©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cooperativity between an Upstream TATA-like Sequence and a CAA Repeated Element Mediates E1A-dependent Negative Repression of the H-2K Class I Gene (*)

(Received for publication, February 7, 1994; and in revised form, October 7, 1994)

Xiaoren Tang (1) Hai-Ou Li (1) Osamu Sakatsume (1) Tomohiro Ohta (1) Hatsumi Tsutsui (1) Arian F. A. Smit (2) Masami Horikoshi (3) Phillipe Kourilsky (4) Alain Israël (5) Gabriel Gachelin (4) Kazushige Yokoyama (1)(§)

From the  (1)Tsukuba Life Science Center, RIKEN (Institute of Physical and Chemical Research), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan, the (2)Department of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010-0269, (3)Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, (4)Unité de Biologie Moléculaire du Géne, Département d'Immunologie and (5)Unité de Biologie Moléculaire de l'Expression Génique, Département Biologie Moléculaire, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cédex 15, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In primary rodent cells transformed by the E1A region of the highly oncogenic adenovirus type 12, repression of transcription mediated by the far upstream TATA-like element was observed only in conjunction with either possible juxtaposition of a CAA repeated element in the presence of E1A and was dependent upon the relative arrangement of both the TATA-like and CAA repeated motifs in both homologous and heterologous promoter constructs. A gel shift competition study demonstrated that the TATA-binding protein (TBP) or a TBP-like protein can bind to both the upstream TATA-like sequence and the regular TATA box on the H-2K basal promoter. Moreover, employing immunoselection and cyclic amplification and selection of targets (CASTing) methods with nuclear extracts derived from Ad12-E1A transformants, we have identified a high affinity binding site in the H-2K class I promoter for E1A-associated DNA-binding proteins. The sequences of the binding sites were identified and were found to contain both the upstream TATA-like motif and the CAA repeated motifs. Our results suggest that the TATA-like sequence in the far upstream region of the H-2K gene is one of the elements that is required for Ad12-E1A-mediated negative repression.


INTRODUCTION

Primary rodent cells transformed by the E1A region of the highly oncogenic adenovirus type 12 (Ad12) but not by the same region of the non-oncogenic virus Ad5 (or Ad2) (1) express the reduced level of the products of major histocompatibility complex (MHC) (^1)class I genes(2, 3) . The lower levels of MHC class I mRNA in Ad12-transformed cells reflect decreased rates of transcription of the endogenous class I genes. This down-regulation of transcription is mediated by the 1,266-amino acid 13S product of the Ad12-E1A region(4, 5, 6, 7, 8, 9, 10) . The resultant decrease in cell-surface levels of MHC class I antigens is reflected by the lower susceptibility of Ad12-transformed cells to allogenic cytotoxic T cells and NK cells, suggesting a model by which Ad12-transformed cells escape immune surveillance and develop into proliferating and evasive tumors(4, 5, 11, 12) .

Control of the initiation of transcription of the mouse H-2K^b MHC class I gene has been studied extensively, and the regulatory region has been shown to contain the common CCAAT and TATA regions as well as multiple cis-acting regulatory elements. The best characterized of these elements are RI, RI`, and R2 in the CRE/IRS region(3, 13, 14, 15) , which have also been defined by a footprinting study in vivo(16) . Several transcriptional factors have been shown to bind to these elements(17, 18, 19, 20, 21, 22, 23, 24, 25, 26) . The level of binding to the R1 element has been shown to be higher in the case of extracts from Ad5-transformed cells than in the case of extracts from Ad12-transformed cells(27, 28, 29) . The R2 binding activity is significantly higher in extracts from Ad12-transformed cells than in extracts from Ad5-transformed cells or parental lines(7, 28, 30) . The poor enhancer activity of the R1 site in Ad12-transformed cells is correlated with increases in the extent of binding of nuclear factors to the R2 element, suggesting the presence of a repressor whose effects are mediated via the R2 site. Kralli et al.(31) demonstrated recently that the putative R2-binding repressor protein, designated R2BF, is similar to members of the family of thyroid hormone/retinoic acid receptors.

We showed previously that a distal 5`-element (-1,521 to -1,837 relative to the start site of transcription) that contributes to E1A-mediated negative regulation is included in the promoter region of the H-2K gene(9) . The CAA repeated sequences in regions -1,736 to -1,689 and -1,616 to -1,535 are both necessary for full negative regulation of the H-2K gene by E1A. We report here that an additional element, a TATA-like sequence, in the far upstream region of the 5`-flanking sequence (-1,773 to -1,767) is also a key element in the negative regulation of expression of the MHC class I H-2K gene by E1A in conjunction with either upstream or downstream CAA repeats. A gel shift competition assay demonstrated that the factor that binds to the far upstream TATA-like sequence can also bind to the TATA-box sequence in the basal promoter. Using an immunoselection and cyclic amplification and selection of targets (CASTing) method, we also obtained direct evidence that Ad12-E1A can associate with the proteins that bind to the TATA-like sequence and to the CAA repeats in Ad12-E1A-transformed cells.


MATERIALS AND METHODS

Plasmids-The deletion and substitution CAT mutants, namely, del-4ml, del-4m2, del-5ml, and del-5m2 were constructed as follows. The BspHI/RsaI DNA fragment of a distal 5`-flanking sequence of the H-2K gene, including the element CTGTAAGCCAGACCC or the TATA-like sequence TATTAAA, was mutated to either ATGTAAGAAAGAAAC or TAGCGAA, respectively, and ligated to the RsaI/DdeI DNA fragment (-1,735/-1,188) or the BstNI/Sau3A fragment of a distal 5`-flanking sequence of the H-2K gene (-1,615/-1,534) with 4-6-bp BamHI or HindIII linkers of both ends of the DNA. The resultant fragments were inserted into the BamHI site or the HindIII site of pH-2K^b(367)CAT (32) to generate appropriate CAT derivatives in the correct or reversed orientation. The TATA-like sequence in the BspHI/RsaI DNA fragment was converted to various TATA-like sequences found in the promoters of the H-2K, adenovirus major late promoter (AdML), hsp70, c-fos, histone, beta-globin, adenoviruses E3 and E4, and SV40 early genes (see (33) ) by site-directed mutagenesis (using a kit from Amersham Japan, Tokyo, Japan) and the appropriate primers. In each case, overhang was generated by EcoRI, and the DNA fragment was either inserted into the EcoRI site of pH-2K^b(367)CAT(CAAu) or coinserted with the DNA fragment RsaI/DdeI (-1,735/-1,688) into either the EcoRI site of pSV2CAT (34) or the HindIII site of pBLCAT2 (35) to generate various CAT plasmids ( Fig. 4and Fig. 5).


Figure 4: Relative promoter activity of pH-2K^bCAT constructs with various TATA-like sequences and a CAA repeated motif. The histogram represents the CAT activity of the pH-2K^bCAT constructs in CYpAdC3 cells or Ad5-E1A-transformed 3Y1 cells, as indicated. All CAT values are the averages of results from at least five transfections. Normalized CAT activity associated with pH-2K^b(367)CAT was taken arbitrarily as 1.0. The standard deviation for each result is indicated by a bar.




Figure 5: Effects of combinations of various TATA-like sequences in the upstream region and various proximal promoters in Ad12-E1A- or Ad5-E1A-transformed cells. Average results of five independent experiments are shown in the histogram as CAT activities relative to activity associated with pH-2K^b(367)CAT in CYpAdC3 cells, which was arbitrarily taken as 1.0. P+E is the H-2K^b promoter/enhancer region. The standard deviation for each result is shown by a bar.



Separate plasmids containing the H-2K minimum promoter (pH-2P) and the H-2 enhancer (pH-2E) joined to the basal promoters of the early SV40, thymidine kinsase, and conalbumin genes were generated as described elsewhere(10) . Sequences of constructs were verified by the dideoxy chain termination method(36) . Plasmids pE1A, pE1A12-12S, pE1A12-13S, pE1A5-13S, pE1A5-12S(37, 38) , pH-2K^b(2015)CAT(9) , and pRSVbeta-gal (39) have been described previously.

Cell Culture, DNA Transfection, and CAT Assay

Rat 3Y1 cells were cultured as described elsewhere(32, 40) . The primary cells, such as BALB/c baby mouse kidney cells, baby hamster kidney cells, and baby rat kidney cells, were infected by Ad12 or Ad5 virus and cultured as described elsewhere(6) . Transfections for long term expression were carried out as described elsewhere(13) . The activities of CAT and beta-galactosidase were assayed as described elsewhere(9, 13) . The ratio of the activity of CAT to that of beta-galactosidase and the number of copies of the transfected genes that had been integrated into genomic DNA were used for normalization of results.

Preparation of Nuclear Extracts and Gel Shift Assay

Nuclear extracts were prepared from various cell lines essentially as described by Yano et al.(26) . Gel shift and competition assays were carried out as described elsewhere(41) . The probes were the AccI/RsaI DNA fragment (-1,837 to -1,736) and the AccI/DdeI DNA fragment (-1,837 to -1,689) of the H-2K^b promoter. Effects on antibodies against TBP, Ad12-E1A, or Ad5-E1A were analyzed in the gel shift assays.

Gel Shift Assays with Products of Translation in Vitro

The transcription and translation of the gene for TBP were carried out in vitro using the expression plasmid referred to as human TBP/pGEM1 (42) as recommended by the manufacturer (Promega, Madison, WI). Gel shift assays with products of translation in vitro were carried out as described(20) . The DNA-protein complex and free probe were separated by polyacrylamide gel electrophoresis on a 5% gel in 0.5 times Tris-borate buffer (1 times TBE buffer: 0.089 M Tris, 0.089 M boric acid, 2 mM EDTA). The gel was dried and exposed to two sheets of x-ray film. The film closest to the gel recorded signals from both S and P and the second film recorded only signals from P.

Immunoselection and CASTing

Immunoselection and CASTing were performed by a combination of the methods of Kinzler et al.(43) and Perkins et al.(44) with slight modifications. A DNA fragment containing the promoter region of H-2K^b (HindIII-NruI) was sheared to an average size of 200-400 bp (mass average) by sonication on ice. The ends of the DNA fragments were blunted with the Klenow fragment of DNA polymerase I from Escherichia coli and then ligated to ``catch linkers'' (5`-GAGTAGAATTCTAATATCTC-3`) using T4 ligase. To separate catch linkers that had become ligated to themselves, the ligated linkers were digested with XhoI. The reaction mixture for the first cycle of immunoselection and CASTing included 84 µg of DNA fragments and 40 µl of a nuclear extract of 3Y1 cells (400 µg of protein) in a total volume of 1.0 ml of buffer (100 mM NaCl, 20 mM Hepes (pH 7.5), 1.5 mM MgCl(2), 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin, 10 µg/ml leupeptin, 0.1% Triton X-100, 20% glycerol). After a 25-min incubation at 20 °C, 10 µl of a solution of rat IgG were added, and then the mixture was slowly agitated at 20 °C for 1 h after the addition of 100 µl of a suspension of protein A-Sepharose (Sigma). The mixture was centrifuged, and the supernatant was further incubated with 40 µl of a nuclear extract of CYpAdC3 cells or Ad5-E1A-transformed 3Y1 cells (400 µg of protein) for another 25 min at 20 °C. Then 10 µl of a solution of monoclonal antibodies specific for Ad12-E1A or Ad5-E1A and 100 µl of the suspension of protein A-Sepharose were added to the reaction mixture, and the final mixture was treated according to the same protocol as described above. The beads were washed five times with 1.0 ml of isotonic saline that contained 0.1% Triton X-100 and 0.1 mg of bovine serum albumin per ml and then twice with 1.0 ml of immunoprecipitation buffer (50 mM Hepes (pH 7.5), 150 mM KCl, 5 mM MgCl(2), 10 mM ZnSO(4), 1% Triton X-100, 0.05% SDS). DNA was freed from complexes by incubation of beads for 50 min at 50 °C in dissociation buffer (500 mM Tris (pH 9.0), 20 mM EDTA, 10 mM NaCl, 0.2% SDS). Protein A-Sepharose was removed by centrifugation, the supernatant containing DNA was extracted with phenol and chloroform, and the DNA was precipitated with ethanol. Various amounts of recovered DNA (1/2, 1/20, and 1/200 dilutions) were amplified using the catch linkers as primers (25 cycles; 94 °C for 60 s, 55 °C for 60 s, and 72 °C for 120 s) as described elsewhere(43, 44) . Amplified products were analyzed by agarose gel electrophoresis (1.2% agarose, 1 times TBE), and 10 µl of a solution of the amplified material (typically containing 30-50 ng of DNA) were used to initiate the second and subsequent rounds of CASTing. After six cycles of CASTing, the amplified DNA was radiolabeled by the Klenow fragment of DNA polymerase I with [alpha-P]dTT(45) . Preparative gel shift assays were performed as described elsewhere(45) . Each shifted band, representing a protein-DNA complex, was excised from the gel and eluted from the gel by overnight agitation at 37 °C in 0.6 ml of a solution of 0.5 M ammonium acetate, 1 mM EDTA, 0.1% SDS, and 10 mM Tris (pH 7.5). DNA was extracted with phenol and chloroform, precipitated in ethanol. The DNA pellet was resuspended in 10 µl of 10 mM Tris (pH 8.0), 1 mM EDTA. PCR was then performed under the conditions described above. After the fourth cycle of gel shifting and PCR, the resultant DNAs were digested with EcoRI and ligated into pBluescript KS() (Stratagene, La Jolla, CA). The products were used to transform competent XL-1 Blue E. coli cells.

Nucleotide Sequencing

Nucleotide sequencing was performed by the dideoxy chain termination method as described elsewhere(36) .


RESULTS

The Upstream TATA-like Sequence and the CAA Repeated Motif

Ad12-E1A-transformed CYpAdC3 cells and baby mouse kidney primary cultured cells, infected with either Ad12 or Ad5 virus, were used for the promoter-screening assay. A distal 5'-end element between -1,521 and -1,837 that contributes to negative regulation mediated by the E1A gene products was previously defined within the H-2K^b gene(9) . In an attempt to define the precise controlling sequence in the 316-bp DNA fragment, we constructed a set of fusions of partially deleted and substituted regulatory regions with the pH-2K^b(367)CAT construct, which contained the promoter and enhancer regions of the mouse H-2K^b gene, as described previously(32) . To our surprise, the TATA-like sequence (-1,773 to 1,767) in the 316-bp far upstream region was found to contribute to the E1A-mediated repression in Ad12-E1A-transformed cells. The plasmid del-3CAT (lane 2 (Fig. 1, lane 2)), which contained only the TATA-like sequence, did not show this negative activity. However, when the DNA fragment from -1,809 to -1,735 was ligated with either an upstream CAA repeated element (-1,735 to -1,688) or a downstream CAA repeated element (-1,615 to -1,534), repression was detected at significant levels in Ad12-E1A-transformed cells, but not in Ad5-infected cells (lanes 3-6). The results obtained with substitution mutations of the TATA-like sequence in this region showed that this negative regulation was caused by the TATA-like sequence (lanes 7 and 8). Similar results were obtained in adenovirus-infected baby hamster kidney and baby rat kidney primary cells (data not shown). Collectively, the results indicate that the far upstream TATA-like sequence is one of the negative elements involved in the E1A-mediated control of expression of the H-2K^b MHC class I gene. The same results were obtained in co-transfection experiments using 3Y1 cells and NIH3T3 cells, with CAT reporter genes and an E1A expression vector (data not shown).


Figure 1: Deletion study of the E1A-responsive negative element in the upstream region of the promoter of the H-2K gene. A schematic representation of the CAT constructs derived from pH-2K^b(367)CAT (32) and their relative CAT activities is shown. Dotted lines represent internal deletions. Relative CAT activities of mutants were measured in Ad12-E1A-transformed CYpAdC3 cells(9) , in Ad12-infected or Ad5-infected baby mouse kidney cells, and in uninfected baby mouse kidney cells. Normalized CAT activity associated with pH-2K^b(367)CAT was taken arbitrarily as 1.0. The boxes represent TATA-like sequences and the upstream and downstream CAA repeated sequences (TATTAAA, CAAu, CAAd). Shaded boxes represent a mutated TATA-like sequence (TAGCGAA). Empty oval, upstream element, 5`-CTGTAAGCCAGACCC-3` (-1,788 to -1,774); striped oval, mutated upstream element, 5`-ATGTAAGAAAGAAAC-3` (-1,788 to -1,774). The construction of CAT plasmids is described under ``Materials and Methods.''



Specificity in Terms of the Type of Adenovirus and the Type of mRNA

We studied the effects of the upstream TATA-like element together with the CAA repeated motif in the presence or absence of either pE1A12S or pE1A13S cDNA constructs from adenovirus type 12 and type 5 (Fig. 2). Only the 13S mRNA from Ad12 had the ability to regulate negatively the expression of the MHC class I promoter (panel B). Thus, the negative effect exerted by the TATA-like sequence together with either an upstream or a downstream CAA repeated element appears to be specific to the mRNA for Ad12-13S E1A.


Figure 2: Relative promoter activities of H-2K-CAT constructs with the upstream TATA-like sequence and a version of the CAA repeated motif in 3Y1 cells (panel A) and E1A-transformants obtained with Ad12-13S E1A (panel B), Ad12-12S E1A (panel C), Ad5-13S E1A (panel D), and Ad5-12S E1A cDNA (panel E). Normalized CAT activity associated with pH-2K^b(367)CAT in 3Y1 was arbitrarily taken as 100. All CAT activities are the averages of results from at least five transfections, and the standard deviation for each is indicated. A, pH-2K^b(2015)CAT ((9) ); B, pH-2K^b(367)CAT; C, del-4m1CAT; D, del-5m1CAT.



Study of the Influence of the Spatial Relationship between the Upstream TATA-like and CAA Repeated Sequences

The effects of the position and the orientation of the upstream TATA-like sequence were studied in transfection experiments (Fig. 3A). The TATA-like sequence was placed in front of the upstream CAA repeated motif, upstream of pH-2K^b(367)CAT (promoter/enhancer) in both the original and the reverse orientation. The CAT plasmid, which had the original orientation but in which the TATA-like sequence was separated from the H-2K^b promoter/enhancer by a CAA upstream sequence (lanes 4 and 5), displayed about the same level of suppression as the wild-type CAT construct (see Fig. 1, lane 1). By contrast, the CAT plasmid with the opposite orientation of the upstream TATA-like element was not associated with a negative effect (lanes 6 and 7). When the upstream TATA-like sequence was placed at the downstream BamHI site of the vector in the original orientation, a significant decrease in CAT activity was observed (lanes 8 and 10). However, when the upstream TATA-like sequence was placed at the same BamHI site of the plasmid in the opposite orientation, no suppression was detected (lanes 9 and 11). When the position of the upstream TATA-like element was exchanged with that of the CAA repeated motif in the original or the reverse orientation, no significant repression of H-2K^b promoter activity was detected (lanes 12 and 13). Thus, the TATA-like element was active at an increased distance from its original position, but the repression of H-2K^b promoter activity was sensitive to the orientation of the TATA-like element. These results were obtained in Ad12-E1A-transformants and were not obtained at similar significant levels in Ad5-E1A-transformed cells.


Figure 3: Effects of the nature of the upstream TATA-like sequence in the promoter region of the H-2K gene. A, effects of the relative positions and the orientations of the upstream TATA-like sequence and the CAA repeated motif on the activity of the H-2K promoter. The orientation of each upstream TATA-like sequence and CAA repeated motif is indicated by an arrow. Average results of four independent experiments are shown in the histogram as CAT activities relative to that expressed by pH-2K^b(367)CAT. Normalized CAT activities of pH-2K^b(367)CAT in CYpAdC3 cells and Ad5-E1A-transformed 3Y1 cells (lane 1) were taken arbitrarily as 1.0. B, activity of the H-2 enhancer/silencer linked to heterologous promoters or to the homologous basal promoter in Ad12-E1A- or Ad5-E1A-transformed cells. All CAT values are the averages of results from at least four transfections. Normalized CAT activity associated with pH-2K^b(367)CAT in CYpAdC3 cells was taken arbitrarily as 1.0. The orientations of the upstream TATA-like sequence and the CAA repeats are indicated by arrows. TATA, upstream TATA-like sequences (BspHI/RsaI); CAAu, upstream CAA repeated motif (RsaI/DdeI); CAAd, downstream CAA repeated motif (BstNI/Sau3A). H-2E, H-2 enhancer; H-2P, H-2 promoter; H-2P/E, H-2 promoter/enhancer; TK, thymidine kinase promoter; SV, SV40 promoter; Cona, conalbumin promoter. The standard deviation for each result is indicated by a bar.



Previous studies have demonstrated that monomers and dimers of the CAA repeats do not function as negative elements in Ad12-E1A-mediated repression of the H-2K^b gene(10) . Therefore, we next examined whether the upstream TATA-like sequence might affect the H-2K^b promoter activity of CAT plasmids with oligomerized CAA repeated motifs. To our surprise, the effect of the TATA-like sequence on the H-2K^b promoter activity was apparent in conjunction with monomeric or dimeric CAA motifs (data not shown). This effect was also sensitive to the relative orientations of the TATA-like sequence and the CAA motifs but not to the relative positional effects of the two elements (data not shown).

The Negative Effect of the TATA-like Element Is Independent of the Promoter

We analyzed the influence of the promoter on the negative effect of the upstream TATA-like element (Fig. 3B). The repression of three different constructs driven by SV40, thymidine kinase (TK), and conalbumin (Cona) promoters was clearly observed (5.0-7.0-fold repression), similar to the repression associated with the homologous promoter (10-fold repression), although the extent of repression was variable (lanes 3, 6, 9, and 12). Thus, this negative effect was not dependent upon the specific promoter tested in the case of Ad12-E1A-transformed cells. By contrast, the upstream TATA-like element had only a moderate negative effect (1.5-2.0-fold repression) in Ad5-E1A transformants.

Effects of Alterations in the Upstream TATA-like Sequence

To determine the relative abilities of various TATA-like sequences to repress the E1A-dependent activity of the H-2K^b promoter, we constructed a series of CAT plasmids with various TATA-like sequences, such as those of the adenoviruses E3 and E4, Ad-ML, hsp70, SV40 early genes, histone H1, c-fos, and beta-globin, by site-directed mutagenesis (Fig. 4). When the upstream TATA-like sequence (TATTAAA) was changed to the basic TATA sequence of promoters (TATAAA), we observed significant repression (lanes A and B). When the upstream TATA-like sequence was converted to the Ad-ML (TATAAAA), hsp70 (TATAAAA), or c-fos (TATAAAA) sequences, significant repression was detected (lane C). The conversion to E4 (TATATATA) and SV40 early (TATTTAT) sequences, did not result in negative repression (lanes G and H). Conversion to the histone H1 (TATATAA), the beta-globin (CATATAA), or the E3 (TATAACT) sequences resulted in moderate negative repression (lanes D, E, and F). These results imply that full negative activity of this upstream element requires the sequence TAT (A/T) AA. The sequence specificity of the TATA-like element was seen only in the case of Ad12-E1A-transformed cells. No significant similar result was obtained in Ad5-E1A-transformed cells.

Effects of Combinations between Upstream TATA-like Sequences and the Basal TATA-box

We next examined the combined effects of the upstream TATA-like sequence and the TATA-box of the basal promoter on the E1A-dependent negative repression of the MHC class I H-2K^b promoter. Various upstream TATA-like sequences were generated by site-directed mutagenesis, as indicated above, and were introduced into either Ad12-E1A- or Ad5-E1A-transformed cells. We constructed CAT plasmids driven by the SV40 early (TATTTAT) promoter or the thymidine kinase promoter (TATTAA), which are known to be negatively and positively affected by Ad5-E1A-mediated transactivation, respectively ((33) ; Fig. 5). In the case of H-2K^b promoter/enhancer constructs, as already shown in Fig. 4, the negative effects of the H-2K^b upstream TATA-like motif (lane 3), the TATA-box (lane 2), and the TATA-box of the Ad-ML promoter sequences (lane 4) were significant. The TATA sequence of the SV40 early promoter did not cause any significant reduction in the promoter activity (lane 5). In the case of the SV40 early promoter/enhancer or the thymidine kinase promoter constructs, the same pattern was observed (lanes 6-17). It was noteworthy that the repression observed with SV40 and thymidine kinase reporter constructs was specific to Ad12-E1A-transformed cells as compared with Ad5-E1A-transformed cells (8-10-fold repression versus 1.5-2.0-fold repression) even though it has been shown that the SV40 promoter is negatively regulated by E1A without serotype specificity(33) . It is clear that the extent of negative repression in Ad12-E1A transformants is much greater than that observed in Ad5-E1A-transformed cells. Taken together, the results suggest that the trans-repression activity is dependent upon a sequence preference that is associated with the upstream TATA-like element and not upon the sequences of the TATA-box close to the basal promoter together with the CAA repeated motif in the presence of Ad12-E1A.

Mutual Competition between Binding Proteins

In an attempt to determine whether similar proteins might bind to the upstream TATA-like sequence and the basal TATA-box in Ad12-E1A-transformed cells, we performed a band shift assay using 31-mer oligonucleotides that corresponded to the upstream TATA-like sequence and the proximal TATA-box sequence in the promoter of H-2K^b. As shown in Fig. 6A, both DNA probes generated three major bands with extracts derived from CYpAdC3 cells (lanes 2 and 3). Cross-competition resulted in the disappearance of the three bands (lanes 4-7). Thus, similar or identical nuclear proteins seemed to associate with the TATA-like sequence and the TATA-box sequence in the promoter region of H-2K^b. To determine whether TBP is directly associated with the binding proteins, a band shift assay, using the 149-bp AccI/DdeI DNA fragment as a probe, was carried out in the presence of TBP-specific antiserum. As shown in Fig. 6B, the two retarded bands (B1 and B2) were shifted still further, and the one retarded band (B3) disappeared upon addition of rabbit antibody against human TBP (lanes 3-5) and mouse antibody against human TBP (lanes 6-8) but not upon addition of the control preimmune rabbit serum (lanes 11 and 12). Antisera that had been preincubated with purified human TBP protein from HeLa cells had no effect on the retarded bands (lanes 9 and 10). To confirm this result, we used a recombinant protein, encoded by the TBP gene, that had been translated in vitro from the TBP gene in a reticulocyte lysate system (Fig. 6C, lanes 2 and 3) to characterize the protein(s) that binds to the upstream and proximal TATA sequences. The DNA probe specific for the upstream TATA-like sequence gave rise to a shifted band, which could be competed out by an excess of unlabeled oligonucleotides specific for the downstream TATA-box (Fig. 6D, lanes 3 and 4) but not by an excess of mutated competitors (lane 5). Taken together, the results indicate that similar or identical proteins, including TBP, associate with the upstream TATA-like sequence and the proximal TATA-box sequence.


Figure 6: Gel shift studies. A, competition in gel shift assays with oligonucleotide DNA probes that correspond to the upstream TATA-like sequence (-1,755 to -1,776) and the proximal TATA-box sequence (-8 to -28), using nuclear extracts from CYpAdC3 cells. Lane 1, DNA probe labeled with T4 kinase; [P]TATAu, 5`-ggacgctggaTATAAAgtccacgcagcccgc-3`; lanes 2, 4, and 5, [P]TATAd DNA probe, 5`-agccagacccTATTAAAtgtctccctttaga-3`; lanes 3, 6, and 7, [P]TATAu DNA probe; lanes 4 and 6, 500 ng of TATAu competitor oligomer; lanes 5 and 7, 500 ng of TATAd competitor oligomer. An arrowhead indicates the protein-DNA complex. B, effects of TBP-specific antiserum on the shifted complex in the gel shift assay. Lane 1, free DNA probe (AccI/DdeI); lane 2, nuclear extract from CYpAdC3 cells; lanes 3-5, 10, 10, and 10 dilutions of rabbit antiserum against human TBP (referred as anti-24-28); lanes 6-8, 10, 10, and 10 dilutions of mouse antiserum against human TBP; lanes 9 and 10, anti-24-28 (10 and 10 dilutions, respectively) that had been preincubated with TBP protein (24 µg), which had been purified as described elsewhere(42) ; lanes 11 and 12, preimmune rabbit control serum (10 and 10 dilutions, respectively). Arrowheads indicate protein-DNA complexes (B1, B2, and B3). C, in vitro translation of [S]methionine-labeled TBP in a rabbit reticulocyte lysate. Lane 1, pGEM1 without the rTBP sequence; lane 2, 500 ng of rTBP/pGEM1; lane 3, 1 µg of rTBP/pGEM1. The arrowhead on the right indicates the recombinant TBP. D, binding properties of in vitro translated rTBP. Gel shift analysis used P-radiolabeled oligonucleotides (0.1 pmol) that corresponded to the upstream TATA-like sequence in the H-2K promoter, as described elsewhere(42) . Approximately 20 fmol of [S]methionine-labeled TBP (lanes 2-5) were used in gel shift assays in the presence or absence of a 500-fold molar excess of synthetic oligonucleotide duplexes that corresponded to TATAu, TATAd, and a mutated TATA sequence (TATAm; 5`-agccagacccTAAATTAtgtctccctttaga-3`). An arrowhead indicates the rTBP-DNA complex.



Immunoaffinity Selection and Amplification by PCR of the Binding Sites of Ad12-E1A-associated Nuclear Proteins

Immunoaffinity selection and CASTing experiments were carried out to identify the binding site on the DNA at which the Ad12-E1A- or Ad5-E1A-associated proteins bind. After a preparative gel shift assay using DNA fragments isolated by immunoselection with antiserum specific for Ad12-E1A or Ad5-E1A, we isolated the material in the shifted bands obtained with extracts of Ad12-E1A or Ad5-E1A-transformed cells (Fig. 7A). We purified the DNA fragments extracted from the shifted bands and amplified the fragments by PCR using both primers of the catch linkers. After amplification of the DNA fragments by PCR, the DNA fragments were again recycled for incubation in a reaction mixture that contained an extract of Ad12-E1A or Ad5-E1A transformants. The amplified DNA fragments obtained by immunoaffinity and CASTing experiments (Fig. 7B) were subcloned into pBluescript for DNA sequencing. The mean size of DNAs cloned into the vector was approximately 300 bp, which is the same as that of the DNAs used as starting materials. Among 280 clones isolated by this CASTing method, 195 clones (70%) were shown to include DNA sequences such as CAA repeated sequences and the upstream TATA-like sequence by use of an extract of Ad12-E1A transformants (Fig. 8, A and B). By contrast when we started with an extract of Ad5-E1A transformants, we found that most of the clones (178 clones; 64%) included DNA sequences such as the TATA box of the H-2 promoter and H-2 enhancer elements (CRE/IRS) (Fig. 8B). To examine whether these isolated clones represented DNA fragments specific for the binding protein(s) present in extracts of Ad12-E1A-transformed cells, we performed a gel shift assay using the DNA fragments, isolated from this CASTing experiment, as DNA probes. As shown in Fig. 7C, when five representative DNA fragments that included the upstream TATA-like motif and both CAA repeated motifs were used as probes, all exhibited binding capacity specific for proteins from Ad12-E1A transformants (lanes 6-10), but not for proteins from Ad5-E1A-transformed cells (lanes 11-15). Constructs mutated in the TATA-like element did not show any significant binding (lanes 1-5). We next examined the effect of the addition of antiserum specific for Ad12-E1A. As shown in Fig. 7D, when the DNA fragment that included the upstream TATA-like sequence was used as probe, the shifted bands disappeared after addition of the specific antibodies (lanes 3-6). By contrast, antibodies raised against Ad5-E1A did not affect the mobility of bands (lanes 7 and 8). Moreover, the antiserum against Ad12-E1A that had been preincubated with Ad12-E1A protein did not affect the shifted bands on the gel (lane 9). Thus, we conclude that the far upstream TATA-like sequence is one of the targets for binding by the Ad12-E1A-associated complex, which is specific for E1A-mediated negative repression.


Figure 7: Immunoselection and CASTing studies. Cloning of the binding sites in the H-2K promoter region by immunoselection and CASTing is shown. A, preparative gel shift assay using DNA fragments isolated by immunoaffinity precipitation with Ad12-E1A-specific or Ad5-E1A-specific antiserum and PCR cycling. P-labeled DNA fragments (5 times 10^4 cpm) isolated from the sonicated DNA from the H-2K promoter region by immunoselection and PCR cycling were incubated with an extract from CYpAdC3 cells or Ad5-E1A-transformed 3Y1 cells. B1 and B2, DNA-protein complexes; F, free DNA probe. Lane 1, extract of CYpAdC3 cells; lane 2, extract of 3Y1 cells; lane 3, extract of Ad5-E1A-transformed 3Y1 cells after passage through a heparin column (see (10) ). B, agarose gel 1.2% electrophoresis of the DNA fragments after immunoselection with E1A-specific antibody and the CASTing assay. Lanes 1-6, isolation of binding site DNAs; lane 7, HaeIII digest of times 174; lane 8, HindIII digest of . C, gel shift assays of representative DNA fragments isolated by immunoselection and CASTing. Lanes 1-5, DNA fragments with mutations in the TATA-like sequence (TATTAAA) to TAGCGAA as the gel shift probe and the nuclear extract from CYpAdC3 cells; lanes 6-10, representative examples of DNA fragments used as DNA probes for gel shift assays with a nuclear extract from CYpAdC3 cells; lanes 11-15, DNA probes used in lanes 6-10 as probe and an extract from Ad5-E1A-transformed 3Y1 cells. An arrowhead indicates the protein-DNA complex. D, effects of Ad12-E1A-specific antiserum on the shifted complex. One of the representative DNA probes (5 times 10^4 cpm) isolated as described above was incubated with an extract of CYpAdC3 cells in the presence of increasing levels of Ad12-E1A-specific or Ad5-E1A-specific antiserum.^2Lane 1, free DNA probe; lane 2, nuclear extract from CYpAdC3 cells; lanes 3-6, 10, 10, 10, and 10 dilutions of Ad12-E1A-specific antiserum; lanes 7 and 8, 10 and 10 dilutions of Ad5-E1A-specific antiserum (M73); lane 9, 10 dilution of Ad12-E1A-specific antiserum that had been preincubated with Ad12 E1A protein (10 µg).




Figure 8: Nucleotide sequence analysis of DNA clones isolated by immunoselection and CASTing. A, the major or minor binding sites indicate the nucleotide sequences of the putative cis-element of the DNA fragments cloned in pBluescript. The frequency of the clones containing insert DNAs of the major binding sites is more than 25% in total recombinant clones obtained. A number indicates the distance from the cap site of the H-2K gene. B, size distribution of the DNA fragments isolated by immunoselection and CASTing. The sizes of DNA fragments cloned in pBluescript were analyzed statistically. Upper panel, for the extract from CYpAdC3 cells; lower panel, for the extract from Ad5-E1A-transformed 3Y1 cells. Putative cis-elements are represented in the figure.




DISCUSSION

We previously identified the CAA repeats in the far upstream region of the mouse MHC class I H-2K^b gene as negative regulatory elements by using the product of E1A gene of adenovirus type 12(9, 10) . We have now provided evidence that the TATA-like sequence located between -1,773 and -1,767 is also required for the E1A-dependent negative regulation of the MHC class I H-2K^b gene in conjunction with one of two CAA repeated elements. A TATA-like element with either an upstream CAA repeat or a downstream CAA repeat is functional (see Fig. 1). However, whereas such a TATA-like element can function independently of its relative position and distance from the promoter, its function is dependent on its relative orientation with respect to the CAA repeats and the TATA-like sequence. A TATA-like sequence in an inverted orientation plus one of two CAA repeats is associated with decreased E1A-dependent negative regulation (Fig. 3A). We failed to observe such a negative function of a TATA-like element in an orientation-dependent but position- and distance-independent manner in normal cells and Ad5-E1A-transformed cells (Fig. 3A). Moreover, we found that the combined negative activity of the upstream TATA-like sequence with CAA repeats appeared to be specific to the 13S E1A from Ad12 and not to the 12S E1A or the 13S E1A from Ad5 (Fig. 2).

To determine the way in which the repression of the MHC class I gene functions in the context of heterologous basal promoters, a DNA fragment containing one CAA repeated sequence was linked, in separate constructs, to the SV40 early, herpes simplex virus type 1, and chicken conalbumin basal promoters. We did not detect any major difference in the suppressive activity on these heterologous promoters as compared with the H-2 basal promoter-enhancer combination in Ad12-E1A transformants (Fig. 3B). Our results are consistent with the previous report by Ge et al.(30) . Thus, the H-2 TATA-box sequence per se may not be important since its conversion to other TATA-box sequences in the promoter failed to affect transcription of the H-2 gene in Ad12-E1A-transformed cells. However, the suppressive activity of these elements on heterologous promoters was less significant in Ad5-E1A-transformed cells (Fig. 3B).

We next examined the nature of the optimum TATA-like sequence in the upstream region for E1A-mediated negative regulation. Examples of endogenous cellular and viral genes that are activated as a result of the E1A trans-activation process include the heat-shock gene for hsp70(46, 47) , the gene for hsp89(48) , the gene for beta-tubulin (49) , the c-fos gene(50) , the viral E1B gene(51) , and the long terminal repeat of human HIV(52) . A binding study in vitro demonstrated that Ad-E1A binds to TFIID(53, 54) . The correlation between the much greater trans-activation by the 13S E1A protein than by the 12S E1A protein (56, 57) and the greater affinity of TFIID for 13S-E1A (53, 54, 55, 56) suggests that the E1A-TFIID interaction plays a fundamental role in E1A trans-activation in nononcogenic E1A-transformed cells. Therefore, we attempted to change the far upstream TATA-like sequence of the H-2 promoter to the TATA-box sequences from heterologous genes to examine the effects of these elements on E1A-mediated negative repression. As shown in Fig. 4, TATA-box sequences from H-2K^b and Ad-ML (as well as from hsp70 and c-fos genes) were all able to function as upstream TATA-like elements in mediating the E1A-dependent repression of the H-2K^b promoter (lanes A, B, and C). Moreover, it is clear that the upstream TATA-like sequence is preferentially functional in conjunction with either an upstream or a downstream CAA element ( Fig. 1and Fig. 5). In addition, the results of our immunoselection and CASTing studies suggest that nuclear proteins present in Ad12-E1A-transformed cells can bind to DNA fragments that contain the TATA-like sequence and the basal TATA-box sequence in cooperation with E1A molecules ( Fig. 7and Fig. 8). To our surprise, we found that the majority of DNA fragments isolated by the immunoaffinity and CASTing method by use of an extract from Ad12-E1A transformants contained the upstream TATA-like sequence and the CAA repeated motif. By contrast, most of the DNA fragments isolated by use of an extract of Ad5-E1A transformants contained the basal TATA-box and H-2 enhancer (for example, CRE/IRS (R1/R2) sequences) (Fig. 8B). Addition of antiserum against Ad12-E1A to the reaction mixture used for the gel shift assays with a DNA fragment that contained the upstream TATA-like sequence as probe resulted in the disappearance of shifted bands (Fig. 7D). Addition of antiserum against Ad5-E1A did not have such an effect (Fig. 7D).

What is the nature of the binding proteins that associate with the DNA sequence of the far upstream CAA repeats and the TATA-like element, and how do these proteins interact with TBP (or a TBP-like protein) to achieve negative regulation of the H-2K^b gene in Ad12-E1A transformants but not in Ad5-E1A transformants? Gel shift experiments using competitive oligonucleotides designed on the basis of binding sites, recombinant TBP, and TBP-specific antiserum revealed that similar or identical molecules, possibly including TBP, bound to both the upstream TATA-like sequence and the basal TATA-box sequence in vitro and in vivo (Fig. 6). We speculate that the similar or identical TBP (or TBP-like protein) can bind to both TATA sequences in parent 3Y-1 cells and Ad5-E1A-transformed cells. Several lines of experimental evidence have suggested that E1A-CR3 of nononcogenic adenovirus binds in vitro specifically and stably to the isolated TBP of holo-TFIID(53, 54, 55, 56) . Recent studies support this model and provide direct evidence that trans-activation is mediated through a direct physical association between the E1A-CR3 domain and TBP in the holo-THIID complex(55, 56) . We also observed that the affinity of Ad12-13S E1A for TBP is much stronger than that of Ad5-13S E1A protein for TBP in vitro (data not shown). The affinity of TBP (or TBP-like protein) for the region of the upstream TATA-like element and the CAA repeated motifs is weak in Ad5-transformed cells, about 10-fold lower than that in Ad12-E1A transformants (see (10) ). Recently we succeeded in characterizing the recombinant proteins that bound to the CAA repeats and were associated with Ad12-E1A protein in vitro (data not shown). In the presence of Ad12-E1A, E1A associates with TBP on the upstream TATA-like element and the TATA-box to form a protein complex that includes TBP, proteins that bind to CAA repeats, and E1A. Alternatively, E1A may modify the binding affinities within complexes that include TBP to allow more efficient binding to the upstream TATA-like sequence. In this way, the basal transcriptional activity of the H-2K^b gene can be decreased. The formation of a large protein complex including TBP and proteins that bind to CAA repeats might be critical for association with E1A in Ad12-E1A transformants but not in Ad5-E1A transformants. We also cannot exclude the possibility that the distinct sequences of respective E1A proteins may be involved in the binding to the TBP or to a TBP-like protein to cause the conformational changes that alter the transcriptional machinery of the H-2K^b gene. Preliminary studies by glycerol gradient centrifugation indicate that a large complex of these components derived from Ad12-E1A transformants but not from Ad5-E1A transformants or parent cells is formed in vitro. (^3)We speculate that the ability to form such a large complex, which includes TBP, the proteins that bind to CAA repeats, and E1A, might be critical for regulation of the H-2K^b gene. Kralli et al.(31) and Ge et al.(30) reported that the R2 class I enhancer element is involved in Ad12-E1A-mediated and not in Ad5-E1A-mediated repression in transformed cells. We cannot rule out such a possibility since we detected moderate repression of the promoter activity of H-2 constructs with the R1/R2 region in the presence of Ad12-E1A (see (10) ). At this time we cannot explain the possible functional relationship between R2BP(31) , COUP-TF, which has been identified more recently by Liu et al.(58) , and the factors with which we have been dealing here. It is possible that dual regulation, involving proximal and far upstream elements, might be important for the full negative control of the activity of the H-2 promoter by Ad12-E1A.

The Ad12-E1A-dependent negative repression was independent of the nature of the heterologous promoters tested but was preferentially observed with the specific common sequence TAT(A/T)AA. Recent studies by Brou et al.(59) demonstrated that distinct TFIID complexes mediate the effects of different types of transcriptional activation. Similarly, different complexes that included TBP may be formed and may bind to the upstream TATA-like sequence or to the proximal TATA sequence in different ways. Further characterization of the genes that encode the proteins that bind to the TATA-like sequence and to the basal TATA-box should help us to elucidate the mechanism of regulation of transcription of the MHC class I H-2K^b gene.


FOOTNOTES

*
This work was supported by the special coordination funds of the Science and Technology Agency, by the Life Science Project of RIKEN, and by the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X15104[GenBank].

§
To whom correspondence should be addressed. Tel.: 298-36-3612; Fax: 298-36-9120.

(^1)
The abbreviations used are: MHC, major histocompatibility complex; TBP, TATA-binding protein; bp, base pair(s); CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; hsp, heat shock protein.

(^3)
X. Tang, H.-O. Li, O. Saiatsume, T. Ohta, H. Tsutsui, A. F. A. Smit, M. Harikoshi, P. Kourilsky, A. Israël, G. Gachelin, and K. Yokoyama, unpublished data.

(^2)
K. Yokoyama, manuscript in preparation.


ACKNOWLEDGEMENTS

The authors thank H. Guo for the excellent technical assistance and A. Fujita and S. Shimizu for preparing the manuscript.


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