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The Journal of Immunology, 2003, 171: 4219-4226.
Copyright © 2003 by The American Association of Immunologists

Transcriptional Control of Murine CD94 Gene: Differential Usage of Dual Promoters by Lymphoid Cell Types 1

Brian T. Wilhelm*,{dagger}, Josette-Renée Landry*,{dagger}, Fumio Takei*,{ddagger} and Dixie L. Mager2,*,{dagger}

* Terry Fox Laboratory, British Columbia Cancer Agency, and Departments of Medical {dagger} Genetics and {ddagger} Pathology, University of British Columbia, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The CD94 gene product is involved in controlling NK cell activation, and is one of a family of immune receptors that is found in the NK gene complex in both humans and mice, adjacent to members of the NKG2 family. CD94 forms a heterodimeric complex with several members of the NKG2 family on the surface of NK, T, and NKT cells. These complexes recognize the nonclassical MHC class I molecules HLA-E and Qa-1b in humans and mice, respectively. The mechanism for cell type-specific expression of CD94 and other genes from the NK gene complex has not yet been elucidated. In the current study, we show that the murine CD94 gene has two promoters, one of which is upstream of a previously unidentified exon. We illustrate by quantitative real-time PCR that lymphoid cell types use these two promoters differentially and that the promoter usage seen in adult cells is already established during fetal development. We determined that the differential promoter usage by NK cells appears to be susceptible to perturbation, as both the murine NK cell line LNK, as well as cultured C57BL/6 NK cells showed altered promoter usage relative to fresh NK cells. Furthermore, the promoter activity observed in transfection assays did not correlate with expression of the endogenous CD94 gene, suggesting the involvement of chromatin structure/methylation in transcriptional regulation. Our detection of DNase I hypersensitive sites at the CD94 locus that are present only in a cell line expressing endogenous CD94 supports this hypothesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer cells are cytolytic cells that function as part of the innate immune system. Target cells become susceptible to killing by NK cells in part by their down-regulation of cell surface MHC class I expression (1, 2, 3, 4). The receptors that regulate the activity of NK cells have been identified in both humans and mice and can be grouped into two main classes based on protein structure. In mice, the largest family of mouse receptors belong to the lectin-like Ly-49 multigene family (5), whereas in humans, members of the killer Ig-like receptors (KIRs) 3 that are part of the Ig superfamily perform the same role (5, 6). Although C-type lectin receptors are present in humans and Ig-like receptors are present in mice, these two species appear to favor the predominant use of only one of the two groups of receptors for interaction with MHC class I molecules, based on the number and variation of genes in each family. In apparent agreement with this theory, it was believed that the only receptors that functioned as homologues between the two species were the CD94/NKG2 family of C-type lectin genes, which in both species interact with nonclassical MHC class I molecules (7, 8). Two recent publications, however, have shown that the KIR receptors are also present in mice (9, 10), and therefore may also function in a similar fashion in both species.

Because of their initial characterization, experimental evidence has demonstrated a role for the heterodimeric CD94/NKG2 receptors in the regulation of immune responses in NK cells (11, 12, 13, 14) as well as subsets of T cells (15, 16). Interestingly, while some of the NKG2 gene products, such as NKG2A, form inhibitory heterodimers with CD94, others such as NKG2C and E form activating complexes in association with CD94 and a signaling adaptor molecule DAP12 (14, 17). Despite the data collected on their function, relatively little is known about how the expression of these genes is regulated. Previous studies have shown that nearly all fetal mouse NK cells express CD94/NKG2 on their surface (18) before there is any detectable expression of the Ly-49 genes, with the exception of Ly-49e (19, 20). This high frequency of expression decreases after birth, when Ly-49 expression begins to approach adult levels (21, 22), and so it appears that there is a developmental switch between the predominate receptors expressed by mouse NK cells. It has been subsequently demonstrated that CD94/NKG2 expression in fetal NK cells is responsible, at least partially, for their ability to distinguish between MHC Ilow and MHC Ihigh target cells (23, 24). The requirement for CD94/NKG2 expression for self-tolerance by NK cells is not absolute; however, as a recent report noted, the in-bred mouse strain DBA2/J, which develops normally, does not express CD94/NKG2 on the cell surface (25).

Aside from changes in expression during development, it has been shown that altered expression of CD94/NKG2 may lead to, or exacerbate, pathological conditions. In experiments using the oncogenic polyomavirus, it was found that mice susceptible to tumor development had mature antiviral CD8+ T cells whose CD94/NKG2A expression was up-regulated as a result of the infection (26). This up-regulation was sufficient to impair the cytotoxic action of T cells allowing tumor development. A similar loss of NK-mediated cytotoxicity and up-regulation of CD94/NKG2 expression was seen with NK cells cocultured with renal carcinoma cells (27).

At the level of transcriptional regulation, the promoter of the human CD94 gene has been previously characterized by primer extension to the region just upstream of the translation start codon in the first exon (28), and our group previously reported that the sequence in this region shows a high level of conservation with the mouse CD94 gene (29). In the current study, we extend these findings by demonstrating that a short region upstream of the translation start codon of either the mouse or human CD94 gene can act as a strong lymphoid-specific promoter. We have also identified a novel upstream promoter of mouse CD94 that is active in various lymphoid cells. Our results indicate that the two CD94 promoters are used differentially by lymphoid cell types beginning during fetal development. A role for altered chromatin/methylation patterns in the transcriptional regulation of CD94 is also suggested by our DNase I hypersensitive site (HSS) experiments. Finally, in the course of our investigation, we have precisely determined the nature of the CD94 defect in DBA2/J mice. The present work therefore provides insight into how CD94 is transcriptionally regulated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse strains

Fetal C57BL/6J pups and 1- to 2-mo-old C57BL/6J mice and DBA2/J mice (The Jackson Laboratory, Bar Harbor, MI) were used for all experiments.

The 5' RACE for CD94 and genomic PCR

The 5' RACE was performed using a splenocyte Marathon RACE kit from Clontech (Palo Alto, CA) and from cultured splenocytes cells, as previously described (30), to isolate the complete transcript for the murine CD94 gene, and a total of 16 clones was sequenced. Genomic sequences corresponding to the 3-kb region upstream of the CD94 genes of both C57BL/6 and DBA2/J mouse strains were PCR amplified and sequenced and can be found under GenBank accession numbers AY331148 and AY331149, respectively. The MatInspector V2.2 program and the Transfac database (http://transfac.gbf.de/TRANSFAC/) were used to map predicted transcription factor binding sites. The region spanning the deletion in the DBA2/J mouse strain was PCR amplified using the primers shown in Table I, cloned, and sequenced, and is available under GenBank accession AY331150.


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  		Table I. Primers used

 
Constructs

Reporter constructs were generated using the tailed primers listed in Table I by amplification using C57BL/6 genomic DNA as a template and the Pfu enzyme (MBI Fermentas, Burlington, Ontario, Canada). The PCR products were purified using the Qiagen PCR-product purification kit (Missisauga, ON, Canada), digested with the appropriate tail restriction enzyme (KpnI or BglII), and then ligated into KpnI/BglII-digested PGL3B firefly luciferase vector. To eliminate a high background activity of PGL3B in lymphoid cells observed in our laboratory, additional poly(A) sites were inserted upstream of the multiple cloning site of the PGL3B vector. All constructs were similarly modified to eliminate experimental differences caused by sequence differences between vector backbones. HSS enhancer constructs were generated by digesting the constructs created above with BamHI and ligating the PCR product of the entire first intron of the CD94 gene (using primers in Table I) into the BamHI site. All constructs were sequenced to confirm sequence integrity.

Transfections and cell culture

The EL-4 cell line was cultured in DMEM supplemented with 5% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin. The LNK cell line was obtained from K. Nakanishi through the laboratory of S. Anderson (National Cancer Institute, Frederick, MD) and was cultured in RPMI 1640 containing 50 µM 2-ME, nonessential amino acids, 5% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, sodium pyruvate, L-glutamine, HEPES, and IL-2 (8000 IU/ml; Chiron Canada, Ville St-Laurent, Quebec, Canada). An IL-2-independent version of the CTLL-2 cell line was obtained from T. Gonda (Hanson Centre for Cancer Research, Adelaide, Australia) and was grown in RPMI 1640 containing 50 µM 2-ME, nonessential amino acids, 5% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, sodium pyruvate, and L-glutamine. NIH 3T3 cells were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin. LNK and CTLL-2 cell lines were transfected using the DEAE-dextran transfection kit and protocol supplied (Amersham Biosciences, Baie d’Urfé, Quebec, Canada), and EL-4 and NIH 3T3 cells were transfected using Lipofectamine (Invitrogen Life Technologies, Burlington, Ontario, Canada), as previously described (31) or according to the manufacturer’s protocol, respectively. All transfections were performed twice in duplicate for each cell line tested. To control for variations in transfection efficiency, dual transfections with the PRL-TK Renilla luciferase vector were performed, and all firefly luciferase values were normalized by the Renilla activity. The activity of the constructs was made relative to an empty PGL3B vector (modified with additional upstream poly(A) signals) with the SV40-promoted PGL3P as a positive control.

Cultured splenocytes

Spleens were homogenized, and RBC were removed from single suspensions by a 20-s lysis with ice-cold distilled water. Remaining cells were washed twice with PBS and incubated for 1 h in a medium-filled packed nylon wool column. Cells were slowly eluted from the column, washed, and cultured in RPMI 1640 containing 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, sodium pyruvate, L-glutamine, and IL-2 (8000 IU/ml). After 3 days, the medium was changed and all nonadherent cells were removed by washing twice with PBS. Cells were cultured for another 3–6 days before RNA extraction.

Flow cytometry

Adult splenocytes and fetal (18–20 days postcoitus) liver samples were prepared as single-cell suspensions, stained with labeled Abs, and analyzed by flow cytometry. Cell suspensions were pretreated with a culture supernatant containing anti-FcR{gamma}III/IIR, 2.4G2 to prevent FcR binding by labeled Abs. Primary mAbs directed against the following cell surface markers were used: NK1.1 (PK136) PE, CD94 (18d3) biotin, NKG2A/C/E (20d5) FITC, CD49b (DX5) PE, CD3{epsilon} (145-2c11) FITC, and a secondary reagent, streptavidin-PE, used with the CD94 Ab, were all purchased from BD Biosciences (Palo Alto, CA). All of the flow cytometry and sorting was performed on a FACSVantage SE (BD Biosciences) using CellQuest software.

Northern blot analysis

RNA was isolated from EL-4 and LNK cells using TRIzol, as described by the supplier (Invitrogen Life Technologies). Following the elimination of remaining genomic DNA with DNase I (Invitrogen Life Technologies), 10 µg of RNA from EL-4 and LNK cells was electrophoresed on a 1.2% agarose, 5% (v/v) formaldehyde, 1x MOPS buffer gel and transferred to a Zetaprobe membrane (Bio-Rad, Mississauga, Ontario, Canada). The Northern blot was hybridized in ExpressHyb (BD Biosciences) at 68°C with a CD94 cDNA fragment corresponding to the nearly full-length transcript. The membrane was washed twice for 20 min in 2x SSC, 0.05% SDS at room temperature, followed by two washes of 20 min in 0.1x SSC, 0.1% SDS at 50°C. To confirm the amounts of mRNA loaded in each lane, the blots were rehybridized with a human 1.9-kb actin cDNA fragment.

Quantitative real-time PCR

RNA to be used for real-time PCR was isolated, as described above, and treated with DNase I (Invitrogen Life Technologies) to remove any possible DNA contamination and reverse transcribed, as previously described (30), using random hexamers. The RNA from FACS-sorted NK, T, and NKT cells was diluted based on the absolute cell numbers collected (5 x 104–7.5 x 105) so that the RNA concentrations were identical before reverse transcription. Quantitative real-time PCR was performed using 1 µl of the prepared cDNA along with 22 µl of water, 1 µl of each of the two primers, and 25 µl of the 2x Sybr green PCR master mix (PE Applied Biosystems, Foster City, CA) and the following amplification conditions: 30 s at 95°C, 30 s at 65°C, and 30 s at 72°C for 40 cycles on a Bio-Rad iCycler. Primers were designed according to PE Applied Biosystems recommendations, and differences in primer amplification efficiency were calculated, as recommended. For each experiment, dissociation curve analysis was performed to verify the presence of only a single PCR product. The relative quantification of the transcripts was derived using the comparative threshold cycle method (PE Applied Biosystems User Bulletin 2, ABI PRISM 7700 Sequence Detection system) supplied by the manufacturer. All quantitative real-time PCR experiments were performed twice in duplicate.

DNase I HSS scan

The DNase I HSS assay was performed, as previously described (32). Briefly, DNA was collected from LNK and EL-4 cell lines, in which cell pellets were lysed using a Dounce homogenizer in RSB Nonidet P-40 (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40). Nuclei were collected and further homogenized. The nuclei were then treated with varying concentrations of DNase I before inactivation and proteinase K digestion overnight. DNA was then cleaved with the appropriate restriction enzyme, electrophoresed, Southern blotted, and probed. A DNA fragment from the proximal promoter region of the Lck gene previously shown to indicate HSS in the EL-4 cell line (33) was used as a positive control for the technique. The CD94 probes used were the 1.2-kb proximal promoter construct insert and the 600-bp distal promoter construct insert. Hybridization conditions were performed as per the ExpressHyb (BD Biosciences) protocol.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The murine CD94 gene has a novel upstream exon

To identify the transcriptional start point of the CD94 gene, we performed 5' RACE on splenocyte cDNA. The clones that were isolated contained sequence that extended beyond the previously presumed 5' end of the gene. We determined that the sequence originated from a previously unidentified exon in the 5' untranslated region (UTR) of CD94. The novel 278-bp exon, called exon 1a, is ~3.3 kb upstream of the previously identified first exon, hereafter named exon 1b (Fig. 1). No canonical promoter sequences such as TATA or CAAT boxes could be identified upstream of the novel exon. Although rat expressed sequence tags were found that contained sequence highly similar to the novel upstream CD94 exon, no homologous region could be identified upstream of the human CD94 for a distance of 10 kb using BLAST software. Fig. 1 shows a scale drawing of the revised genomic structure of the mouse CD94 gene as well as the sequence and predicted binding sites for several transcription factors in the region. The figure also includes locations of the fragments used below to test for promoter activity.



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  		FIGURE 1. A, Sequence of the novel upstream exon and surrounding region. The sequence of the 5' region of the mouse CD94 gene is shown where the translational start codon in exon 1b is underlined and the bold sequence corresponds to the upstream exon 1a region. Bent arrows above and below the sequence lines represent the end points of one or more sequenced 5' RACE products that originated in exon 1a or 1b, respectively. Splice donor and acceptor sites are shown by vertical lines marked by SD and SA, respectively, and predicted transcription factor binding sites are shown as boxed regions of sequence with the factor names below. B, A schematic diagram of the mouse CD94 gene showing the exons of the gene as large numbered boxes with the novel 5' exon shown as 1a. The distance between the two alternative first exons is shown, and the regions of DNA cloned for promoter constructs are shown to scale below.

 
The novel 5' UTR sequence (Fig. 1A, shown in bold) created from the exon 1a promoter contains four upstream ATGs, two of which are in the same reading frame as the previously determined CD94 peptide, that could potentially encode additional amino acid sequence at the N terminus for the protein. This possibility, while not formally excluded, is highly unlikely as none of the four ATGs are in a favorable context and the in-frame ATGs present in the novel UTR include stop codons before the previously identified translation start site. It therefore seems most likely that the UTR does not alter the amino acid sequence of CD94, although changes in the secondary structure of the mRNA might influence the translation of the various transcript forms.

The mouse CD94 gene promoters have activities that do not correlate with expression of the endogenous gene

To test for promoter activity of the region upstream of the novel exons 1a and 1b, we first attempted to identify a cell line that expresses CD94 endogenously, so as to have a permissive environment for promoter characterization. Only one NK cell line, LNK, which had previously been described (34), was positive for CD94/NKG2 expression at the cell surface by FACS (Fig. 2A).



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  		FIGURE 2. CD94 is expressed by the cell line LNK, but not EL-4. A, FACS profiles of the EL-4 and LNK cell lines stained with anti-CD94 and anti-NKG2A/C/E Abs. B, Northern blot of RNA collected from the EL-4 and LNK cell lines. In the top panel, a fragment of the actin cDNA was used as a probe. In the bottom panel, a portion of the CD94 cDNA was used as a probe.

 
We generated reporter constructs (as described in Materials and Methods) containing the sequence 5' of exon 1a or 1b, which were then transfected into the CD94+ LNK cell line, as well as other lymphoid and nonlymphoid cell lines. The results of the luciferase transfection assays are shown in Fig. 3. Although none of the CD94 constructs tested had activity in the NIH 3T3 cell line, the 5' promoter (upstream of exon 1a) exhibited limited activity, and the constructs from the 3' promoter (upstream of exon 1b) had high activity in all lymphoid cell lines tested. The promoter activity of the region 5' of exon 1b appears to be conserved between the human and mouse genes. The human CD94 construct that contains the 800 bp upstream of the human CD94 translational start codon exhibited high activity in all lymphoid cell lines tested. We have previously characterized the similarity between the human and mouse genes in this region (29), but it is not clear which, if any, of the regions conserved between species are involved in transcriptional regulation.



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  		FIGURE 3. Results of reporter construct transfections. The cell lines used for the transfections are indicated in the legend on the right-hand side. The vertical axis indicates the fold induction of luciferase activity over the basic construct (PGL3B) that lacks promoter activity. The constructs and their sizes, relative to the start of exon 1a or the ATG in exon 1b, are indicated under the horizontal axis. Each transfection as performed twice in duplicate and the transfection results were normalized by the cotransfected Renilla activity to eliminate variations in transfection efficiency. The error bars shown on the graph represent the pooled SD of the four transfection samples.

 
Interestingly, the promoter activity of the CD94 fragments did not correlate with cell surface expression of the endogenous gene. Both the CTLL-2 and EL-4 cell lines that were negative for CD94 expression by FACS (Fig. 2A and data not shown) had high luciferase activity. Through Northern blots (Fig. 2B) and quantitative real-time PCR (data not shown), we determined that this lack of CD94 expression in EL-4 cells is the result of a transcriptional defect rather than a problem with cell surface presentation. The high promoter activity observed, combined with the lack of endogenous gene expression, indicates that the trans-acting factors that bind to the promoter region are present, but that alterations in chromatin structure or methylation patterns might repress expression of the endogenous CD94 gene. This possibility is supported by the DNase I HSS scans discussed below.

The two CD94 promoters have lymphoid cell type-specific usage that is established before birth

In an effort to characterize the usage patterns of the two CD94 promoters, freshly isolated adult splenocytes and fetal liver cells from C57BL/6 mice were FACS sorted into NK, NKT, and T cell fractions from which RNA was collected. Real-time quantitative PCR was performed using primers that would distinguish between CD94 transcripts that originated from the upstream promoter (containing exons 1a and 1b) and those originating from both promoters (containing exons 1b and 2). The latter was used to represent total CD94 transcripts encoding functional proteins, as the translational start codon is in exon 1b.

As illustrated in Fig. 4A, the usage of the upstream CD94 promoter varied by lymphoid cell type, with adult NK cells using it almost exclusively. Conversely, T cells that expressed CD94 did so primarily from the exon 1b promoter. NKT cells, which express markers characteristic of both NK and T cells, had expression from both promoters. Furthermore, the trends in promoter usage seen in the adult cell types were present in the same late-stage fetal cell types, albeit to a lesser extent. Based on this observation, it appears the cell type-specific promoter usage begins at an early stage of development, with adult patterns being reached shortly after birth.



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  		FIGURE 4. A, Quantitative real-time PCR results of differential promoter usage by cell type. The cell types tested are indicated along the horizontal axis, while the vertical axis shows the percentage of the total transcripts that contain exon 1a. Fetal and adult cell types are indicated by shading patterns in the legend on the bottom of the figure. B, Quantitative real-time PCR results of differences in promoter usage in fresh cells compared with cultured cells. The figure indicates the percentage of CD94 transcripts that contain exon 1a in freshly isolated NK cells, NK cells cultured for 7 days (as described in Materials and Methods), or the LNK cell line. The error bars in A and B represent the pooled SD for the four samples (two reactions performed in duplicate) for each cell type.

 
Discrepancies between the promoter usage observed by real-time PCR and 5' RACE clones obtained from cultured NK cells led us to investigate the promoter usage by the LNK cell line as well as cultured NK cells. Adult mouse NK cells were isolated from bulk splenocytes using a nylon wool column and cultured in medium containing IL-2 for 7 days before collection of RNA. Real-time quantitative PCR was performed on these samples as well as on RNA collected from the LNK cell line, to compare the promoter usage of the cultured cells. Fig. 4B shows that, relative to freshly isolated NK cells, both cultured cells and the LNK cell line have significantly decreased exon 1a promoter usage. The decrease in exon 1a promoter usage in cultured NK cells was coordinate with a general down-regulation of CD94 expression after culturing (data not shown). Promoter usage of other cultured lymphoid cell types was not determined.

DNase I HSS correlate with endogenous CD94 expression

The fact that transient transfections of constructs containing the downstream CD94 promoter (exon 1b) had high activity in lymphoid cell lines regardless of endogenous expression led us to look for DNase I HSS in expressing and nonexpressing cells. We first verified that our procedure could identify DNase I HSS by detecting the previously described HSS at the distal promoter of the Lck gene in EL-4 cells (31) (data not shown). The region around CD94 was then examined in LNK vs EL-4 cells, and evidence for HSS in the expressing LNK cell line was obtained (Fig. 5). Initial CD94-specific hybridizations performed with the 1.2-kb fragment from the exon 1b promoter region (Fig. 5A) indicated that there were two HSS. The exon 1b promoter fragment initially used as a probe did not precisely locate the HSS, although the two sites could be qualitatively described as major and minor. To clarify the position of these sites, further hybridizations were conducted, using XhoI genomic digests and the short exon 1a promoter insert as a probe (data not shown). The positions of the sites derived from these hybridizations are indicated in Fig. 5B. The presence of these sites appears to correlate with CD94 expression, as they are detectable in the CD94-expressing cell line LNK, but not EL-4, which does not express CD94.



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  		FIGURE 5. DNase I HSS analyses of the EL-4 and LNK cell lines. A, Shows Southern blots prepared from DNase I-treated genomic DNA from EL-4 and LNK cell lines, digested with BglII. The expected size of the BglII fragment detected by the probe used is 9.3 kb, and the size ladder for the Southern blot is shown between the two blots. Numbered arrows on the right-hand side of the blot indicate the hypersensitive fragments generated in LNK DNA. B, Shows a scale diagram of the mouse Klre-1 and CD94 locus with the 9.3-kb BglII fragment indicated by the line under the exons of the genes. The positions of the major (1) and minor (2) HSS are indicated by numbered vertical arrows.

 
To further characterize the nature of the HSS, the region containing the stronger HSS (Fig. 5, site 1) corresponding to the second intron of the CD94 gene was placed into the enhancer position of the PGL3B vector of the previously tested shorter exon 1a (–600 bp) and exon 1b (–500 bp) constructs. In transfections of the four cell lines previously tested (Fig. 3), none of the new enhancer constructs showed activity higher than the original construct. Additionally, a multiple sequence alignment of this region in the human, rat, and mouse genes did not reveal any significant region of sequence conservation. Given the lack of effect of this region on reporter gene expression, and as it has been reported that transient transfections of DNase I HSS can show enhancer activity (35), our results suggest that if the CD94 DNase I HSS has a functional role, it may be necessary for it to be in the proper genomic context to have activity.

The CD94 gene in the DBA2/J mouse strain is partially deleted

Because of our interest in the transcriptional regulation of CD94 expression, we sought to characterize the nature of the defect in a recently reported mouse strain (DBA2/J) that lacked detectable protein and RNA expression of CD94 by FACS and Northern blotting, respectively (25). Surprisingly, quantitative real-time PCR products from either of the sets of exon 1a/b and 2 primers were generated in the same ratio in the DBA2/J substrain as in a CD94-expressing strain (C57BL/6) relative to an internal control (GADPH) (data not shown). In response to this finding, new primers were designed to test for the presence of the last two exons (5 and 6) of the CD94 transcript. Real-time PCR indicated that the product from these primers could not be amplified from the DBA2/J strain, whereas a product was easily detected from B6 mice. The results of a series of RT-PCR amplifications from FACS-sorted (DX5/NK1.1+, CD3-) adult NK cells from DBA2/J and B6 mice are shown in Fig. 6A. Although products from exons 1a to 3 were generated from both strains, products amplified from exons 4–6 or exons 2–6 could only be generated using B6 cDNA. These results suggest that there is some defect in the 3' end of the CD94 gene in DBA2/J that does not prevent transcription, but does prevent translation and cell surface presentation of the protein. To clarify the nature of the defect, we PCR amplified, cloned, and sequenced a section of the 3' end of the DBA2/J CD94 gene. This sequence, a portion of which is shown in Fig. 6B, shows that there is a {approx}2.4-kb deletion of sequence (in comparison with B6) that includes exon 6 of the CD94 gene (Fig. 6C). The sequenced region spanning the deletion is available under GenBank accession AY331150. Why the CD94 transcripts were not detected in the previous report is not clear; however, at least one of the probes used in the published Northern blot was from the 3' end of the CD94 transcript that would not be able to detect the deleted transcripts. As the defect in this mouse strain was not related to transcriptional regulation, the expression of CD94 in these mice was not investigated further.



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  		FIGURE 6. Characterization of the deletion in the CD94 gene in the DBA2/J mouse strain. A, RT-PCR performed on FACS-sorted NK cells in the B6 (CD3-NK1.1+) and DBA2/J (CD3-DX5+) strain using primers that would amplify various regions of the CD94 transcript. The exons amplified in each reaction, and the mouse strain used, are indicated at the top. B, Nucleotide sequence of the region of the deletion in C57BL/6 and DBA2/J. The horizontal line with arrows indicates the deletion region, and the size is indicated between the arrows (2385 bp). C, A schematic representation of the region of the CD94 gene deleted in the DBA2/J mouse strain. The figure is not shown precisely to scale.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous work on NK receptor genes has suggested that their transcriptional control is complex. Even in cases in which trans-acting factors involved in NK gene cluster (NKC) gene regulation have been identified (36, 37), the effects seen have not been the same for all members of a given gene family. Our current work with the mouse CD94 promoter provides further evidence that genes within the NKC have a complicated pattern of transcriptional control.

The identification of a novel upstream CD94 promoter and the observation that various lymphoid cells use the two promoters differentially also offer a theoretical mechanism for the expression of NK receptors on non-NK cells.

It has been observed that small subsets of T cells express receptors usually found on NK cells in both humans (38) and mice (39), and that this expression has functional consequences. For instance, the expression of CD94/NKG2 proteins has been shown to influence the CD8+ T cell activity in viral infections (26), although this effect appears to be virus specific (40, 41). It has also been shown that several cytokines, including IL-12 (42), TGF-{beta} (43), IL-15 (44), and IL-10 (45), are capable of inducing expression of CD94/NKG2 in human T cells. In one of these reports, the signal for induction of CD94 gene expression was independent of the activity of IL-15 (42), similar to a recent report in which a majority of pathogen-specific CD8+ T cells up-regulated expression of CD94/NKG2A independent of the activity of IL-15 (40). In the past, IL-15 has been demonstrated to be necessary to induce expression of CD94/NKG2 on developing human (46) and murine (47) NK cells; however, high dose IL-2 in the mouse system has also been found to have similar activity as IL-15 (47). It is therefore possible that the downstream promoter may have high activity only in T cells in vivo and it may be regulated differentially through cytokine signaling in the microenvironment. As the full signaling pathways for many cytokines have not been defined, it is not possible to confirm the theoretical prediction of the presence of transcription factor binding sites or response elements in the promoter regions of these genes.

Such a simplistic scenario for regulation, in which NK cells use one promoter and T cells use another, does not explain our transfection results in which the downstream (exon 1b) promoter constructs had high activity in lymphoid cell lines that lack expression of the endogenous CD94 gene. A complete explanation of the function of each promoter would therefore need to address issues of chromatin structure or histone modifications in each region. Although murine NK cells appear to go through a switch in receptor system usage during development (18, 39), potential chromatin modifications or alterations in methylation that may influence mouse NKC expression have not been previously investigated. In contrast, the expression of KIR genes, which are the functional homologues of the Ly-49 genes in humans, has recently been shown to be dependent on alterations of the methylation status of the promoter region of the genes (48). Our experiments were able to detect the presence of DNase I HSS within the CD94 locus, which would suggest that some alteration in chromatin structure allows expression of the endogenous gene in the LNK cell line (Fig. 5). Two HSS, one major and one minor, were detected on either side of exon 1b of CD94. The existence of DNase I HSS near the distal promoter, where our promoter constructs had high activity in all lymphoid cell lines, regardless of endogenous CD94 expression, also supports the idea that chromatin structure may prevent access of available transcription factors to the endogenous CD94 genes. The transcription pattern in the LNK line is also consistent with this hypothesis as, despite the fact that the LNK cell line is of NK origin, the majority of CD94 transcripts arise from the downstream promoter (Fig. 4B) similar to T cells.

It is also of interest that constructs using sequence from the previously defined promoter region upstream of the translational start codon of the human CD94 gene also showed high activity in all murine lymphoid cell lines tested. We have previously shown that this region has a fairly high level of identity between the two species (29), and so the shared promoter activity is perhaps not surprising. Interestingly, unpublished results have shown that there is a novel promoter upstream of the human CD94 gene that is also used differentially between lymphoid cell types (J. Coligan, personal communication). This finding suggests that the complex dual promoter system of the CD94 gene is conserved between species.

Unlike the exon 1b promoter, and despite an apparent conservation of function, there does not appear to be sequence conservation between the upstream promoter (exon 1a) regions of the two species. The nature of the upstream promoter region of the murine CD94 gene is further complicated by our recent characterization of a novel gene located in close (2.3 kb) proximity to the upstream promoter of CD94 (49) that is not present in the human genome. This new gene, Klre-1, was identified based on expressed sequence tag evidence and gene prediction programs from the sequence of the completed mouse genome (50). We found the expression of Klre-1 and CD94 to be coordinate, but this does not necessarily mean that they are transcriptionally regulated in the same manner. Indeed, the NKG2D gene, which is on the other side of CD94, has a significantly different expression pattern (51, 52).

The possibility of developmentally coordinated expression is also consistent with the detection of DNase I HSS in the vicinity of the promoter regions (Fig. 5). The fragment generated by restriction digest in the DNase I HSS analysis encompassed not only the 5' end of the CD94 gene (from exon 4), but also the last exon of the Klre-1 gene, which is 2.3 kb upstream of exon 1a of CD94. It is therefore possible that the detected DNase I HSS in the promoter region of the CD94 gene may influence the expression of Klre-1 as well as CD94. Our finding does not, however, rule out the possibility that there are other HSS further away from the CD94 promoter region that control the developmental expression pattern of CD94 and Klre-1. Such a genomic organization would suggest that, if the CD94 upstream promoter activity is in fact conserved between species, it might be functionally defined by a short stretch of sequence not detectable in strict homology-based searches.

The observation that promoter usage in fetal NK, T, and NKT cells shows a bias that increases in cells from mature mice indicates that promoter usage changes during development. The comparison of CD94 promoter usage in cultured vs fresh NK cells also shows that, even in adult cells, the promoter usage pattern is not fixed. Because the decrease in exon 1a promoter usage occurs at the same time as an overall down-regulation of CD94 expression, it suggests that culturing NK cells with IL-2 (or NK cell activation in vivo) leads to the shutdown of the upstream promoter, resulting in a reduction in CD94/NKG2 expression. Functionally, the down-regulation of receptors that inhibit cytotoxicity would make sense if the immune system needs to activate NK cells against pathogens.

Our experiments show that the mouse CD94 gene clearly has a complex promoter structure that appears to regulate cell type-specific activity. Previously published reports of immune system genes with multiple promoters illustrate that the complex structure of the mouse CD94 promoter is not unique (33, 53, 54). Indeed, the usage of dual promoters has been described for several of the Ly-49 genes in a situation that might be analogous to CD94. The distal promoter of some Ly-49 genes was shown to have promoter activity in fetal cells and in bone marrow cells, possibly linking its usage to the initiation of Ly-49 expression in NK cells (55). In addition, the mouse NKR-P1C gene has also recently been shown to have a novel upstream noncoding exon that is differentially used during NK cell development, as well as a DNase I HSS upstream of the gene (35). It is very interesting that many NK receptor genes, at least in mice, share several common characteristics, including alternative first exons whose use varies during development as well as the presence of DNase I HSS, although this has not yet been demonstrated to occur in the case of Ly-49 genes. It will be interesting to characterize the transcriptional mechanisms of the murine NKG2 genes to see whether their promoter structures and usage are similarly complex or whether transcriptional regulation of CD94 is predominant in assuring proper cell surface expression of CD94/NKG2 dimers.


   Acknowledgments
 
We thank Dr. Tom Gonda and Dr. S. Anderson for the donation of cell lines used in this work, Dr. S. Porter for technical assistance, and Dr. J. Coligan for helpful discussion and for sharing unpublished results.


   Footnotes
 
1 This work was supported by a grant from the Canadian Institutes for Health Research (CIHR) to D.L.M. Core support was provided by the British Columbia Cancer Agency. B.T.W. and J.-R.L. are recipients of a scholarship from the Michael Smith Foundation for Health Research, and J.-R.L. is a recipient of a CIHR studentship. Back

2 Address correspondence and reprint requests to Dr. Dixie L. Mager, Terry Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada. E-mail address: dmager{at}bccrc.ca Back

3 Abbreviations used in this paper: KIR, killer Ig-like receptor; HSS, hypersensitive site; NKC, NK gene cluster; UTR, untranslated region. Back

Received for publication February 26, 2003. Accepted for publication August 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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