Department of Pathology and Kaplan Cancer Center, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA
Correspondence to: D. Meruelo
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Abstract |
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Keywords: H-2 BF1, H-2Dd, CREB, cis-regulatory element, trans-activating factor
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Introduction |
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We previously demonstrated that in Radiation leukemia virus (RadLV)-infected thymocytes, increased cell surface expression of H-2Dd results from elevated levels of mRNA transcription and that the transcriptional increase correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognized a 5' flanking cis-sequence, 5'-TGACGCG-3', of the H-2Dd gene (9,10). This cyclic adenosine-3',5'-monophosphate (cAMP) regulatory element (CRE)-like sequence, named H-2 BF1 binding motif, is highly conserved among species in the 5' flanking region of MHC class I genes (11,12). Recently, we also demonstrated that H-2 BF1 consists of homo- and heterodimers of activation transcription factor 1 (ATF-1) and CRE binding protein (CREB), and that the expression of mRNA encoding these factors was increased in the thymocytes following RadLV infection (12,13). Moreover, transfection experiments showed that ATF-1 and CREB activated the transcription of a reporter gene, which followed the H-2Dd promoter containing the H-2 BF1 motif (12,13). Increased H-2Dd expression on the RadLV-infected thymocytes also correlated with the DNA binding activity of H-2 BF1 (13). However, it is not known whether the positive transcriptional regulation mediated by the H-2 BF1 complexes, consisting of the H-2 BF1 binding motif and ATF-1/CREB trans-acting factors, has universal validity in the regulation of constitutive and/or variable MHC class I expression.
In the present studies, we have chosen to utilize a dominant negative molecule of ATF-1, which had been originally isolated and designated ATF-1DN. Through its stable expression in a human epidermoid carcinoma cell line, A431, ATF-1DN could quench factors capable of binding to the H-2 BF1 motif and impacted on MHC antigen expression in these cells. A comparative study of class I antigen levels between the stable transfectants and control cells lacking ATF-1DN can provide clues to the physiological role of H-2 BF1 complexes in the regulation of MHC gene expression.
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Methods |
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In vitro protein synthesis
The plasmids, pSG5/ATF-1 and pSG5/CREBD (CREB-Delta), were described elsewhere (13). A fragment containing the ATF-1DN coding sequence was obtained by digestion of pBK-CMV/ATF-1DN with SacII and HindIII, and was subcloned into the corresponding sites of pSG5/ATF-1, resulting in construction of pSG5/ATF-1DN. Using these expression plasmids as templates, ATF-1DN, ATF-1 and CREBD were synthesized in vitro by TNT T7-coupled Wheat Germ Extract System (Promega, Madison, WI). For labeling of proteins, [35S]methionine (0.04 mCi) (NEN/Life Science, Boston, MA) was included in the reaction.
Western blot analysis
The in vitro translated products in sample buffer containing 1% 2-mercaptoethanol were boiled for 5 min, electrophoresed through 12% SDSpolyacrylamide gel and then transferred to a nitrocellulose membrane. Non-specific binding of the following antibodies was blocked by incubation of the filter in 20 mM TrisHCl buffer (pH 7.5) containing 150 mM NaCl, 0.05% Tween 20, 5% non-fat dry milk and 2% BSA for 8 h at 4°C. The filter was then incubated with the buffer in the presence of 2.5% non-fat dry milk, 1% BSA and 1 µg/ml of a mouse monoclonal anti-ATF-1 antibody, 25C10G (Santa Cruz Biotechnology, Santa Cruz, CA), for 16 h at 4°C. After being washed with PBS for 1 h at room temperature, the filter was incubated with the buffer containing 2.5% non-fat dry milk, 1% BSA and 1:20,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG antiserum (Pierce, Rockford, IL) for 4 h at 4°C. After removing the excess secondary antibody by washing with PBS for 1 h at room temperature, the immobilized peroxidase activity was visualized using SuperScript Substrate (Pierce).
Far-Western blot analysis
The nitrocellulose filter which the in vitro products were transferred to was washed with 20 mM HEPES buffer (pH 7.4) containing 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 0.1 mM ZnCl2, 1 mM DTT and 10% glycerol for 4 h at 4°C. The filter was then incubated with the buffer in the presence of 5% BSA for 4 h at 4°C and probed with in vitro made [35S]methionine-labeled ATF-1DN in the buffer containing 2.5% BSA for 16 h at 4°C. Excess probe was removed by washing the filter in the buffer containing 0.1% Nonidet P-40 for 1 h at room temperature. The filter was then dried and autoradiographed.
Electrophoretic mobility shift assays (EMSA)
Preparation of the H-2 BF1 probe was described previously (12,13). In brief, an oligonucleotide containing the H-2 BF1 motif (5'-CACTGATGACGCGCTG-3') and its complement were synthesized, and then annealed. The double-stranded oligonucleotide was 5' end-labeled with [-32P]ATP (0.15 mCi) (NEN/Life Science) by T4 polynucleotide kinase (New England Biolabs, Beverly, MA). The labeled oligonucleotide (2x105 c.p.m.) was incubated with the in vitro products or nuclear extracts (described below) in the presence of 5 µg Double-Strand POLY(dIdC) (Amersham Pharmacia Biotech, Piscataway, NJ) and 5 µg BSA for 40 min on ice, and then electrophoresed through non-denaturing 6% polyacrylamide gels with 0.5xTBE (Trisborate/EDTA).
Selection of ATF-1DN stable transfectants
A human epidermoid carcinoma cell line, A431 (ATCC, Rockville, MD), was grown in DMEM (Gibco/BRL, Gaithersburg, MD) with 10% FBS. Sequences between the NheI and SpeI sites were removed from pBK-CMV/ATF-1DN to avoid translational initiation for ß-galactosidase. A431 cells (5x105) in a 6-cm dish (Becton Dickinson, Franklin Lakes, NJ) were transfected with 2 µg of the modified pBK-CMV/ATF-1DN vector accompanied with 6 µl of LipofectAMINE (Gibco/BRL). In parallel, the modified pBK-CMV was used to generate control transfectants. The transfected cells were selected with G418 at 0.6 mg/ml for 2 weeks.
Primers for PCR
For PCR, a sense primer, s-ATF (5'-ATGGAAGATTCCCACAAGAGTA-3'), and three antisense primers, a-ATF (5'-TCAAACACTTTTATGAGAAT-3'), a-1 (5'-ACGGTCTGCGGGAGAGACGT-3') and a-DN (5'-ACTGTCTGTGGGAGTAACGT-3'), were synthesized. The primers s-ATF and a-ATF correspond to the nucleotides from 1 to 22 and 791 to 810 respectively of the coding sequence for ATF-1 (see Fig. 1 and Table 1
). The other two primers, a-1 and a-DN, reflect the different sequences between ATF-1 and ATF-1DN corresponding to the ORF nucleotides from 556 to 575.
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Set-up for PCR
All PCR were carried out using Ready To Go PCR beads (Amersham Pharmacia Biotech). After heating for 5 min at 95°C, the reaction tubes proceeded to 30 cycles of the amplification step (95°C 1 min, 55°C 1 min and 72°C 2 min) in the DNA Thermal Cycler (Perkin-Elmer, Norwalk, CT). The PCR products were electrophoresed through 1.5% agarose gels and visualized by the ethidium bromide staining.
Hybridization with ATF-1 oligonucleotide probe
An oligo DNA corresponding to the nucleotides from 151 to 170 of the ORF for ATF-1, which is identical to ATF-1DN, was synthesized and then labeled with [-32P]ATP (0.15 mCi) (NEN/Life Science) by T4 polynucleotide kinase (New England Biolabs). The labeled oligonucleotide (1x106 c.p.m./ml) was hybridized to the nitrocellulose membrane, to which the RT-PCR products were transferred, for 16 h at 42°C.
Extraction of nuclear proteins
A431 cells and the neomycin-resistant clones were lysed in 10 mM TrisHCl buffer (pH 7.4) containing 150 mM NaCl, 0.5% NP-40 and 0.2 mM PMSF. Nuclear pellets were washed once with PBS, collected by microcentrifugation, and then lysed in 25 mM Tris-HCl buffer (pH 7.9) containing 50 mM KCl, 12 mM MgCl2, 0.5 mM EDTA, 10% glycerol, 1 mM DTT and 0.4 mM PMSF using a 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA). Tubes were kept on ice during the operation to avoid heating. Protein concentration was determined as described (12,13) and the EMSA were performed as above. For a supershift assay, the anti-ATF-1 (25C10G; Santa Cruz) and anti-ATF-2 (C-19; Santa Cruz) antibodies were admixed in the reaction.
Flow cytometry
The stable transfectants and control cells were stained with or without a mouse monoclonal anti-HLA ABC antibody, W6/32 (Biodesign International, Kennebunk, ME), followed by FITC-conjugated rabbit anti-mouse IgG antiserum (Southern Biotechnology Associates, Birmingham, AL). To rule out the non-specific binding of mouse Ig, another unrelated mouse mAb of subclass IgG2a (Santa Cruz) was used as an isotype-matched control for W6/32. After manipulating 1x104 cells, live cells defined by scatter gates were analyzed by the FACScan (Becton Dickinson). Alteration of MHC class I expression was calculated as (A' B')/(A* B*) and represented as Class I Expression Index. A and B represent the mean intensity of the MHC class I expression and of the background staining for the secondary antibody respectively. The apostrophe and asterisk indicate that the data belong to the ATF-1DN expressing and control cells respectively. Experiments were repeated 4 times and then the data were statistically analyzed. Difference was considered to be significant when P < 0.05.
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Results |
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During mouse genomic screening for ATF-1, we isolated a 1.3 kb fragment which consisted of a similar sequence to the ATF-1 cDNA (15). The genomic sequence, designated ATF-1DN, contained the whole ORF for ATF-1. Since there was no intron in the cDNA-mimicking sequence, it appears that ATF-1DN may be a pseudogene, although its constitutive and/or inducible expression in vivo has not yet been ruled out. In the coding sequence, there were 13 nucleotide replacements, resulting in 7 amino acid substitutions (Table 1). Five of the changed amino acids were in the transactivation domain and two others were found in the DNA binding domain. The P-Box in the transactivation domain, containing putative phosphorylation sites for PKA and possibly other protein kinases, was not affected. A mutation in the basic region of the DNA binding domain caused alteration of arginine 217 to glutamine. Arginine 217 is conserved in the homologous regions of CREB and CRE modulators (14), which are closely related to ATF-1, suggesting its importance for DNA binding activity. Another amino acid mutation appeared between leucines 239 and 246 in the leucine zipper motif. Based on the mutated sequence, we presumed that ATF-1DN, if translated, could dimerize with wild-type factors through the intact leucine zipper motif and block their ability to bind to DNA due to the alteration in the DNA binding region.
ATF-1DN encodes an analogous protein of ATF-1
At first, we examined whether ATF-1DN encodes a viable protein, by the in vitro transcription and translation system using [35S]methionine. Autoradiography of the in vitro translated products separated through SDSpolyacrylamide gel indicated that a protein slightly smaller than wild-type ATF-1 was made from pSG5/ATF-1DN (Fig. 2A). Figure 2
(A) also shows that there was not much difference in the transcription and translation efficiency among ATF-1DN (Fig. 2A
, lane 2), ATF-1 (Fig. 2A
, lane 3) and CREBD (Fig. 2A
, lane 4). Immunoblotting by an anti-ATF-1 antibody revealed that the product derived from pSG5/ATF-1DN contained a reactive epitope for the antibody (Fig. 2B
, lane 2). These results indicated that an analogous protein of ATF-1 could be made from the ATF-1DN gene.
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A preliminary EMSA showed that when H-2 BF1 complexes were formed in advance in vitro, ATF-1DN could not separate ATF-1 and CREBD from the H-2 BF1 oligonucleotide (data not shown). This means that transient transduction of ATF-1DN would not fully display the repressor activity for the endogenous ATF-1/CREB factors, if the H-2 BF1 complexes are already formed in the cells. Thus we planned to produce transfectants that constitutively express the ATF-1DN gene. To generate stable transfectants, A431 cells were transfected with the ATF-1DN gene linked with a neomycin-resistant gene. The transfected cells were exposed to G418 (0.6 mg/ml) for 2 weeks, resulting in establishment of two clones, named A431/DN2 and A431/DN4. As a control, A431 cells were also transfected with a parent vector that lacks the ATF-1DN gene.
ATF-1DN expression in the neomycin-resistant clones
Genomic DNA and total RNA was extracted from the neomycin-resistant clones including the control cell line, A431/Neo. The quality of RNA preparations was assessed by RT-PCR for the wild-type ATF-1 (using s-ATF and a-ATF as a sense and antisense primer respectively, data not shown). Expression of the dominant negative ATF-1 in the selected clones was assayed with antisense primers that could distinguish between ATF-1 and ATF-1DN. The antisense primers were synthesized on the basis of differences in their sequences (a-1 and a-DN for ATF-1 and ATF-1DN respectively). Specificity of the primers in PCR was examined, using plasmids containing the wild-type and mutant ATF-1 as templates. When utilized with a same sense primer (s-ATF), a-DN but not a-1 could distinguish ATF-1DN from ATF-1 (Fig. 4A). According to these results, cDNA pools were synthesized by reverse transcriptase reaction from the total RNA and then the portion of ATF-1DN was amplified using a-DN. To confirm specific amplification, the RT-PCR products were hybridized with an oligonucleotide probe designed for the predicted fragment. Specific hybridization of the probe was seen in A431/DN2 and DN4 cells but not in A431/Neo cells (Fig. 4B
). DNase treatment of the RNA templates revealed that the predicted PCR products were generated from the synthesized cDNA and not from possible genomic DNA contamination (data not shown).
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Correlation between the H-2 BF1 activity and cell surface MHC class I expression
To clarify the correlation between the H-2 BF1 activity and cell surface MHC class I expression, A431/Neo, DN2 and DN4 cells, growing under normal conditions, were stained with an anti-HLA-ABC antibody, followed by an fluorescence-conjugated secondary antibody for the flow cytometry. Experiments were repeated 4 times, and alteration of MHC class I expression on A431/DN2 and DN4 cells was assessed as described in Methods, and then compared with A431/Neo cells. The MHC class I expression of A431/DN4 cells, which showed a remarkable reduction of H-2 BF1 activity, was significantly higher than A431/Neo cells (Table 2), while no significant increase was observed in A431/DN2 cells. Contrary to our expectations, these observations clearly indicate that the H-2 BF1 complexes negatively regulate the constitutive expression of MHC class I in these cells.
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Discussion |
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Detailed analysis of the mouse MHC class I promoter has revealed that several cis-regulatory elements in the 5' flanking regions are important for the expression and regulation of these genes through the sequence-specific binding of trans-activating nuclear proteins (reviewed in 17). Some combinations of the cis- and trans-factors elicit positive signals for the gene transcription, while others provoke negative signals. It seems likely that the fine tuning of MHC class I gene expression is modulated by the balance of these dual signals under physiological and pathological circumstances.
We have demonstrated that ATF-1 and CREB, associating with the 5' flanking cis-sequence, 5'-TGACGCG-3', termed H-2 BF1 binding motif, were involved in the positive regulation of H-2Dd gene expression following RadLV infection of mouse thymocytes (913). In order to examine the physiological role of ATF-1/CREB transcription factors in the regulation of MHC class I gene expression, we utilized a dominant negative molecule of ATF-1 (ATF-1DN) which had been isolated from a mouse genomic library. Recently, Shimomura et al. constructed a mutant of ATF-1 by the site-directed mutagenesis method (18). The targeted mutation was arginine 229 to leucine in the DNA binding domain. The mutant functioned as a dominant negative molecule for CREB, indicating that alteration in the DNA binding domain was critical for the DNA recognition. It seems likely that the mutation of arginine 217 to glutamine in the DNA binding domain of ATF-1DN may also prevent binding to the target DNA, although the significance of other mutations in the transactivation domain remains to be determined.
Our findings indirectly indicate that ATF-1DN is associating with the wild-type factors. (i) Although ATF-1DN itself does not bind to the H-2 BF1 motif (Fig. 3, lane 10), it functions as a dominant repressor for the wild-type ATF-1/CREB transcription factors. (ii) In the EMSA using the in vitro translated products, formation of the DNA/CREBD complex was suppressed with a lower ratio of ATF-1DN than the DNA/ATF-1 complex formation (cf. Fig. 3
, lanes 2, 6 and lanes 3, 7). The latter finding may reflect the fact that ATF-1 molecules prefer to form heterodimers with CREB rather than to form homodimers (13), thus suggesting interaction between ATF-1DN and the wild-type ATF-1/CREB transcription factors. Although Far-Western blot analysis could not show binding of ATF-1DN to wild-type ATF-1 and CREBD transferred to the nitrocellulose membrane (data not shown), this result does not necessarily mean that ATF-1DN is unable to bind to the wild-type factors under native conditions; denaturation of ATF-1 and CREBD through SDSPAGE might prevent association with ATF-1DN.
Murine ATF-1DN could quench human endogenous ATF-1/CREB trans-acting factors which bind to the H-2 BF1 oligonucleotide, reflecting a high homology of mouse ATF-1 to human factors, ATF-1 (19) and CREB (20). The inhibition of ATF-1/CREB trans-acting factors through stable transfection of the dominant negative ATF-1 to A431 cells resulted in an increase of cell surface MHC class I expression. This finding indicates that the endogenous ATF-1/CREB trans-acting factors associate with the H-2 BF1 binding motif in the 5' regulatory region of MHC genes under the normal growth conditions and mediate a suppressive signal for the constitutive class I expression in A431 cells. In the RadLV infection models, an increase of H-2Dd antigen causes the development of cell-mediated immune responses to eliminate the RadLV-infected thymocytes (21). Although the H-2 BF1 binding motif is highly conserved among strains (11,12), RadLV infection does not induce H-2 expression in H-2 haplotypes other than Dd and a high percentage of these strains go on to develop thymomas (22). This strain dependency suggests that the signal through the complexes of H-2 BF1 binding motif and ATF-1/CREB trans-factors does not always enhance the transcription of the gene. In addition, it has been shown that the H-2 BF1 motif in the rat MHC class I genes mediates thyrotropin/cAMP-induced silencer activity in the thyroid cells (23). Therefore, opposite signals are possibly triggered through the H-2 BF1 motif in different haplotypes of MHC genes and/or type of cells.
These data lead us to propose that ATF-1, and its associated proteins including CREB, may contribute to a malignant phenotype via suppression of the MHC class I gene expression in tumor cells. Recently, it has been shown that human melanoma cell lines carrying the dominant negative CREB decreased their radiation resistance and anchorage-independent growth in vitro (24), and their tumorigenic and metastatic potential in nude mice (25). The reduction of malignancy was interpreted to be caused by down-regulation of collagen type IV and cell surface adhesion molecule MCAM/MUC18 mRNA expression; both genes contained the binding site for the CREB transcription factors (25). The dominant negative CREB expression in melanoma cells also rendered them susceptible to apoptosis induced by the cytosolic calcium increases (26). On the other hand, it has been demonstrated that the MHC class I gene transfection to a human glioblastoma cell line resulted in suppression of the anchorage-independent growth (27). It would be interesting to determine whether MHC class I expression is also increased in these melanoma cells. At this time, it has not been determined if ATF-1 and CREB would mediate the increased malignant potential in tumor cells other than melanoma. Our results suggest that transfection of dominant negative ATF-1 into tumor cells would be one strategy to address this issue.
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Acknowledgments |
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Abbreviations |
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ATF-1 activation transcription factor 1 |
cAMP cyclic adenosine-3',5'-monophosphate |
CRE cAMP regulatory element |
CREB CRE binding protein |
EMSA electrophoretic mobility shift assay |
H-2 BF1 H-2 binding factor 1 |
ORF open reading frame |
PKA protein kinase A |
RadLV Radiation leukemia virus |
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Notes |
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Received 5 July 1999, accepted 14 October 1999.
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References |
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