Cis elements for transporter associated with antigen-processing-2 transcription: two new promoters and an essential role of the IFN response factor binding element in IFN-
-mediated activation of the transcription initiator
Yong Guo2,
Tianyu Yang,
Xingluo Liu,
Shengli Lu,
Jing Wen,
Joan E. Durbin1,
Yang Liu and
Pan Zheng
Department of Pathology and Comprehensive Cancer Center, Ohio State University Medical Center, 129 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, USA
1 Department of Pediatrics, Columbus Children's Hospital, Ohio State University, Columbus, OH 43205, USA
2 Present address: Hoechst Marion Rousse, Inc., CNS-Molecular Biology, Bridgewater, NJ 08807, USA
Correspondence to:
P. Zheng; E-mail: zheng-1{at}medctr.osu.edu
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Abstract
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Expression of cell surface MHC class I:peptide complex requires coordinated expression of multiple genes such as MHC class I heavy chain, ß2-microglobulin (ß2m), transporters associated with antigen-processing (TAP)-1 and TAP-2, and proteosomal components low-molecular weight polypeptide (LMP)-2 and LMP-7. All of these genes are expressed at defined and distinct levels in normal tissues, and are inducible by IFN-
. While the cis elements involved in transcription of the MHC class I heavy chain, ß2m, TAP-1 and LMP-2 have been analyzed extensively, those for TAP-2 and LMP-7 have not been well studied. Here we systematically analyzed the cis elements for TAP-2 transcription. We found at least two independent elements that are sufficient to activate transcription of a reporter gene. One (hereby called TAP-2 P1) is located 5' to the TAP-2 exon 1, while the other (hereby called TAP-2 P2) is a transcription initiator residing in intron 1. Analysis of the 5' sequence of TAP-2 mRNA indicates that both promoters are active. Moreover, while the TAP-2 promoter region contains cis elements that can mediate TAP-2 induction by IFN-
, such as
-activation site and IFN response factor binding element (IRFE), only the IRFE is required for IFN-
induction of TAP-2 promoter in vitro. The IRFE appears to work as an enhancer for the initiator (P2). Together with another promoter recently identified by others, TAP-2 therefore has three independent promoters that can be differentially regulated.
Keywords: antigen presentation genes, IFN-
activation, transporters associated with antigen-processing-2, transcriptional regulation
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Introduction
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MHC class I antigens are expressed constitutively in the majority of tissues, but the levels of expression differ significantly. While most leukocytes express high levels of MHC class I, other organs express these genes at much reduced levels, with almost no detectable expression in the central nervous system (1). In addition, MHC class I antigens are highly inducible by a number of cytokines, such as IFNs (2) and tumor necrosis factors (3). Since MHC class I is the target for the majority of cytotoxic T cells (4), the expression level of these proteins determines the efficiency of immune surveillance by CD8 T cells. Indeed, both viruses (5,6) and tumors (711) can evade the immune system by down-regulating cell surface MHC class I expression.
Expression of MHC class I antigen on the cell surface requires expression of multiple genes (1214), such as MHC class I heavy chain, ß2-microglobulin (ß2m), peptide transporters associated with antigen-processing (TAP)-1 and TAP-2, and immune proteosomal components low-molecular-weight polypeptide (LMP)-2 and LMP-7, all of which are inducible by IFN-
(15). Cell surface MHC class I expression is controlled by both transcriptional and post-translational mechanisms. Most studies on transcriptional regulation of MHC class I antigen presentation genes have focused on MHC class I heavy chain and ß2m genes. More recently, the bi-directional promoter that controls both LMP-2 and TAP-1 has been studied in detail (12,16,17), but very little information is available on the promoters for LMP-7 and TAP-2. Here we have carried out a detailed deletion analysis in order to characterize the TAP-2 promoters. Our analysis has revealed two independent promoters for TAP-2 expression. Moreover, we report here that IFN response factor binding element (IRFE), but not the
-activation site (GAS) element, is required for IFN-
-mediated induction of the TAP-2 promoter and that the IRFE acts as an enhancer for the transcription initiator.
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Methods
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Cells and experimental animals
A number of murine cell lines were used for the study. The NIH3T3 cell line and the embryonic fibroblast cell lines, prepared from either wild-type (B6WT) or STAT-1(/) (B6STKO) C57BL/6j mice were kindly provided by Dr David E. Levy (New York University Medical Center, New York, NY) (18). HeLa and COS cells were obtained from ATCC (Rockville, MD). In some experiments, wild-type or STAT-1(/) C57BL/6j mice were used as sources of splenocytes.
Cloning of DNA fragment between known exons of LMP-7 and TAP-2 genes
The DNA fragment between LMP-7 and TAP-2 genes was amplified by PCR using genomic DNA from 129/sv spleen cells as the template. The LMP7.F1 was used as the forward primer and TAP2.R1 as the reverse primer. All primer sequences are listed in Table 1
. PCR was carried out for 35 cycles: 94°C 1 min, 55°C 1 min and 72°C, 4 min. The 3.8-kb PCR product was cloned into pBluescript-KS vector (Stratagene, La Jolla, CA). Its identity was verified by partial sequencing from both directions. The restriction enzyme mapping of the cloned fragment was identical to the published sequence.
Construction for luciferase reporters and dual-luciferase assay
A large panel of luciferase reporter constructs was made. Each construct consisted of a portion of the TAP-2 gene 5' sequence inserted 5' to the open-reading frame of the luciferase gene. TAP-2 5' gene fragments were generated by PCR using primers listed in Table 1
. Since the forward primer contains a XhoI site and the reverse primer has a HindIII site, the PCR products were subcloned into the XhoIHindIII sites in pGL2-Basic vector (Promega, Madison, WI).
For the IRFE enhancer activity study, P1F5/s was cut out using XhoI and HindIII, and blunt-ended using T4 DNA polymerase (Life Technologies, Grand Island, NY) before being cloned into the SmaI site 5' to the P2 promoter in P2F9 construct. Copy number and direction of the inserts were confirmed by sequencing.
The luciferase reporter constructs were co-transfected with pRL-SV40 (Promega) as a control for transfection efficiency. The promoter activity was determined using a dual-luciferase assay kit from Promega and is expressed as fold induction, calculated according to the following formula: fold induction = (sample luciferase/sample renila luciferase)/(basic luciferase/basic renila luciferase).
Data presented as means of triplicates with variations among replicates always <20%.
Characterization of 5' sequence of the TAP-2 cDNA by PCR
cDNA libraries prepared from either RAW8.1 leukemia cell line or primary splenocytes were used as a source of TAP-2 cDNAs (19). The T7 primer was used as a forward primer and the reverse sequence corresponding to the 4570 bp down-stream of the translation initiation codon of the TAP-2 gene was used as the reverse primer (TAP2.Rev). The PCR products obtained were either sequenced directly or were cloned into the pBluescript vector prior to sequencing.
Characterization of the 5' TAP-2 mRNA sequence by 5' rapid amplification of cDNA ends (5' RACE)
Total cellular RNA was isolated from splenocytes of either wild-type or STAT-1(/) C57BL/6j mice with Trizol reagent (Life Technologies, Grand Island, NY). (5' RACE) was carried out with the 5' RACE system (Life Technologies). Briefly, 2 µg of total RNA was used for first-strand cDNA synthesis using random hexamer primers. The oligo(dC) tailed cDNA was amplified by PCR using Pfu DNA polymerase according to the protocol from the manufacturer (Promega; 35 cycles of 95°C for 1 min, 55°C for 0.5 min, 72°C for 3 min and final extension at 72°C for 5 min) with an abridged anchor primer (AAP, 5' RACE system; Life Technologies) and a TAP-2-specific primer (TAP2P1) complementary to nucleotides 454478 of the TAP-2 coding sequence (5'-CCACAAGGAAGAAGAAGGCAGCTAT) (GenBank M90459). A dilution of the original PCR product served as template for nested PCR using the abridged universal amplification primer (AUAP, 5' RACE system) and a second TAP-2-specific primer (TAP2P2) complementary to nucleotides 422436 of the TAP-2 coding sequence (5'-GGCAGGTCCGGCCTGGACAGCTTCA). PCR products were separated by agarose gel electrophoresis, transferred to nylon membranes and hybridized to a TAP-2 cDNA probe. At the same time, larger DNA fragments (>500 bp) were isolated and directly cloned into the pCNTR shuttle vector system (Eppendorf Scientific, Westbury, NY) for the analysis. Ten TAP-2 cDNA clones were sequenced using a TAP-2-specific primer (TAP2P4) complementary to nucleotides 301323 of the TAP-2 coding sequence (5' GCCCCATAGCCAGCCAGCAGCCA).
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Results
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Deletional analysis reveals two independent TAP-2 promoters
The murine TAP-2 cDNA available at the time of this study did not contain a 5' untranslated region (UTR) sequence upstream of the translation initiation codon (ATG). In contrast, data from GenBank shows both human (13) and rat (20) TAP-2 cDNA sequences contain a 5' UTR encoded by a distinct exon 1. As attempts to characterize the 5' UTR by primer extension and RNase protection were unsuccessful, we used RT-PCR to determine the 5' sequence of the TAP-2 gene. Two cDNA libraries cloned into the pCDM8 vector were used as templates: one was prepared from mouse splenocytes, the other from the RAW8.1 leukemia cell line. The T7 primer was used as the forward primer and the reverse primer consisted of the nucleotides 4570 down-stream of the ATG initiation site (Fig. 1a and b
). The predominant PCR products from both libraries were ~160 bp in length. We cloned the PCR products and sequenced three independent clones from both directions. In addition, both PCR products were subjected to bulk sequencing. The sequences of the three clones and the bulk PCR product were identical, and revealed a 119 bp sequence 5' of the ATG codon. When compared to the genomic DNA sequence, the last 8 bp preceded the ATG and were separated from the remaining 111 bp by a 663 bp gap. Since the junction followed the GT/AG rule, it was concluded that the 663 bp insertion is an intron, which we called intron 1, while the 111 bp sequence was tentatively assigned as exon 1 (Figs 1b and 2a
). This assignment is in agreement with a recent report of Aron et al. (21) and is consistent with the sequence of rat TAP-2 cDNA (X75305, X75306 and X75307). The high GC contents in exon 1 (76/111) may explain the difficulties we encountered in primer extension and RNase protection assay.

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Fig. 1. Searching for TAP-2 exon 1 by RT-PCR. (a) Flow-chart of PCR and cloning of the TAP-2 5' fragment. Two pCDM8 (Invitrogen, Carlsbad, CA)-based cDNA libraries, one from the murine splenocytes and the other from the murine leukemia cell line RAW8.1, were used as templates. The T7.F forward primer and the TAP2.Rev reverse primer, which was based on the sequence of TAP-2 cDNA 4570 bp down-stream of the translation initiation site, were used to amplify TAP-2 cDNA. The PCR products were cloned into the pBluescript vector. (b). Sequence of TAP-2 cDNA and alignment with genomic DNA. The inserts of three clones were sequenced and found to be identical. The sequence is also confirmed by bulk sequence of the PCR products. The first 111 bp were separated from the remaining cDNA fragment by a 663-bp fragment. The DNA encoding the 111 bp is hereby assigned as exon 1.
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Fig. 2. Cloning the TAP-2 promoter region. (a) Diagram of the 5' region of the TAP-2 gene in the context of the mouse MHC region in the chromosome 17. The bold arrows indicate the direction of transcription, while the small arrows indicate the primers used to amplify the 5' region of TAP-2. (bd) A 1.7-kb fragment of the TAP-2 5' sequence has optimal promoter activity in three different cells lines. The full-length 3.8-kb DNA fragment between LMP-7 and TAP-2 (P1P2) and the 1.7-kb DNA fragment (P1F1) were cloned into the pGL2-Basic luciferase vector and transiently transfected into NIH3T3 cells (b), or embryonic fibroblast cell lines prepared from either wild-type (B6WT) (c) or STAT-1(/) (B6STKO) (d) C57BL/6 mice. The luciferase activity in cell lysates was analyzed by dual-luciferase assay after 48 h. The pGL2-Basic (Basic) and pGL2-SV40 (SV40) from Promega were used as control.
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The DNA sequence 5' of the TAP-2 open reading frame was not published when our analysis was initiated. Since the LMP-7 and TAP-2 genes are closely linked within the MHC class II region (Fig. 2a
), we decided to isolate the intervening sequence using a pair of PCR primers based on available murine LMP-7 and TAP-2 sequences. The forward primer consisted of the 3' UTR of LMP-7 (LMP7.F1), while the reverse primer sequence corresponded to the 5' coding exon of TAP-2 (TAP2.R1). Using these primers, a 3.8-kb PCR fragment was amplified from 129/sv mouse spleen DNA and cloned into pBS-SK vector. Partial DNA sequence of ~600 bp from either terminus revealed that the 5' sequence of the 3.8-kb fragment was identical to the LMP-7 3' UTR, while the 3' sequence was identical to the 5' portion of TAP-2. While this work was in progress, 138 kb of sequence from the murine MHC class II region was submitted to GenBank by Hood and colleagues (AF027865). The partial sequence we obtained was identical to the region between LMP-7 and TAP-2. Further restriction mapping indicated that the DNA fragment we had cloned was identical to the published sequence (data not shown).
As a first step towards characterizing the promoter region that controls TAP-2 expression, we compared the promoter activity of a 1.7-kb 3' fragment (P1F1) with that of the full-length 3.8-kb fragment (P1P2). The two fragments were cloned into the pGL2 luciferase reporter vector and transiently transfected into NIH3T3 or embryonic fibroblast cell lines prepared from either wild-type or STAT-1(/) C57BL/6j mice. After 48 h, the cell lysates were analyzed by dual-luciferase assay. As shown in Fig. 2
(bd), both P1F1 and P1P2 fragments had strong and comparable promoter activity. We therefore restricted further analysis on the 1.7-kb P1F1 fragment.
As illustrated in Fig. 2
(a), the P1F1 fragment contained ~1 kb of sequence 5' to exon 1, all of exon 1 and intron 1, as well as part of the exon 2 sequence. Analysis of P1F1 revealed multiple potential transcription factor binding sites, as depicted in Fig. 3
(a). These cis elements were scattered in the region 5' of exon 1 and in intron 1. To determine the potential contribution of these cis elements, we generated a series of promoter deletion mutants and inserted these fragments into the pGL-2 luciferase vector (Fig. 3b
). Upon transfection of these constructs into both murine (NIH3T3) and human (HeLa) cell lines, we consistently observed undiminished promoter activity when all but 70 bp of the sequence 5' of the exon 1 was deleted (Fig. 3c
). Further deletion in the 5' region, as in the case of P1F5, resulted in a 3-fold reduction of the promoter activity. Moreover, removal of an additional 32 bp 5' of exon 1 (P2Sac) reduced promoter activity by another 5- to 10-fold. Note here that the P2Sac fragment that contains exon 1 and intron 1 still maintains significant promoter activity. Nonetheless, the 70-bp fragment 5' of exon 1 plays an important role in transcriptional activation of the TAP-2 promoter.

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Fig. 3. Identification of the first promoter sequence 5' of exon 1. (a) Known cis elements in the 1.7-kb DNA fragment 5' to TAP-2 (P1F1). Numbering of the sequence is relative to the ATG codon of TAP-2 (thick bent arrow). The sequences of potential cis elements are boxed, the sequences of exon 1 and part of exon 2 are underlined, and three transcription starting sites (TSS) are indicated by bent arrows. (b) Diagram of the deletion mutants. (c) Deletion analysis revealed the critical function of a 70-bp fragment, 5' of exon 1, for the promoter activity of the 1.7-kb fragment. Constructs with deletions upstream of exon 1 were transfected to either NIH3T3 or HeLa cells and the promoter activity was determined by the dual-luciferase assay.
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To test whether the 70- and 32-bp fragments are sufficient to activate transcription, we inserted the 70 (P1F4/s)- or the 32 (P1F5/s)-bp element into the luciferase vector (Fig. 4a
), and compared their activity to that of the SV40 promoter. As shown in Fig. 4
(b), both had significant promoter activity but the 30 (P1F5/s)-bp fragment was much less potent. These fragments had neither TATA box nor SP1 sequence, but were remarkably G/C rich.

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Fig. 4. The 70- and 32-bp fragments 5' of exon 1 have significant promoter activity. (a) Diagram of the cis elements in the P1 region and the constructs used. (b) Promoter activity. Note detectable promoter activity of the IRFE sequence (P1F5/s) and the significant enhancement of the promoter activity due to an additional 40-bp fragment upstream (P1F4/s).
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Nevertheless, the 70 (P1F4/s)-bp fragment had a substantially lower promoter activity than the entire P1F4 fragment and the significant promoter activity observed in the P2Sac fragment (Fig. 3b
) suggested the existence of additional cis elements within the intron 1. We therefore generated another series of reporter constructs in which partial intron 1 sequences were inserted 5' to the luciferase gene (Fig. 5a
). Since two potential cAMP response element (CRE) sites (in reverse orientations) could be recognized in the intron 1, we designed our deletion fragments to test the function of these known elements. In three cell lines, we observed that all four fragments tested had measurable promoter activity (Fig. 5b
d). Removal of either one (P2F4 in Fig. 5b
) or two (P2F5 in Fig. 5c and d
) of the CRE sites reduced the promoter activity by 2- to 5-fold, suggesting that these CRE sites have a positive role in TAP-2 activation. Interestingly, promoter activity was restored when an additional 32-bp fragment was deleted (P2F8). It is therefore possible that this fragment may have a negative regulatory role that can be neutralized by the CRE sites. Since the minimal 38-bp fragment (P2F9) had activity virtually identical to that of the longest fragment (P2F1), all the promoter activity of the intron 1 must therefore reside within this 38-bp fragment (P2F9). Consistent with this conclusion, the 38-bp fragment contains a typical initiator (Inr) sequence TCA(+1)TTTC.

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Fig. 5. Characterization of TAP-2 promoter 2. (a) Diagram of the cis elements in P2 region and the constructs used. (bd) The constructs were transfected into HeLa (b), NIH3T3 (c) and COS (d) cells to test their promoter activity 48 h after transfection.
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5' RACE revealed that the transcription initiator is functional in vivo
The deletional analysis described above revealed that TAP-2 gene has two promoters: one resides just 5' of exon 1 and the other is potentially a transcription initiator (Inr) residing within intron 1. A unique feature of the Inr is that transcription starts at the +1 site of the CTA(+1)TTTC, which serves as a useful fingerprint of the Inr utilization (22,23). Since utilization of the two promoters should result in distinct products, we set out to characterize the 5' UTR of the TAP-2 transcripts. As discussed, we were unable to identify the 5' transcription start sites by primer extension and therefore took the 5' RACE approach. We made a reverse primer using TAP-2 exon 2 sequence and amplified the 5' sequence using RNA isolated from either wild-type or STAT-1(/) splenocytes. The PCR products were cloned into the pBlusescript vector and the TAP-2 clones obtained were individually sequenced. These sequences were aligned with the genomic DNA sequence (Fig. 6a
) and the results are summarized in Fig. 6
(b). In addition to the 5' sequence shown in Fig. 1
, which we called TAP-2-1, the 10 clones sequenced fell into three groups. Group 1 (TAP-2-2) has all but 28 bp of exon 1 sequence [1/10, from the STAT-1(/) spleen cells]. The close proximity and the spacing between the 5' of the TAP-2 cDNA (TAP-2-1 and TAP-2-2) and that of the promoter 1 identified in this study strongly suggest that the promoter 1 (TAP-2 P1) is responsible for transcription of this product.

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Fig. 6. Identification of the 5' termini of TAP-2 RNA by 5' RACE: evidence for utilization of the initiator in spleen cells. (a) Alignment of four groups of TAP-2 transcripts with the TAP-2 genomic DNA sequence. (b) Summary of the clones isolated by two different strategies. (c) Relative abundance of the four species of TAP-2 RNA. The 5' RACE products were separated by 1.5% agarose gel electrophoresis and then transferred to a nylon membrane. The membrane was probed with TAP-2 cDNA.
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Half of the clones sequenced belong to group 2 (TAP-2-3), which started precisely at the +1 site of the Inr (Fig. 6a
). This precise initiation strongly suggested that they are the products of transcription using Inr as the promoter. Group 3 clones (TAP-2-4) have a 5' terminus 9 bp down-stream of the putative translation initiation ATG codon. Because a second in-frame ATG start codon was present at position 49 after the first, it is possible that TAP-2-4 mRNA can still encode a truncated TAP-2 protein. Nevertheless, it is unclear whether this last group of cDNA reflects real mRNA product in vivo and, if so, whether this truncated TAP-2 protein can be functional.
In order to measure the relative abundance of the three groups of the TAP-2 cDNA, the 5' RACE products were separated by agarose gel electrophoresis and then analyzed by Southern blot using TAP-2 cDNA as the probe. Inserts from all three groups of cDNA clones were isolated and used as markers. As shown in Fig. 6
(c), four major species of the 5' RACE products were detected by Southern blot. The molecular weights of these bands were consistent with the four transcription start sites we have predicted. It is therefore likely that the 5' termini of the major TAP-2 mRNA species have now been identified. Moreover, since all species were present in the RACE products from both wild-type and STAT-1(/) spleens, both promoters must have been functionally independent of STAT-1.
IRFE as an essential IFN-
responsive element that acts as an enhancer for Inr
An important feature of genes involved in antigen presentation is their induction by IFN, especially IFN-
. The TAP-2 promoter region contains a GAS and an IRFE. To determine whether any of these cis elements are required for IFN-
-mediated induction of TAP-2, we carried out a systematic deletion analysis. The deletion mutants were cloned into the luciferase reporter constructs, as illustrated in Fig. 7
(a), and then used to transfect either wild-type or STAT-1(/) embryonic fibroblasts. Transfected cells were left untreated or treated with 1000 U/ml of IFN-
and lysates were tested for luciferase activity at 48 h. The ratios of luciferase activity in IFN-treated over untreated cultures are presented in Fig. 7
(b). The data showed that deletion of all but 32 bp 5' of exon 1 had no effect on IFN-
-induced TAP-2 promoter activity. Since the deletion of GAS had no effect on IFN-
responsiveness, the GAS was not necessary for induction by IFN-
. In contrast, IFN-
induction was eliminated after a 32-bp sequence was removed. Regardless of the constructs used, IFN-
function depends on the STAT-1 gene, as the STAT-1(/) fibroblasts showed no induction of TAP-2 activity by IFN-
.
Since the essential 32-bp fragment contains an IRFE, it is most likely that this element is required for IFN-
responsiveness. To confirm this, we generated a mutant that contained all of the TAP-2 5' sequence except the IRFE (Fig. 8a
). As shown in Fig. 8
(b), the response to IFN-
was completely eliminated by deletion of the IRFE. Thus IRFE is required for IFN-
-response of the TAP-2 promoter. Again, despite the fact that promoter activity was unaffected by deletion of GAS, IFN-
function was strictly dependent on the STAT-1 gene as TAP-2 was not induced by IFN-
in fibroblasts derived from the STAT-1(/) mice.
To test if the P1, which contains the IRFE, is responsive to IFN-
, we compared P1F1, P1F4/s and P1F5/s activity in cells with and without IFN-
stimulation. As shown in Fig. 9
(a), while the P1F1 promoter was highly responsive to IFN-
stimulation, the response of P1F4/s and P1F5/s to IFN-
was not significant. This result suggests that the IFN-
cannot stimulate IRFE to enhance P1 activity. An alternative hypothesis is that IRFE works in concert with the Inr. To evaluate this possibility, we linked, in different copy numbers and orientations, the P1F5/s fragment, which contains the IRFE, to the P2F9 fragment, which contains the Inr. Since the P2F9 by itself was not responsive to IFN-
(data not shown), we defined the activity of P2F9 as 1 to better illustrate the function of IRFE. As shown in Fig. 9
(b), a single copy of P1F5/s significantly increased the P2F9 activity and additional copies of the P1F5/s increased the promoter activity. The reverse orientation appears somewhat more active. These results suggest that IRFE can act as an enhancer for the Inr residing in the intron 1.
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Discussion
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The TAP-2 gene encodes an essential subunit for the transporter associated with antigen processing. While it is clear that transcriptional regulation of TAP-2 is an important aspect of antigen presentation, very little information is available on the gene structure and regulation of TAP-2 transcription. Our results presented here, and a recent publication by Arons et al. (21), provide the much-needed initial characterization. Taken together, the two studies reveal three distinct promoters responsible for expression of TAP-2 mRNA, two of which are responsive to the IRFE-mediated induction by IFN-
.
The first promoter, P1, resides immediately upstream of exon 1. Deletion analysis revealed that essentially all of the promoter activity is contained in a 70-bp fragment 5' of exon 1. A lower, but significant promoter activity can be detected in an even smaller 32-bp fragment containing the IRFE sequence. Interestingly, while these short fragments have promoter activity, they contain neither a TATA box nor an Sp1 consensus sequence. The high G/C content of this region may allow binding of RNA polymerase to initiate transcription. The existence of this promoter is supported by Arons et al. (21), who reported a significant promoter activity of a 95-bp fragment encompassing both the 32- and 70-bp fragments. The second promoter, P2, resides 38 bp 5' of the translation start codon. Sequence analysis of this region indicates that a transcription initiator (Inr) is located within the region. A signature of Inr function is that transcription starts at the +1 position of the Inr (22,23). Analysis of the 5' sequence of TAP-2 transcripts identified using 5' RACE indicates that the Inr is indeed employed as a second promoter. However, Arons et al. (21) were not able to observe any TAP-2 transcripts initiated in the intron 1 by RNase protection assay. This is most likely due to technical difficulties associated with the high G/C content of this region, as the majority of the 5' initiation sites were not identified by this method (21). The third promoter, identified by Arons et al. (21) (hereby called TAP-2 P3), encompasses 111 bp of exon 1. This region contains two multiple starting site down-stream element (MED1) sequences which may explain the multiple initiating sites identified by Arons et al. (21). It is unclear whether the difference in our results is due to technical difficulties or reflects the use of different cell lines in our respective studies.
It is worth noting that while all three promoters can function independently, in a physiological context they are most likely to function in concert. This is underscored by the requirement of the first intron for optimal functioning of the first promoter, P1. The precise sequence in the intron 1 that enhances the P1 activity remains to be characterized. Sequence analysis revealed that the DNA that encodes intron 1 contains two CRE elements. As a group, these cis elements do not enhance the function of the Inr. It remains to be tested if they are responsible for increasing the efficacy of the promoters 1 and 3.
Although the initiator alone has strong promoter activity, this activity appears to be negatively regulated by the additional sequence in the 5'. Since the initiator was preceded by two CRE elements that have been implicated in suppression of MHC class I heavy chain transcription (24), we tested the effect of deleting CRE on the activity of the initiator. Our results demonstrated that deletion of the two CRE elements reduces the initiator activity, thus the CRE elements are not negative regulators for the TAP-2 initiator.
Our data and that of Arons et al. (21) have shown that IRFE is required for IFN-
-mediated induction of TAP-2 promoters 2 and 3 respectively. In addition, the IRFE is required for optimal constitutive activity of promoter 3 (21), as is generally true for MED1 promoters (25). However, linking the IRFE to the Inr did not appreciably increase the Inr's constitutive activity. Since the IRFE is part of P1, it obviously plays a role in its constitutive activity.
Most, if not all, transcriptional activation by IFN-
is mediated by the JakStat pathway (18,26,27). The IFN-
signaling process is initiated by binding of dimeric IFN-
to the ligand-binding IFN-
receptor
subunit chain. This leads to receptor dimerization, which is followed by activation of the Janus protein tyrosine kinases, Jak-1 and Jak-2, associated with the IFN-
receptor subunits. Subsequently, this leads to the phosphorylation of latent cytoplasmic transcription factor Stat-1 and the translocation of activated dimeric Stat-1 to the nucleus. The GAS response element TTCC(C or G)GGAA present within responsive promoters is bound directly by the Stat-1 dimer, thereby leading to transcriptional activation. Several GAS-like elements that possess the palindromic core sequence TTN5AA are also bound by Stat-1 (2629). Our study revealed that in the STAT-1(/) fibroblast the TAP-2 promoter is completely resistant to IFN-
-mediated induction. However, the critical involvement of the IRFE, but not the GAS element, in IFN-
-mediated activation of the TAP-2 promoter argues for a different mechanism. Several lines of evidence support this. First, Arons et al. (21) showed that the IRF-1, but not IRF-2 and IFN consensus sequence binding protein, is sufficient to induce the TAP-2 promoter. Second, Stat-1 is required for optimal expression of IRF-1 (18,30). In this regard, it is most likely that activation of TAP-2 promoter requires both Stat-1 and IRF-1, much as what has been suggested for LMP-2 (31). Recent studies revealed an alternative pathway in which IFN-activated Stat-1 complexed with IRF-9 (p48) can activate transcription through interaction with the IFN-stimulated response element (3234). The function of IRF-9 in the binding of TAP-2 promoter is not clear, as our preliminary studies indicated that anti-p48 did not super-shift the IRFEprotein complex from IFN-
-stimulated cells (data not shown).
In summary, our analysis and that of Arons et al. (21) indicate that, despite the presence of numerous cis elements, relatively few are involved in both constitutive and IFN-
inducible expression of the TAP-2 gene. The constitutive expression is controlled by three promoters, a 70-bp fragment 5' to the exon 1, the MED1 sequence within exon 1 and an initiator located 32 bp 5' of the translation start codon. Identification of these cis elements will facilitate characterization of the transactivating element that controls TAP-2 gene expression. Moreover, since the promoter activity dissected in our study is based on analysis of five different cell lines from three different species, murine, monkey and human, it is most likely that the elements identified may function in a number of contexts. The existence of distinct promoter and transcription initiation sites allows independent mechanisms for expression of TAP-2. This apparent redundancy will likely make it more difficult to inactivate transcription by virus and tumors, while providing ample opportunities for interplay of multiple pathophysiological factors.
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Acknowledgments
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We thank Jennifer Kiel for secretarial assistance. This study is supported by NIH grants (CA82355, CA69091 and CA58033), Department of Defense grant (DAMD17-00-1-0041), a seed grant from The Ohio Cancer Research Associates and by Ohio State University Comprehensive Cancer Center.
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Abbreviations
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5' RACE rapid amplification of 5' cDNA end |
CRE cAMP response element |
GAS -activation site |
IRF interferon regulatory factor |
IRFE interferon response factor binding element |
LMP low-molecular-weight polypeptide |
MED1 multiple start site element down-stream |
TAP transporter associated with antigen processing |
UTR untranslated region |
 |
Notes
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Transmitting editor: R. A. Flavell
Received 15 June 2001,
accepted 24 October 2001.
 |
References
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