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The Journal of Immunology, 1998, 160: 5907-5914.
Copyright © 1998 by The American Association of Immunologists

The Regulation of Murine H-2Dd Expression by Activation Transcription Factor 1 and cAMP Response Element Binding Protein1

Nobuhisa Ishiguro, G. Dalon Brown, Akihiro Ishizu and Daniel Meruelo2

Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, NY 10016


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Resistance to radiation leukemia virus (RadLV)-induced leukemia is correlated with an increase in H-2Dd expression on the thymocyte surface. It has been shown that elevated H-2Dd expression on infected thymocytes is a result of elevated mRNA transcription and that the transcriptional increase is correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognizes the 5'-flanking sequence (5'-TGACGCG-3') of the H-2Dd gene. Recently, it has been shown that the activation transcription factor 1 (ATF-1) homodimer is one form of the H-2 BF1 complex. Here we demonstrate that the cAMP response element binding protein (CREB) homodimer and the heterodimer of CREB/ATF-1 also recognize the cis regulatory motif and are two additional forms of the H-2 BF1 complex. The levels of mRNA encoding ATF-1 and CREB were both increased in RadLV-infected thymocytes that showed increased levels of H-2 mRNA. Also, all three H-2 BF1 binding activities, ATF-1 homodimer, CREB homodimer, and ATF-1/CREB heterodimer, were increased in RadLV-infected thymocytes that expressed high levels of H-2Dd Ag on the cell surface. Transfection experiments demonstrated that ATF-1 and CREB activated a reporter plasmid containing the H-2 BF1 motif. These observations strongly suggest that both ATF-1 and CREB are involved in the regulation of H-2 gene expression following RadLV infection of mouse thymocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The MHC encodes highly polymorphic cell surface Ags that play an important role in recognition and elimination of virus-infected or neoplastic cells by the immune system (1, 2). Murine H-2 class I gene expression has been shown to be significantly altered in thymic cells after both radiation leukemia virus (RadLV)3 infection and RadLV-induced transformation (3). Very soon after infection of mice, there is a dramatic increase in the levels of MHC Ag on the surface of infected thymocytes. Resistant strains of mice (H-2Dd haplotype) develop a cell-mediated immune response, while no such reaction is detectable in susceptible hosts (4). Subsequently, the infected cells are eliminated from the resistant mice, and only a very small percentage of these animals develop tumors (4). Susceptible mice (strains other than the Dd haplotype) fail to recognize the infected cells immunologically, and a high percentage go on to develop thymomas. These findings clearly indicate the importance of immunosurveillance in the phenotypic response (resistance vs susceptibility) to RadLV-induced tumorigenesis.

An examination of the stimulation in H-2Dd expression at the molecular level revealed that infection by RadLV resulted in the increased transcription of H-2 genes in the thymocyte (5, 6). Nuclear extracts of infected thymocytes contained an increased DNA binding activity, termed H-2 binding factor 1 (H-2 BF1), which specifically recognized the sequence 5'-TGACGCG-3' in the 5'-flanking region of the H-2Dd gene (5, 6). This cAMP response element (CRE)-like cis sequence was recognized earlier as being involved in class I regulation (6, 7, 8, 9). In these studies, however, the trans factor(s) involved was not identified. Recently, we have demonstrated that activation transcription factor 1 (ATF-1) is one component of the H-2 BF1 complex in vivo (10).

Originally, ATF-1 was cloned by screening cDNA libraries for binding to dsDNA probes containing the CRE sequence (11, 12). Both ATF-1 and CRE binding protein (CREB) have been grouped functionally into the bZIP family of trans-activating proteins, which is characterized by the response to cAMP-dependent protein kinase (13, 14, 15), and have been shown to form either homodimers or a heterodimer and to bind to the CRE motif (13). The CRE binding motif is known to be essential for basal transcriptional activity of many promoters, and in some instances direct evidence has been obtained that CREB can activate the promoter (16). ATF-1 has also been shown to be involved in the expression of numerous genes via the CRE motif; these genes include calcitonin (17), Na,K-ATPase {alpha}1 subunit (18), and IL-1ß (19). In the experiments described here, we show that CREB is another component of the H-2 BF1 complex, forming CREB homodimers and CREB/ATF-1 heterodimers and functioning as a potential regulator of MHC class I expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, virus, and cells

B10.T(6R) mice resistant to RadLV were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred at New York University Medical Center. RadLV was prepared as cell-free extracts of thymomas (20). Preparation of infected thymocytes for RNA preparation and nuclear extraction was previously described (6). Cell lines were derived from RadLV-induced thymomas (21). F9 cells were maintained on gelatin-coated dishes in DMEM with 10% calf serum.

Antisera and FACS analysis

H-2 alloantiserum 056 ((B10.BRxA.SW)F1 anti-B10.S(7R)) has been shown to react specifically with H-2Dd (20, 22). FITC-conjugated rabbit anti-mouse IgG was purchased from Pierce (Rockford, IL). Samples were prepared for FACS as previously described (6) and were analyzed using the FACScan (Becton Dickinson Immunocytometry System, Mountain View, CA).

Northern blot analysis

Total RNA isolation, Northern blotting, and hybridization were conducted as previously described (23, 24). The hybridization probes used were H-2 IIa (sub), a broadly cross-reactive cDNA subclone of H-2 IIa lacking the repetitive sequences (25, 26); ATF-1, excised by BamHI and HindIII from the ATF-1A clone (10); and ß-actin (27).

RT-PCR

Complementary DNA was synthesized from 5 µg total RNA in 20-µl reactions using 200 U of Superscript II RNase H- RT (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. After incubation for 1 h at 42°C, the samples were heated at 70°C for 15 min and diluted to 100 µl with low TE (10 mM Tris-HCl (pH 7.5) and 0.1 mM EDTA). Five microliters were used for the 50-µl PCR solution containing 50 mM KCl, 100 mM Tris-HCl (pH 8.3), 1.5 mM (for the ß-actin primer set) or 1.0 mM (for the CREB primer set) MgCl2, 0.001% (w/v) gelatin, 0.2 mM of each dNTP, 0.4 µM of each primer (see below), and 2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT). In experiments in which PCR products were quantified, 4 µl of [{alpha}-32P]dCTP (370 MBq/ml; New England Nuclear Research, Boston, MA) was also added, and the complete reaction mixture was divided into 10-µl aliquots. Primers were designed as follows; mouse ß-actin (28), 5'-TCAGAAGGACTCCTATGTGG-3' (sense) and 5'-TCTCTTTGATGTCACGCACG-3' (antisense); and mouse CREB (29), 5'-CCAGTCTCCACAAGTCCAAACAG-3' (sense) and 5'-GGCACTGTTACAGTGGTGATGG-3' (antisense). The ß-actin primer cycling conditions used after heating for 5 min at 94°C were 45 s at 94°C, 45 s at 62°C, and 2 min at 72°C. For CREB primers, after 5 min at 92°C, cycling conditions were 1 min at 92°C, 1 min at 50°C, and 1.5 min at 72°C. After 15, 18, 21, 24, and 27 cycles (for the ß-actin primer set) or after 25, 27, 29, 31, and 33 cycles (for the CREB primer set), aliquot tubes were removed from the DNA Thermal Cycler (Perkin-Elmer), and 20% of the aliquot was electrophoresed through either 1% agarose or 10% polyacrylamide gels. Autoradiograms were analyzed, and bands were quantified by the National Institutes of Health Image Program, version 1.60 (Bethesda, MD). Aliquots were also removed, and 32P-labeled PCR product was measured in duplicate by TCA precipitation (28).

Gel mobility shift assays

Nuclear extracts were prepared according to the method of Dignam et al. (30). The protein concentration was determined using the Bio-Rad protein assay (Richmond, CA). The following oligonucleotide and its complement containing the H-2 BF1 motif were synthesized: 5'-CACTGATGACGCGCTG-3'. Complementary oligonucleotides containing the mutant H-2 BF1 motif were also synthesized: 5'-CACTGATGAAGAGATG-3'. Equimolar amounts of these oligonucleotides were annealed with their complement and 5' end labeled by T4 polynucleotide kinase (New England Biolabs, Beverly, MA). The labeled oligonucleotides were incubated with either the nuclear extracts or the in vitro translation products and electrophoresed through nondenaturing 5 or 6% polyacrylamide gels with 0.5x TBE.

Gel mobility supershift

For gel supershift assays, specific Abs were mixed with binding reactions as described above, incubated for 1 h on ice, and loaded onto the gel. Specific mAbs against ATF-1 (product no. sc-270x and sc-243x), ATF-2 (product no. sc-187x), and c-Jun (product no. sc-45x) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Specific polyclonal Ab against CREB (product no. 06-504) was purchased from Upstate Biotechnology (Lake Placid, NY).

In vitro protein synthesis

ATF-1A and ATF-1B (10), CREB{alpha} and CREB{Delta} (29), and CREM{alpha} (31) cDNAs were subcloned into pSG5 (Stratagene, La Jolla, CA), and the corresponding proteins were synthesized in vitro by the TNT T7-coupled Wheat Germ Extract System (Promega, Madison, WI) according to the manufacturer’s instructions. Unmodified pSG5 was also used for in vitro translation as a negative control. The pmcCREB{alpha} and pmcCREB{Delta} plasmids were provided by Dr. G. Schütz (29).

Western blot analysis

Nuclear extracts from the RadLV-induced thymoma-derived cells and the in vitro translated ATF-1 and CREB{Delta} were treated with or without calf intestinal alkaline phosphatase (New England Biolabs) as previously described (32, 33). Samples containing 1% 2-ME were boiled for 5 min, electrophoresed through 12% SDS-polyacrylamide gel, then transferred to a nitrocellulose membrane. Immunodetection for ATF-1 was performed using the sc-270x Ab.

Plasmid construction and transfection assays

The expression plasmids pRc/RSV/ATF-1 and pRc/RSV/CREB{Delta} were generated by subcloning ATF-1A and CREB{Delta} cDNA, respectively, into pRc/RSV (Invitrogen, Carlsbad, CA). The plasmid pRc/RSV/1-FTA contained the ATF-1A cDNA in the antisense orientation. Plasmid {Delta}-122 CAT, containing sequences 5' of the mouse H-2Dd gene (-122 to +20), which includes the H-2 BF1 binding motif 5'-TGACGCG-3' (-99 to -93), linked to the gene for chloramphenicol acetyltransferase (CAT), was a gift from Dr. I. Stroynowski (34). Since it has been shown that ATF-1 and CREB are activated upon phosphorylation by cAMP-dependent protein kinase A (PKA) (13), we obtained the mammalian expression vector for the catalytic subunit of the cAMP-dependent protein kinase, RSV-CHO-PKA-C{alpha}, version 2, from Dr. R. A. Maurer (35). F9 cells (5 x 105/10-cm dish) were transfected using lipofectamine (Life Technologies) according to the manufacturer’s instructions. The DNAs for each transfection contained 3 µg of reporter plasmid ({Delta}-122 CAT); 2 µg of RSV-CHO-PKA-C{alpha}, version 2, or pBluescript II SK+ (Stratagene); 2.5 µg of pRc/RSV/ATF-1, pRc/RSV/CREB{Delta}, or pRc/RSV/1-FTA; and 1 µg of pSVß-galactosidase control vector (Promega) as an internal reference. The total amount of plasmid DNA was adjusted to 11 µg/transfection. After 40 h, extracts were prepared, and CAT assays were performed as previously described (36). Acetylated chloramphenicol was measured using the National Institutes of Health Image Program from autoradiographed film, and results were corrected for ß-galactosidase activity to normalize for transfection efficiency (37).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ATF-1 and CREB bind to the H-2 BF1 binding motif

Binding to the dsH-2 BF1 oligonucleotide (wt) was detected in the nuclear extracts from the RadLV-induced thymoma-derived cell line (Fig. 1Go, lane 1) and resulted in three bands (a–c). No translational by-product bound nonspecifically to the oligonucleotide. In vitro translation products were incubated with the oligonucleotide and electrophoresed on the same nondenaturing polyacrylamide gel. Lane 2 shows the results where only the ATF-1 cDNA translation product was used in the binding assay. Band c has been shown to be made up of ATF-1 homodimers (10). The faster migrating smear observed in lane 2 (also seen in lane 4) is believed to be a result of truncated forms of the ATF-1 protein; these truncated proteins were evident when [35S]methionine-labeled in vitro translation reactions were analyzed by denaturing SDS-PAGE (data not shown). Lane 3 shows the resulting band when CREB{Delta} cDNA was translated alone. This band comigrated with band a in lane 1 and is believed to be CREB homodimers. Likewise, the newly formed band seen when the two translation products were mixed (lane 4) comigrated with band b in lane 1. This band would be expected to represent ATF-1/CREB heterodimers. These results are in agreement with previous studies in which homodimer and heterodimer formation of the ATF-1 and CREB proteins were documented (13).



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  		FIGURE 1. Gel mobility assays using oligonucleotides corresponding to the H-2 BF1 binding motif. End-labeled oligonucleotide (1 x 105 cpm) was incubated with either nuclear extracts or in vitro translation products. Lane 1, 1 µg of nuclear extract from RadLV-induced thymoma-derived cell line; lane 2, in vitro products of ATF-1A cDNA subcloned into pSG5; lane 3, in vitro products of CREB{Delta} cDNA subcloned into pSG5; lane 4, mixture of in vitro products of ATF-1A and CREB{Delta}. The mixture of the in vitro translated ATF-1A and CREB{Delta} was heated to 42°C, cooled slowly to 4°C, and used for the gel shift. Samples were electrophoresed through nondenaturing 6% polyacrylamide gels at 150 V for 7 h.

 
Specificity of the binding was demonstrated by competition assays in that the addition of unlabeled wt oligonucleotide at 50-, 100-, and 150-fold molar excesses resulted in a reduction of all bands in lane 1 (Fig. 2Go, A–D, lanes 2–4, respectively), while the addition of unlabeled mutant oligonucleotide had little effect on the binding (lanes 5–7).



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  		FIGURE 2. Gel mobility and competition assays. End-labeled H-2 BF1 binding motif (wt) oligonucleotide (1 x 105 cpm) was incubated with the following: A, 1 µg of nuclear extract from RadLV-induced thymoma-derived cell line; B, in vitro translation products of ATF-1A; C, in vitro translation products of CREB{Delta}; and D, a mixture of in vitro translation products of ATF-1A and CREB{Delta} (see Fig. 1Go). Lane 1, No competitor added; lanes 2 to 4, unlabeled wt oligonucleotide (WT) at 50-, 100- and 150-fold molar excesses, respectively, was added before the radiolabeled probe; lanes 5 to 7, unlabeled mutant (MUT) oligonucleotide at 50-, 100-, and 150-fold molar excesses, respectively, was used as a competitor. Samples were electrophoresed through nondenaturing 5% polyacrylamide gels at 150 V for 2 h (B and C) or 7 h (A and D).

 
Among three forms of H-2 BF1 complex in the nuclear extracts, band b representing the ATF-1/CREB heterodimers was most abundant (Fig. 1Go, lane 1, and Fig. 2GoA, and also seen in Figs. 3Go and 6B), thereby indicating that the majority of the H-2 BF1 in the cell was present in the heterodimer form. A similar observation has been reported using other type of cells (38). As seen in the in vitro mixture of ATF-1 and CREB (Fig. 1Go, lane 4, and Fig. 2GoD), it seems likely that they easily form heterodimers under these conditions.



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  		FIGURE 3. Gel supershift assay. Nuclear extracts (1 µg) from the RadLV-induced thymoma-derived cell line were incubated with end-labeled ds wt oligonucleotide (1 x 105 cpm). Lane 1 had no Ab added. Abs against ATF-1 (lane 2, sc-270x; lane 3, sc-243x), c-Jun (lane 4, sc-45x), ATF-2 (lane 5, sc-187x), and CREB (lane 6, 06-504) were mixed with parallel binding reactions and loaded onto a 6% polyacrylamide gel at 150 V for 7 h. Supershifts of the H-2 BF1 complex are indicated by asterisks.

 
ATF-1 and CREB are components of H-2 BF1 in RadLV-induced thymoma cell extract

To demonstrate that ATF-1 and CREB are indeed components of H-2 BF1 in the thymocyte, we examined the effects of Abs (two anti-ATF-1 and one anti-CREB) on the mobility of the H-2 BF1:DNA complex. Abs to two other closely related, but distinct, members of the leucine zipper family of transcriptional activators (anti-c-Jun and anti-ATF-2) were included as negative controls. Supershift of all three bands in Figure 3Go, lane 1, was seen when one anti-ATF-1 Ab (sc-270x) was included in the reaction (lane 2). This Ab, according to the manufacturer’s analysis, cross-reacts somewhat with CREB-1, and therefore the observed shift of all bands is not unexpected. The second anti-ATF-1 Ab (sc-243x) is specific for ATF-1, and, as is seen in lane 3, only band c was shifted upward. Anti-CREB Ab (06-504) resulted in a near quantitative shift of band a and a partial shift of band b (lane 6). This is consistent with bands a and b being CREB homodimers and ATF-1/CREB heterodimers, respectively. These data strongly suggest that the H-2 BF1 complex is composed of ATF-1 homodimers, CREB homodimers, and ATF-1/CREB heterodimers in the thymocyte.

CREB isoforms expressed in RadLV-infected and normal thymocytes

It has been shown that at least six isoforms (CREB{Delta}, {alpha}, {alpha}{gamma}, {gamma}, {Omega}, and {psi}) are generated from the mouse CREB gene by alternative splicing (29). To characterize the expression of CREB isoforms in RadLV-infected and normal mouse thymocytes, RT-PCR was performed as previously described (29). In this experiment, the data are not quantitative, since the amount of cDNA template was not adjusted to the internal ß-actin control as described in later experiments where quantification was necessary. The results shown in Figure 4Go demonstrate that CREB{alpha} (band A) and CREB{Delta} (band B) are expressed in the RadLV-induced tumor-derived cell line, normal mouse thymocytes, and RadLV-infected thymocytes (lanes 3, 4, and 5, respectively), whereas no product was detected in the absence of template cDNA (data not shown). Plasmids pmcCREB{alpha} (lane 1) and pmcCREB{Delta} (lane 2) were used as controls, and they generated fragments of 279 and 237 bp, respectively. The RT-PCR products were of the same size as CREB{alpha} and CREB{Delta}, while no product corresponding to CREB{alpha}{gamma} (392 bp) or CREB{gamma} (350 bp) was seen. CREB{Delta} mRNA was more abundant than CREB{alpha} (also seen in Fig. 5GoB, panel 1). PCR products were electrophoresed on 2% agarose gels, transferred to nitrocellulose membranes, and detected by hybridization to the radiolabeled oligonucleotide 5'-GAAAATTTTGAATGAC-3', which corresponds to amino acid residues 122 to 126 encoded in exon 8 (29). Signals corresponding to CREB{Delta} and CREB{alpha} were detected, thus confirming the specificity of the PCR (data not shown).



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  		FIGURE 4. Expression of CREB isoforms detected by RT-PCR. Lane 1, PCR products of pmcCREB{alpha} (band A, 279 bp); lane 2, those of pmcCREB{Delta} (band B, 237 bp); lane 3, those of cDNA from the RadLV-induced, tumor-derived cell line; lane 4, those from the normal mouse thymocytes; lane 5, those from the RadLV-infected thymocytes. The PCR products were run on 10% polyacrylamide gel and stained by ethidium bromide.

 


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  		FIGURE 5. A, Northern analysis for 1) H-2, 2) ATF-1, and 3) ß-actin of RNA from normal and RadLV-infected thymocytes. Lanes N1 and N2, Two different RNA preparations of normal B10.T(6R) mouse thymocytes; lanes 1 to 6, six different RNA preparations from RadLV-infected B10.T(6R) thymocytes. B, RT-PCR analysis for 1) CREB and 2) ß-actin of RNA from normal and RadLV-infected thymocytes. C, Relative quantities of RNA from normal and RadLV-infected thymocytes, as measured by Northern analysis and RT-PCR. Panels 1 through 3, The intensities of each band in A, Panels 1 through 3. In each panel, the value of lane N2 was set as 1.0, and the relative hybridization strengths of the other lanes are indicated. Panels 4 and 5, The summary of RT-PCR for CREB and ß-actin, respectively. In each experiment the value of TCA precipitation of the N2 sample was set at 1.0, and the strengths of the other samples were expressed as relative values. The averages and SDs of three independent experiments are shown.

 
Expression of ATF-1 in RadLV-infected and normal thymocytes

The highly conserved MHC class I probe hybridized to mRNA of approximately 1.6 kb and showed that the levels of H-2 mRNA were elevated in all six RNA samples extracted from RadLV-infected thymocytes (Fig. 5GoA, panel 1, lanes 1–6) compared with those from normal thymocytes (lanes N1 and N2). Two bands, corresponding to 2.4 and 1.5 kb, were detected by ATF-1A hybridization to RNA of RadLV-infected thymocytes (Fig. 5GoA, panel 2, lanes 1–6), while very low levels were seen in normal thymocytes (lanes N1 and N2). The shorter mRNA species was less abundant, and the two were generated by differential use of polyadenylation signals (10). The level of ß-actin cDNA hybridization was relatively consistent in all RNA preparations (Fig. 5GoA, panel 3). The intensities of each band in Figure 5GoA were quantified using the National Institutes of Health Image Program, and the results are presented in Figure 5GoC, panels 1 through 3. Although the quantity of H-2 mRNA in individual preparations did not precisely reflect that of ATF-1 mRNA, both mRNA species were increased in all six samples from RadLV-infected B10.T(6R) thymocytes. These data are in agreement, therefore, with the observation that ATF-1 stimulated expression from a reporter gene under the control of the H-2 BF1 binding motif following transfection (10).

Expression of CREB in RadLV-infected and normal thymocytes

Since Northern hybridization for CREB resulted in the detection of multiple signals (39), probably due to encoding different lengths of mRNAs of the CREB gene (29), we adopted RT-PCR to quantify the levels of CREB gene expression. Experiments were designed as follows. The quantity of cDNA used as a template in PCR was standardized by use of ß-actin primers. 32P-labeled nucleotide incorporation was measured by TCA precipitation after 15, 18, 21, 24, and 27 PCR cycles. The results were analyzed and were found to increase exponentially between cycles 18 and 24; curves for individual cDNA samples were also parallel between these two points (data not shown). Twenty-one cycles was chosen as the experimental cutoff, and individual samples were diluted so as to equalize the amount of template cDNA. PCR was repeated in triplicate using the standardized cDNAs, and radiolabeled nucleotide incorporation was determined. Shown in Figure 5GoC panel 5, are the results obtained using ß-actin primers. Samples of each were also analyzed by PAGE and autoradiography. An example of this analysis is shown in Figure 5GoB, panel 2. The incorporation as measured by TCA precipitation; the values obtained from the autoradiograms were parallel in general, and the by-products of PCR detected by autoradiography were <5% (data not shown).

The standardized cDNA samples were next used in PCR with the CREB primers. Incorporation was measured as described above and was found to be exponential, and parallel between cycles 25 and 29. Experimental PCR was performed for 27 cycles, and the data are presented in Figure 5GoC, panel 4, and Figure 5GoB,panel 1. As in the case of ATF-1, CREB mRNA was increased in all samples from RadLV-infected B10.T(6R) thymocytes. These data combined with the composition of the H-2 BF1 complex suggest that both ATF-1 and CREB are involved in the stimulation of transcription from the H-2Dd gene following RadLV infection of thymocytes.

Comparison of H-2Dd cell surface expression and H-2 BF1 binding activity in RadLV-infected thymic cells

Twenty-three B10.T(6R) mice received intrathymic injection of in vivo maintained RadLV. At 28 days after injection, thymocytes from individual mice were analyzed by FACScan using anti-H-2Dd alloantiserum. The thymocytes were grouped into two pools expressing 1) low to moderate and 2) high levels of H-2Dd on the surface. Figure 6GoA shows the profile of normal thymocytes (panel 1, mean intensity of H-2Dd expression was 52.80 with 13.58 of background), infected thymocytes expressing low/moderate levels of H-2Dd (panel 2, mean intensity was 182.34 with 13.77 of background), and infected thymocytes expressing high levels of H-2Dd (panel 3, mean intensity was 711.43 with 55.41 of background). These results reproduced our previous data, showing inducible H-2Dd expression of thymocytes after RadLV infection (5, 6).



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  		FIGURE 6. A, FACS analysis of thymocytes isolated from B10.T(6R) normal and RadLV-infected mice stained with anti-Dd antisera. Panel 1, Thymocytes from normal B10.T(6R) mice. Panel 2, Thymocytes with low to moderate H-2Dd expression. Panel 3, Thymocytes with high H-2Dd expression. Nonspecific binding for mouse serum is indicated by the dotted line. The vertical axis denotes the cell number, and the horizontal axis shows the fluorescence intensity. B, Comparison of binding activity among normal thymocytes (lane 1), RadLV-infected thymocytes with low to moderate H-2Dd expression (lane 2), and those with high H-2Dd expression (lane 3). One microgram of nuclear extract from each preparation was incubated with end-labeled wt oligonucleotide (1 x 105 cpm) and electrophoresed through 5% polyacrylamide gel at 150 V for 7 h.

 
Nuclear extract (1 µg) from each of the three samples (normal, lane 1; low/moderate H-2Dd expression, lane 2; high H-2Dd expression, lane 3) was incubated with wt oligonucleotide (1 x 105 cpm). All three components of H-2 BF1, i.e., ATF-1 homodimers, CREB homodimers, and ATF-1/CREB heterodimers, were increased in nuclear extracts from RadLV-infected thymocytes compared with the binding activity in normal thymocytes. Furthermore, the relative levels were a reflection of the strength of H-2Dd Ag expression on the cell surface.

ATF-1 and CREB activate the transcription of genes containing the H-2 BF1 binding motif

To determine whether ATF-1 and CREB could activate the transcription of a gene under the control of the H-2 BF1 cis binding motif in vivo, the reporter plasmid ({Delta}-122 CAT) was transfected into F9 cells in various combinations with expression vectors encoding ATF-1, CREB{Delta}, and the catalytic subunit of the cAMP-dependent protein kinase (PKA). Addition of the PKA expression plasmid alone resulted in no increase in the induction index (see Fig. 7Go). Similarly, the addition of either ATF-1 or CREB expression vector singly or combination resulted in only small increases of 1- to 2-fold. When the PKA plasmid was combined with ATF-1 and CREB, stimulation of CAT expression was 4.2- and 4.9-fold, respectively. These observations are in accord with the fact that ATF-1 and CREB are functionally active when phosphorylated by cAMP-dependent PKA. Previously, we have shown that ATF-1 with PKA activated the {Delta}-122 CAT reporter, but not {Delta}-65 CAT; the latter lacks the H-2 BF1 binding motif (10). The same results were obtained using CREB, either alone or in combination with ATF-1 (data not shown). These data strongly suggest that both ATF-1 and CREB can activate the transcription of H-2Dd in mouse thymocytes after RadLV infection.



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  		FIGURE 7. ATF-1 and CREB can activate a CAT reporter plasmid containing the H-2 BF1 motif. F9 cells were cotransfected with reporter plasmid ({Delta}-122 CAT) and various combinations, as indicated, of ATF-1 expression plasmid, CREB expression plasmid, and cAMP-dependent protein kinase plasmid. The induction index shows induction relative to the value of {Delta}-122 CAT reporter plasmid and the antisense ATF-1 plasmid without the cAMP-dependent protein kinase expression plasmid in each experiment. Indicated are the mean and SD of five independent experiments. The "a" indicates that instead of sense ATF-1 or CREB{Delta} expression plasmid, the antisense direction ATF-1 expression plasmid (pRc/RSV/1-FTA) was used.

 
However, even if PKA, ATF-1, and CREB were expressed together, the induction index was 5.3, thereby indicating that the effect of ATF-1 and CREB was supplementary rather than additive. In other words, the H-2 promotion activity of ATF-1 and CREB in the system is independent of the dimeric combination. Therefore, the observation that ATF-1 and CREB easily form heterodimers does not necessarily imply that the heterodimers are better at inducing H-2 gene transcription. The significance, if any, of the observation that majority of the H-2 BF1 complex in the cell is the ATF-1/CREB heterodimer form will be examined in the future.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been repeatedly observed that MHC expression is altered by viral infection and/or virus-mediated transformation (for a review, see 40 . These changes are highly significant in term of the ability of the host’s immune mechanisms to eliminate or fail to respond to virus-infected/transformed cells (3, 4, 41, 42, 43). These studies suggest that the quantitative expression of MHC molecules is as important to the effectiveness of the immune system as is the nature of the MHC molecule(s) involved. Therefore, it would be expected that immunomodulation via artificial control of MHC expression on virus-infected and/or neoplastic cells might become a novel therapeutic approach for virus/neoplasm-associated diseases.

We previously demonstrated that in the RadLV-infected thymocytes, increased cell surface expression of H-2Dd was a result of the elevated level of mRNA transcription and identified the cis regulatory element (H-2 BF1 binding motif) of the H-2 gene through DNA footprinting analysis (5, 6). It has been known that there are other cis sequences, such as class I regulatory elements (44, 45) and IFN consensus sequence (46, 47), in the 5'-flanking region of the MHC gene. Most of these cis elements, including the H-2 BF1 binding motif, associate with development/tissue-specific expression (7, 48, 49) and cytokine/hormonal regulation (6, 8, 50, 51) of MHC. On the other hand, some of the cis elements have been found to function as negative regulators of the MHC promoter (52, 53). As another suppressive mechanism of the MHC expression, it has been shown that the production of the trans factors that could bind the sequences was down-regulated in the adenovirus 12-induced, transformed cells (53). To sum up, the constitutive and inducible MHC expression was complicatedly regulated by the interaction of DNA-binding trans factors with the cis regulatory elements of the gene. Although several investigators have identified the trans-acting factors, the regulation of MHC expression has not been revealed completely. To clarify the regulatory mechanisms, identification of new trans-acting factors is desirable.

Use of the yeast one-hybrid system for isolating the trans-acting factors resulted in our cloning many cDNAs from an expression library prepared from a RadLV-induced, thymoma-derived cell line (10). One group of these clones was found to encode ATF-1, and in fact, homodimers of ATF-1 have been shown to be one of the three components of the H-2 BF1 complex in vivo (10). Actually, in the present 6% polyacrylamide gel (Fig. 1Go), there is a subtle gap in the mobility of the band c between lanes 1 and 2 or lanes 1 and 4. This gap was not evident when the gel consisted of 5% polyacrylamide (10). In the thymoma cell nuclear extracts, two distinct molecular masses (38 and 36 kDa) of ATF-1 were detected, with an abundance of the 38-kDa species (data not shown). Although the predicted molecular mass of mouse ATF-1 calculated from the cDNA-derived amino acid sequence is 29 to 30 kDa (10, 54), immunodetection of 38-kDa ATF-1 was consistent with the results of Hsueh et al. (55). The 36-kDa species was not documented in the report (55). However, since it has been shown that differential phosphorylation produces alternative forms of ATF-1 in human (32), we considered the two bands as isoforms with different phosphorylations. The phosphatase treatment and Western blotting revealed that the 36-kDa species was the dephosphorylated form of the 38-kDa species (data not shown). On the other hand, the molecular mass of the in vitro translated ATF-1 was 38 kDa (data not shown), suggesting that the products were phosphorylated. To examine the effect of phosphorylation on gel mobility, we treated the in vitro-made ATF-1 with phosphatase and then performed gel shift analysis. As expected, the dephosphorylated ATF-1 with a molecular mass of 36 kDa could bind the H-2 BF1 oligonucleotide with a reduction of the binding activity (data not shown). The migration was relatively faster and consistent with that of band c in the cell nuclear extract (data not shown). These data suggest that ATF-1 in homodimers exists as a dephosphorylated form in the cell, and we interpret the gap of band c between lanes 1 and 2 or4 as a result of difference in migration of phosphorylated and dephosphorylated ATF-1 in nondenaturing gels.

In the screening of the expression library with the one-hybrid system, many clones were isolated that encode the p65 subunit of NF-{kappa}B. Although the in vitro translated protein product of these clones associated with ATF-1 in vitro (our manuscript in preparation), preliminary attempts to demonstrate the association of ATF-1 and NF-{kappa}B p65 in vivo have not been successful to date. The addition of in vitro translation products from NF-{kappa}B p65 clones did not alter migration in gel shift analysis compared with ATF-1 alone, and anti-NF-{kappa}B p65 Abs did not result in a gel supershift (data not shown). For these reasons we conclude that NF-{kappa}B p65 is not a component of the H-2 BF1 complex under the assay conditions currently in use. A cDNA expression library was also screened by the South-Western method of Singh et al. (56), and only clones of ATF-1 were isolated. It should be pointed out, however, that we cannot rule out a role for NF-{kappa}B p65 in the regulation of H-2 gene expression in vivo, especially in light of the fact that numerous clones of p65 were isolated using a system that, by definition, requires that the cDNA product of the isolate regulates in a positive fashion the cis sequences used, in this case the H-2 BF1 binding motif.

The following observations led to the investigation of CREB as a potential factor involved in the regulation of H-2 gene expression: 1) one anti-ATF-1 Ab (sc-270x) shifted all three forms of the H-2 BF1 complex; 2) a more specific anti-ATF-1 Ab (sc-243x) was selective for the ATF-1 homodimer form and shifted only one form of the H-2 BF1 complex; and 3) sc-270x cross-reacts with CREB. The use of CREB-specific Ab showed that CREB is indeed a component of the H-2 BF1 complex. The fact that CREB was not initially isolated in the cDNA screening is explained by the fact that the CREB mRNA has three stop codons in the 5'-noncoding region (29). In the yeast one-hybrid system, the fusion protein between the Gal4 activation domain and a cDNA can be expressed in a functional form provided that the DNA binding domain encoded by the cDNA is intact and functional (57). In the South-Western screening system, bacteria produce a fusion protein of lacZ and the cDNA-encoded amino acid sequence (56). In instances where the cDNA is full length and contains a termination codon(s) in the 5'-noncoding sequence, that cDNA would not be isolated by either screening methodology.

To identify the isoforms present in the RadLV-induced thymoma-derived thymocyte cell line, RT-PCR was performed as described by Ruppert et al. (29). Our results show that both CREB{alpha} and CREB{Delta} are present, and that the latter is more abundant. This is in agreement with the observation that both these isoforms are expressed in almost all tissues, and that CREB{Delta} is the predominant species (29). The migration of the DNA:protein complexes containing CREB{alpha} vs CREB{Delta} in vitro translation products could not be resolved on nondenaturing gels (data not shown). It has been shown that CREB{alpha} and CREB{Delta} have equal activity as CRE-mediated trans-activation factors (29). The results of our preliminary transfection studies using each isoform individually with the {Delta}-122 CAT reporter plasmid are in agreement with this finding (data not shown).

The identification of the CREB protein as one component of H-2 BF1 is based on the following evidence: 1) Ab to ATF-1 that cross-reacts with CREB shifted all three components of H-2 BF1, while specific anti-ATF-1 Ab shifted only the ATF-1 homodimer; 2) Ab specific for CREB shifted band a and partially shifted band b (Fig. 3Go) while not affecting the migration of the ATF-1 homodimer (band c); 3) in vitro translation products of CREB{Delta} cDNA bound to the H-2 BF1 binding motif, comigrated with band a, and were shifted by anti-CREB Ab (data not shown); and 4) in vitro translation products of CREB{Delta} associated with those of ATF-1 and bound to the target DNA, generating the same migration pattern in gel retardation assays as did the nuclear extracts prepared from thymocytes. The CREB protein shows 59% amino acid homology with another trans-acting factor, CREM (31). Anti-CREM polyclonal Ab (product no. 05-350, Upstate Biotechnology) changed the migration of band a (data not shown), but this product is also reported to cross-react with CREB. To rule out the possibility of misidentification of CREM as CREB, the CREM{alpha} cDNA was obtained from Dr. P. Sassone-Corsi (31) and translated in vitro. We found that CREM{alpha} also bound to the H-2 BF1 motif, but that the position in gel shift analysis was totally different from that of the H-2 BF1 in vivo complex (data not shown). From these observations, we conclude that CREM is not a component of H-2 BF1 under these conditions.

In summary, the H-2 BF1 complex has been shown to be composed of at least three forms; ATF-1 homodimer, CREB homodimer, and ATF-1/CREB heterodimer. We conclude that ATF-1 and CREB up-regulate transcription of the H-2Dd gene after RadLV infection of thymocytes. These findings may shed light not only on the mechanism of virus-induced MHC alteration but also on a novel application for therapeutic immunomodulation through artificial MHC gene control.


   Acknowledgments
 
We thank Dr. Lorraine J. Gudas for providing the F9 cell line, Brandi Levin for injection of the mice, John Hirst for FACS analysis, Dr. Iwona Stroynowski for providing the {Delta}-122 CAT plasmid, Dr. Richard A. Maurer for providing the RSV-CHO-PKA-C{alpha}, version 2 plasmid, Dr. P. Sassone-Corsi for providing the pSVCREM{alpha} plasmid, Dr. Gunther Schütz for providing the pmcCREB{Delta} and pmcCREB{alpha} plasmids, and especially Dr. Chris Pampeno for critical comments during the preparation of this manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant CA22247 (to D.M.) and the National Science Foundation through its support of the computing resources (Grant BIR-9318128). Back

2 Address correspondence and reprint requests to Dr. Daniel Meruelo, Department of Pathology and Kaplan Cancer Center, New York University Medical Center, 550 First Ave., New York, NY 10016. Back

3 Abbreviations used in this paper: RadLV, radiation leukemia virus; H-2 BF1, H-2 binding factor 1; CRE, cAMP responsive element; ATF-1, activation transcription factor 1; CREB, CRE binding protein; CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus; PKA, protein kinase A; wt, wild type. Back

Received for publication March 27, 1997. Accepted for publication January 30, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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