by leucine starvation in C/EBP-deficient cells but not in ATF-2-deficient cells. Wild-type (+/+) or mutant (/) C/EBP or ATF-2 MEF were incubated for 16 h in DMEM F12 (C) or in DMEM F12 lacking leucine (L) or for 6 h in medium containing 0.125 µg of tunicamycin (+Tu)/ml. Total RNA was extracted and Northern blot analysis was performed as described in Materials and Methods. The blots were hybridized with human probes corresponding to CHOP and GAPDH.

View larger version (40K): [in this window] [in a new window]   FIG. 8.   Lack of ATF-2 results in a complete loss of amino acid inducibility. (A) Wild-type (+/+) or mutant (/) MEF were incubated in DMEM F12 (+Leu) or in DMEM F12 lacking leucine (Leu) and were harvested for RNA isolation after the indicated incubation times. (B) Wild-type (+/+) or mutant (/) MEF were incubated for 16 h in MEM (control) or in MEM lacking one amino acid or for 6 h in medium containing 0.125 µg of tunicamycin (+Tu)/ml and were harvested for RNA isolation. Northern blot analysis was performed as described in Materials and Methods. The blots were hybridized with human probes corresponding to CHOP and GAPDH. Quantification of the Northern blot analysis is given graphically, below the respective panels.

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View larger version (21K): [in this window] [in a new window]   FIG. 9.   ATF-2 is essential for the induction of the CHOP promoter and AARE activity by leucine starvation. (A) Wild-type (+/+) or mutant (/) C/EBP MEF were transfected with LUC reporter constructs containing either the CHOP promoter region from 649 to +91 [CHOP (649/+91)] or two copies of the 19-bp segment of the CHOP promoter including the AARE (313 to 295) inserted 5' to the TK promoter (2X CHOP AARE). (B) Wild-type (+/+) or mutant (/) ATF-2 MEF were transfected with a mixture containing CHOP (649/+91) or 2X CHOP AARE LUC reporter constructs and a wild-type or a dominant negative form (ATF-2Ala [50]) of ATF-2 expression plasmids. Twenty-four hours after transfection, cells were incubated for 16 h in DMEM F12 (420 µM leucine) or in DMEM F12 lacking leucine (0 µM leucine) and then were harvested for preparation of cell extracts and determination of LUC activity. Relative LUC activities were determined as described in Materials and Methods. The relative fold induction, defined as the ratio of the relative LUC activity of leucine-starved cells to unstarved cells, is indicated in parentheses above the bars.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The mammalian plasma concentration of free amino acids shows striking alterations according to nutritional and pathological conditions (3, 13, 64). In recent years, evidence has accumulated that amino acids play an important role in controlling gene expression (30, 32). Nevertheless, the molecular mechanisms involved in the amino acid regulation of mammalian gene expression have not been elucidated to date. The work presented in this report focuses on the identification of the cis- and trans-acting elements which are involved in the transcriptional activation of CHOP in response to amino acid starvation.

The cis DNA sequence located upstream from the transcription start site (313 to 295) is essential for amino acid activation of the CHOP promoter. This sequence is the first described that can regulate a basal promoter in response to starvation of several individual amino acids, in all human cell lines tested (HeLa, Caco-2, and HepG2). This DNA element is conserved in the CHOP gene in species other than humans, such as the hamster (46). Mutations affecting a stretch of nine nucleotides (AARE core) result in a loss of amino acid responsiveness. From these properties, this DNA sequence can be called an AARE. It appears that sequences outside the AARE core could also play an important role in achieving the full amino acid induction. Indeed, mutation mt7 outside the core sequence (see Fig. 4A) results in a slight decrease in amino acid inducibility, although the effect of this mutation appears to be inhibited in mutation mt9.

Sequences of the CHOP AARE region show some homology with the specific binding sites of the C/EBP and ATF/CREB transcription factor families (15, 63). All members of these families contain a DNA binding domain consisting of a cluster of basic amino acids and a leucine zipper region (b-ZIP domain) (33). They can form homodimers or heterodimers through their leucine zipper regions (17) and then bind to both ATF and C/EBP recognition sites (1, 4). Depending on the composition of the heterodimer, different sequence elements are preferentially recognized, leading to variable transcriptional effects (10, 58). Our present results demonstrate that although the CHOP AARE sequence binds C/EBP and ATF-2 in vitro, only ATF-2 has a critical role in the transcriptional activation of CHOP by leucine starvation. ATF-2 is capable of interacting with other b-ZIP proteins and binding to particular recognition sequences (22, 28, 29, 50). Our data suggest that ATF-2 could bind to the CHOP AARE as homodimer or as heterodimer with an unknown dimerization partner. cis-acting elements which are recognized by ATF-2 in association with other known transcription factors are not able to activate transcription in response to amino acid starvation. For example, the c-jun promoter contains two AP-1-like sites that are critical for its regulation by certain stresses and some other stimuli (2, 44, 57). In vitro binding studies reveal that in most cells examined, proximal (jun1TRE) and distal AP-1 (jun2TRE) sites bind heterodimers composed of c-Jun and ATF-2 (58, 59). Although the CHOP AARE shares significant similarities with the jun2TRE (Fig. 5A), c-Jun is not able to bind to the AARE in vitro (Fig. 6, lane 7). In addition, we demonstrate that jun2TRE sequences do not confer amino acid inducibility (Fig. 5B). Therefore, the association of ATF-2 and c-Jun does not mediate the response to amino acids. Further work will be required to determine whether another partner associated with ATF-2 is important in the mechanism of transcriptional activation by amino acid starvation.

We do not observe differences in the AARE binding activity of ATF-2 between leucine-starved and nonstarved conditions. The transactivating capacity of ATF-2 is activated via phosphorylation of N-terminal residues Thr-69, Thr-71, and Ser-90 by stress-activated protein kinases (19, 38, 59). We show that the ATF-2Ala dominant negative mutant in which these three residues cannot be phosphorylated inhibits full up-regulation of CHOP promoter activity induced by leucine starvation (see Fig. 9B). Moreover, when cotransfected into ATF-2^/ cells, this dominant negative mutant is not able to rescue the amino acid inducibility of the CHOP promoter. Taken together, these data suggest that the specific amino acid pathway that leads to transcriptional activation of CHOP may involve phosphorylation of prebound ATF-2 rather than an increase in ATF-2 binding (19).

The signaling pathways that recognize amino acid availability in mammalian cells have not been investigated as extensively as those in bacteria or yeast. We show that ATF-2 is essential in the amino acid pathway that leads to the induction of CHOP mRNA and confirm that this specific pathway is independent of the unfolded protein response. ATF-2 has been shown to be ubiquitously expressed, with the highest level of expression being observed in the brain (52), but the physiological role of ATF-2 remains poorly understood. Knockout mice generated by gene targeting exhibited lowered postnatal viability and growth, with a severe respiratory distress (39) or with a defect in endochondral ossification and a reduced number of cerebellar Purkinje cells (47). The data presented in this paper suggest that modulation of ATF-2 activity could also play an important role in the process of defense or adaptation to amino acid limitation that occurs in nutritional or pathological conditions.

The concept that amino acids can regulate gene expression has just started to emerge. It is now clear that amino acids can play an important role in the control of gene expression in concert with hormones. However, the underlying mechanisms have only begun to be discovered. Our results show that the transcriptional activity of ATF-2 is essential for the regulation of CHOP expression by amino acids. The physiological meaning of CHOP regulation by amino acids is not yet understood. Through its interaction with C/EBP transcription factors, CHOP may participate in the regulation of several mechanisms (43) during cellular response to amino acid limitation. For example, CHOP could play a crucial role in the regulation of nitrogen metabolism under amino acid control, although a direct role for CHOP in this pathway has yet to be demonstrated. During a cellular stress that perturbs function of the endoplasmic reticulum (ER stress), CHOP expression, in concert with a second signal, was found to be absolutely required for the activation of a set of previously undescribed genes referred to as DOCs (for downstream of CHOP) (62). We have hypothesized that CHOP may also mediate the induction of cellular DOC genes in the context of the amino acid response, but the identification of such genes remains to be done.

Defining the precise cascade of molecular events by which the cellular concentration of an individual amino acid regulates gene expression will be an important contribution to our understanding of metabolite control in mammalian cells. These studies will provide insight into the role of amino acids in the regulation of cellular functions like cell division, protein synthesis, and proteolysis.