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J. Biol. Chem., Vol. 278, Issue 34, 31909-31917, August 22, 2003

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Identification of Upstream cis-Acting Regulatory Elements Controlling Lineage-specific Expression of the Mouse NK Cell Activation Receptor, NKR-P1C^*

Belma Ljutic  , James R. Carlyle ¶ || and Juan Carlos Zúñiga-Pflücker  **

From the Department of Immunology, University of Toronto, Sunnybrook and Women's Health Sciences Centre, Toronto, Ontario M4N 3M5, Canada and the ^¶Cancer Research Laboratory, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200

Received for publication, December 18, 2002 , and in revised form, June 10, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mouse NKR-P1C (NK1.1) is a homodimeric type II transmembrane protein expressed on natural killer (NK) cells, NKT cells, and on CD117⁺ progenitor thymocytes capable of giving rise to cells of the T and NK lineages. Although its physiological ligands remain unknown, NKR-P1C engagement with a monoclonal antibody (mAb) leads to interferon- (IFN-) production and the directed release of cytotoxic granules from NK cells. We have cloned and sequenced a 10-kb genomic fragment corresponding to the 5'-flanking region of the C57Bl/6 mouse NKR-P1C gene. A transcriptional initiation site has been mapped in NK cells and an NK1.1⁺ T cell line by primer extension and rapid amplification of 5'-cDNA ends (5'-RACE) techniques. Although the 5'-flanking region of NKR-P1C is TATA-less, we have identified an initiator region and a downstream promoter element, which together constitute the principal minimal functional promoter. Computational analysis of the 10-kb 5'-flanking region revealed potential regulatory factor binding sites. DNaseI hypersensitivity assays identified a single hypersensitive site (HS) about a 9-kb upstream of the transcriptional initiation site. This site, termed HS1, was able to act as a transcriptional enhancer element in an NK cell line, while minimally affecting transcription in non-NK cell lines. Moreover, the HS1 element was shown to function as a promoter, with a transcript detected only in fetal NK1.1⁺ cells. An additional promoter and two non-coding exons were also characterized. These results identify the minimal upstream cis-acting elements, and point to a complex regulatory mechanism involved in the lineage-specific control of NKR-P1C expression in NK lymphocytes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The classic NK1.1 antigen (NKR-P1C) is one member of a group of disulfide-linked homodimeric type II transmembrane C-type lectin-like receptors encoded by the NKR-P1 gene family (1, 2). The NKR-P1 gene cluster is found within a single genetic region designated the natural killer gene complex (NKC),¹ which is located on chromosome 6 in mice, chromosome 4 in rats, and chromosome 12 in humans (2, 3). The NKC region is strikingly conserved among species, even though not all NKC-encoded molecules have been identified in all species (4).

The NKC can be divided into several families of genes: NKRP1, Ly49, CD69, NKG2, and CD94. The prototype of the NKR-P1 family, NKR-P1A, was first cloned in rats using a mAb screened for its ability to redirect NK cell lysis of Fc receptor (FcR)⁺ targets (5, 6). In mice, the NKR-P1 gene family consists of at least five distinct genes: NKR-P1A, NKR-P1B, NKR-P1C, NKR-P1D, and NKR-P1F (while the NKR-P1E locus represents a pseudogene) (1, 7–9). The initial cloning of the mouse NKR-P1 genes revealed that they consist of six exons separated by five introns (7). The first exon of the NKR-P1C gene encodes the N-terminal region of the protein, corresponding to the cytoplasmic domain. The second exon encodes the transmembrane region, exon 3 encodes a variable stalk region, and exons 4–6 contain the putative carbohydrate recognition domain (CRD) (7). Although NKR-P1C had been originally cloned as the NK1.1 antigen (2), we and others have recently demonstrated that the mouse "NK1.1 antigen" actually represents the products of at least two distinct genes, NKR-P1B and NKR-P1C, differentially expressed across mouse strains (10, 11).

NKR-P1C is expressed by all functional NK cells (7) and by a subset of mature T cells termed NKT cells (12). In selected strains of mice, virtually all NK activity is contained within the NKR-P1C⁺ subset (13), and in vivo administration of anti-NK1.1 mAb eliminates NK activity but has no effect on cell-mediated or humoral immunity (14). Moreover, engagement of NKR-P1C with a mAb mediates NK cell redirected lysis of FcR-expressing targets (15), indicating that NKR-P1C is capable of activating and directing the cytolytic process. To date, however, physiological NKR-P1 ligands on target cells have not yet been identified although recombinant NKR-P1 proteins were suggested to bind synthetic carbohydrates (16–20). Thus, the NKR-P1 proteins remain orphan receptors. In addition, our recent work has shown that a population of CD117⁺ progenitor thymocytes expressing NK1.1 is capable of giving rise to the cells of T and NK cell lineages (21–23). Importantly, a single CD117⁺ NK1.1⁺ cell was shown to give rise to both T and NK cells, establishing that NK1.1 expression can serve to identify a common progenitor for both lineages (24).

In light of the developmentally controlled appearance of the NKR-P1 proteins during lymphocyte lineage commitment (21–24) and their lineage-restricted expression by mature NK cells, we wondered how their expression is regulated. To address this, we investigated the molecular cis-acting elements responsible for their transcriptional regulation. This report describes the cloning and detailed analysis of the upstream region of the mouse NKR-P1C gene. We have identified the location of the major NKR-P1C transcriptional initiation site; two new noncoding exons in the 5'-region of the gene (exons 1.1 and 1.2); three promoters; and putative transcription factor binding sites in the upstream flanking region. Furthermore, we describe the detection and analysis of a DNaseI hypersensitive site (HS1), located 9-kb upstream of the major transcriptional initiation site, which exhibits promoter and enhancer-like activity. Taken together, our present findings identify the minimal functional promoter and a unique enhancer/promoter element, HS1, which appear to confer NK lineage-specific expression of the NKR-P1C gene.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Screening of a Mouse Genomic Library—A mouse -phage genomic library derived from C57Bl/6xC3H F1 mice (generously provided by Dr. Gillian Wu, York University, Toronto, ON, Canada) was screened using an NKR-P1C gene-specific probe corresponding to the B6 NKR-P1C cDNA coding sequence. Three clones were identified spanning various regions within the mouse NKR-P1C locus and were subcloned. Clone P13 was characterized in this study.

Primer Extension Analysis—mRNAs used in primer extension assays were generated from two different cell lines: CTLL-2, and MNK-1^9.76kb/GFP. For the CTLL-2 mRNA, primer extension assays were performed using two 18-bp NKR-P1C antisense primers: E1A (5'-CAGGGTTGGATAAGACAG-3') complementary to bases –403 to –420; and E1B (5'-GTGTCCTGGGTGGCTTTA-3') complementary to bases +28 to +45 (both primer locations are relative to the initiator methionine codon ATG in exon 1, Fig. 2, underlined, bold). For MNK-1^9.76kb/GFP mRNA, the same E1A primer was used in addition to a GFP primer (5'-CTCCTCGCCCTTGCTCACCA-3') complementary to bases +2 to +20 of GFP coding sequence. Primers were labeled using [-³²P]dATP (specific activity 6000 Ci/mmol) and T4 polynucleotide kinase (New England Biolabs, Beverly, MA) to a specific activity of 1 x 10⁹ cpm/µg. 1 x 10⁵ cpm of each primer (0.1 ng) was mixed with 1.5 µg of oligo(dT)-selected RNA from CTLL-2 cells or 2 µg of MNK-1^9.76kb/GFP mRNA. 10 µg of total yeast RNA or 2 µg of MNK-1 mRNA were used as negative controls. The RNA samples were reverse-transcribed at 42 °C for 1 h using AMV reverse transcriptase (Invitrogen). Radioactive primer extension products were resolved in a sequencing gel. The same primers were used for sequencing reactions using Sequenase Version 2 Sequencing Kit (Amersham Biosciences, Piscataway, NJ). The template for sequencing reactions was a genomic fragment corresponding to the upstream region of the NKR-P1C gene.

View larger version (55K): [in this window] [in a new window]   FIG. 2. Nucleotide sequence of the NKR-P1C 5'-flanking region. The sequence shown includes the promoter elements, exon 1.1 (E1.1), exon 1 (E1), part of intron 1, and exon 2. The nucleotides are numbered relative to the major transcriptional initiation site (shown by the large forward arrow). Putative DNA binding sites are underlined, and those in reverse orientation are also indicated by an asterisk. Locations of the Inr, DPE, and TTAA motifs are underlined and in bold letters. The small forward arrow represents the transcriptional initiation site for exon 1.1, with the only in-frame start codon depicted in italics. The intron/exon boundaries between exon 1.1 and 2.1, and exons 1.2 (Fig. 7) and 2.2 are represented by vertical lines (solid and dotted, respectively). Intronic nucleotides and untranslated 5'-flanking region are in lowercase letters. Primers for primer extension reactions (E1A, E1B) and for RT-PCR (F1B, F1C, and R1B/C) are also indicated.

View larger version (37K): [in this window] [in a new window]   FIG. 7. Nucleotide sequence of the NKR-P1C 5'-HS1 site. The nucleotides are numbered relative to the starting ATG codon (Fig. 2, bold, underlined). Putative DNA binding sites are underlined with those in reverse orientation indicated by an asterisk. Locations of the Inr, and TATA motifs are underlined and in bold letters. The putative transcriptional initiation site is indicated by the forward arrow. The intron/exon boundary between exons 1.2 and 2.2 (Fig. 2) is represented by dotted vertical lines. Primers used for PCR amplification of HS1 fragment for cloning into pGL-2 backbone (FHS1 and RHS1) and for RT-PCR of promoter 3-derived transcript (F1A) are indicated.

RT-PCR—mRNA from CTLL-2 cells was prepared using GeneElute Direct mRNA Miniprep Kit (Sigma). Total RNA from C57Bl/6 (B6) day 15 fetal liver, fetal thymus, sorted NK1.1⁺ fetal thymus, or RAG2^–/– splenocytes was isolated using TRIzol (Invitrogen) as described by the manufacturer. 10 µg total RNA or 1 µg of mRNA was reverse-transcribed using AMV reverse transcriptase, 0.5 µg oligo(dT) primer (Invitrogen), 1 mM dNTPs, and the supplied buffer. First strand cDNA was amplified by PCR according to the following protocol: 94 °C, 2 min.; 30 cycles at (94 °C, 30 s; 55 °C, 30 s; 72 °C, 30 s); 72 °C, 10 min.; in a 25 µl final reaction volume cycled using an MJ Research Thermal Cycler (Waltham, MA). After amplification, the PCR products were resolved by agarose gel electrophoresis. The sequences of primers used in the PCR amplification are: F1A, 5'-AACTAGGGGGAAAATAGTTGA-3' (position –8428 to –8448); F1B, 5'-AATCATCTAAGCAGGGCTCAT-3' (position –595 to –615 upstream of ATG in exon 1; Fig. 2, underlined, bold); F1C, 5'-AACCCTCAGAGACAGGAATCA-3' (position –117 to –137 upstream of ATG in exon 1); and R1A/B 5'-TGTGTCCATTTCACAGGA-3' (position –8 to +9 relative to ATG in exon 1). RT-PCR products were normalized using -actin as an internal standard.

Construction of Reporter Vectors—A PCR approach was used to generate all of the reporter constructs from the P13 -phage clone. All forward primers contain an Mlu I linker at the 5'-end, while reverse primers contain a SalI linker at the 3'-end. The following is a list of primers used as well as the size/location of constructs: 9.76-kb construct (position –9746 to –1), 5'-ACGCGTGATTTAGGTGACACTAT-3'; 9-kb construct (position –9013 to –1), 5'-ACGCGTCCACAGAAGGATGGACTG-3'; 8-kb construct (position –8012 to –1), 5'-ACGCGTACACAAAAGTGGAAGGAG-3'; 7-kb construct (position –7012 to –1), 5'-ACGCGTATCTGTAATGGCCAGAG-3'; 6-kb construct (position –6012 to –1), 5'-ACGCGTGCAGGCTCTGTGAAATTC-3'; 5kb construct (position –5012 to –1), 5'-ACGCGTCTGGAATTCAATCTATAA-3'; 4kb construct (position –4012 to –1), 5'-ACGCGTAATACCAGTATGGCTCTA-3'; 3-kb (position –3016 to –1), 5'-ACGCGTAATGGGGCCATCTTTCCC-3'; 2-kb construct (position –2013 to –1), 5'-ACGCGTCTTTCTAACGCATCTCCA-3'; 1-kb construct (position –1013 to –1), 5'-ACGCGTGGAGGATCTTAGAGCTTC-3'; 600 bp (position –600 to –1). The 3'-primer for all of the constructs is 5-GTCGACTTTCACAGGAGATGCAACA-3' (positions –1 to –18). All of the positions are relative to the starting ATG codon in exon 1 (Fig. 2; underlined, bold). PCR fragments were digested with Mlu I and SalI and ligated into pGL-2/Basic (promoterless and enhancerless luciferase construct; Promega, Madison, WI). The -phage genomic clone P13 was used as the template for PCR, and all constructs were sequenced to confirm insert orientation and sequence fidelity. Control plasmids included pGL-2/Basic vector alone (promoterless, enhancerless) and pGL-2/SV40 (containing both the SV40 promoter and enhancer; Promega). The CMV-based -galactosidase reporter vector, pCMV--gal (Stratagene Inc., La Jolla, CA), was used as an internal control for transfection efficiency.

500-bp promoter 2 fragment was generated by PCR using the forward primer (containing a SacI site): 5'-GAGCTCGTGTGAATGTGTGTGACTCG-3' (–1293 to –1310) and the reverse primer (containing an Mlu I site): 5'-ACTTTCATGGGGCTCCTGTGCTCTGC-3' (positions –685 to –704), and it was positionally cloned into the pGL-2/Basic vector backbone (Fig. 5C).

View larger version (26K): [in this window] [in a new window]   FIG. 5. Functional analysis of the NKR-P1C 5'-flanking region. A, luciferase reporter assays comparing the relative activities of the entire 9.76-kb upstream region and the 600-bp core promoter. NKR-P1C upstream regions were cloned in front of the luciferase reporter gene in pGL-2/Basic. Constructs were assayed for luciferase activity upon transfection into MNK-1 cells and SL-12 cells. The mean values (± S.E.) of three experiments are shown. Bar graphs show the relative luciferase activity of each construct, plotted as fold induction relative to that of the promoterless luciferase construct (pGL-2/Basic). B, ten constructs containing NKR-P1C upstream regions ranging from 600 bp to 9 kb as shown were generated in pGL-2 and assayed as in A. C, the generation of constructs containing promoter 2 (500bp-P2) and promoter 1 (600bp) in pGL-2 vector backbone are shown. D, the 500-bp-P2 construct and the 600-bp construct containing promoter 1 were transfected into MNK-1 and CTLL-2 cells and assayed as in A.

The HS1 fragment (500 bp) was generated by PCR using the following primers: forward primer (FHS1; Fig. 7) 5'-TTTCACATCTATCACCAGT-3' (position –8977 to –8995 upstream of starting ATG; Fig. 2, underlined, bold); reverse primer (RHS1; Fig. 7) 5'-TATGTGTTATTTTTATCCCA-3' (positions –8484 to –8502 upstream of starting ATG). This product was cloned into the pGL-2/Basic vector backbone (HS1 construct), the pGL-2/600bp vector backbone (HS1/600bp construct), and in front of a TATA box (TATA construct) in the TATA box containing luciferase construct (MCS-luc; Stratagene).

Luciferase, -Galactosidase, and Protein Assays—Cells transfected with the luciferase and -galactosidase reporter plasmids were assayed using the Dual-Light reporter system (Tropix Inc. Applied Biosystems, Bedford, MA), as previously described (25). Results represent the average luciferase activity indexed for -galactosidase activity and protein concentration per sample.

Cell Lines—CTLL-2 (an IL-2-dependent NK1.1⁺ mouse cytotoxic T cell line) (26), MNK-1 (an IL-2-dependent NK1.1⁺ mouse pre-NK cell line) (27), MNK-1^9.76kb/GFP (MNK-1 cell line stably transfected with a construct containing 9.76 kb of NKR-P1C upstream region cloned in front of the GFP gene), SL-12 (a mouse pre-T cell line), and BW5417 (a TCR-deficient mouse T cell line) were grown at 37 °C in 5% CO₂ in complete DMEM (medium supplemented with 10% FBS, 2 mM glutamine, 10 units/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml gentamicin, 110 µg/ml sodium pyruvate, 50 µM 2-ME, and 10 mM HEPES, pH7.4). Human rIL-2 (10 units/ml) was added to the culture medium for CTLL-2, MNK-1, and MNK-1^9.76kb/GFP cells.

DNA Transfections—Cells were electroporated using a BTX ECM600 Electroporation system (San Diego, CA). Electroporations were performed using 4 mm gap cuvettes (BioRad). Cells in 20% FBS RPMI 1640 media were electroporated using the following conditions: MNK-1 and CTLL-2 (300 V, 186 , and 1600 µF); SL-12 (300 V, 186 , and 450 µF); BW5147 (300 V, 186 , and 1500 µF).

DNaseI Hypersensitivity Assay—MNK-1^9.76kb/GFP cells (10⁸) were pelleted and resuspended in reticulocyte standard buffer (RSB; 10 mM Tris HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl₂). Cells were lysed in 0.5% (v/v) Nonidet P-40, and nuclei were collected and resuspended in RSB buffer. Digestions were carried out at 37 °C with 0.25–8.0 units of DNaseI (Invitrogen) for 3 min then terminated with an equal volume of DNaseI stop buffer (0.6 M NaCl, 1% (w/v) SDS, 20 mM Tris-HCl (pH 8.0), 10 mM EDTA). Samples were treated with proteinase K overnight, and DNA was recovered by phenol/chloroform extraction and ethanol precipitation, then resuspended in 100 µl of dH₂O. 10 µg of each sample was digested with NotI followed by Southern blotting and hybridization using a ³²P-labeled GFP probe overnight at 65 °C. Blots were exposed to PhosphorImager screens (Kodak, Rochester, NY) overnight, and scanned using a PhosphorImager (BioRad).

Computational Analysis of the 5'-Flanking Region Sequence—Putative regulatory elements in the 5'-flanking regions of the mouse NKR-P1C gene were identified by searching the transcription factor binding site data base (TRANSFAC 4.0 and MatInspector V2.2; transfac.gbf.de/TRANSFAC/). Parameters used were: core similarity, 1.0; matrix similarity, 0.85. The numerical position of each cis-element corresponds to the 5'-end of each motif relative to the major transcriptional initiation site (bold arrow, Fig. 2).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Isolation of Genomic Clones Containing the 5'-Flanking Region of Mouse NKR-P1C—A -phage mouse genomic library was screened by Southern hybridization using a PCR-generated genomic probe complementary to the NKR-P1C 5'-coding region. Three positive clones were obtained. One clone, P13, had a 13-kb genomic insert (GenBank^TM accession number AY316227 [GenBank] ), which was further characterized with restriction enzymes. To exclude recombination artifacts, restriction enzyme digestion patterns of mouse genomic DNA from C57Bl/6 splenocytes were compared with those of the P13 phage clone and found to be identical (data not shown). To subclone the 5'-flanking region, subgenomic plasmid libraries were generated and screened with a probe corresponding to the NKR-P1C coding region.

Identification of the Major NKR-P1C Transcriptional Initiation Site—To define the transcriptional initiation site of the NKR-P1C gene, primer extension was performed on RNA from CTLL-2 cells (an IL-2-dependent mouse T cell line that expresses NKR-P1C) (26) and MNK-1^9.76kb/GFP (a mouse pre-NK cell line, MNK-1 (27), expressing GFP under the control of the 9.76 kb NKR-P1C 5'-upstream region; see Fig. 6A). Extended products were separated on a denaturing polyacrylamide/urea gel alongside a sequence of the genomic clone to measure the size of extended products. Results depicted in Fig. 1A shows that transcription in MNK-1^9.76kb/GFP cells is initiated at a single site 156 bp upstream of the starting ATG codon (Fig. 2 underlined, bold). This site is also observed in CTLL-2 cells (Fig. 1B, upper strong band). In CTLL-2 cells, transcription is also initiated, although at a much lower level, from another site (Fig. 1B; lower band). The specificity of the primer was confirmed using untransfected MNK-1 cells and yeast RNA as a negative controls (Fig. 1A, right lane and Fig. 1B, left lane).

View larger version (41K): [in this window] [in a new window]   FIG. 6. Analysis of NKR-P1C DNaseI hypersensitive sites in MNK-1 cells. A, MNK-1 cells were stably transfected with the 9.76-kb NKR-P1C upstream fragment driving GFP and analyzed for GFP expression after selection. B, nuclei from MNK-19.76kb/GFP stable transfectants were isolated and treated with increasing concentrations of DNaseI. DNA isolated from these nuclei was subsequently digested with NotI, and Southern blotted using a GFP-specific probe. The upper arrow corresponds to the NotI restriction endonuclease fragment, while the lower arrow identifies the DNaseI hypersensitive site, termed HS1.

View larger version (67K): [in this window] [in a new window]   FIG. 1. Mapping of the mouse NKR-P1C transcriptional initiation site. A, primer extenstion was performed using MNK-19.76kb/GFP mRNA. An oligonucleotide corresponding to the 5'-end of the GFP gene was hybridized to MNK-1–9.76/GFP mRNA and extended with reverse transcriptase. Extension products were size fractioned on a denaturing polyacrylamide gel alongside a Sanger dideoxynucleotide sequencing ladder primed as above. mRNA from untransfected MNK-1 cells was used as a negative control. The major transcription initiation site is indicated with an arrow placed next to the corresponding genomic sequence. B, primer extension was performed using CTLL-2 mRNA. An oligonucleotide corresponding to the 5' end of the mouse NKR-P1C cDNA (shown in Fig. 2) was hybridized to CTLL-2 mRNA, and extended as described above. Total yeast RNA was used as a control for the specificity of the reaction. The major and minor transcription initiation sites are indicated with the arrows placed next to the corresponding genomic sequence. C, mapping of the transcription initiation sites by 5'-RACE. The nucleotide sequence for the genomic portion of mouse 5'-flanking region and part of exon 1 (starting ATG is underlined) is shown. The symbols * and # denote the transcription initiation sites of unique clones obtained by 5'-RACE and characterized by DNA sequence analysis from CTLL-2 cell line, and RAG-2-deficient splenocytes (NK cell-enriched), respectively. The major transcription initiation site as defined by primer extension analysis (A and B, bold) is shown and numbered +1.

Similar results were obtained utilizing the 5'-RACE technique (Fig. 1C). Here, RNA sources included CTLL-2 cells and splenocytes (NK cell-enriched) obtained from RAG-2-deficient mice (Fig. 1C). Although both major and minor initiation sites were detected by this technique, a set of shorter transcripts was also detected. These shorter fragments are likely due to inefficient reverse transcriptase function during the first strand synthesis.

Thus, two complementary techniques independently confirmed that the major NKR-P1C transcriptional initiation site is located at position –156 (relative to the translational initiator methionine ATG codon).

Analysis of the NKR-P1C Promoter Region for Putative DNA Binding Sites—The 5'-flanking region of mouse NKR-P1C is TATA-less (Fig. 2). The transcriptional initiation site, containing the sequence 5'-TCA⁽⁺¹⁾GTCA-3', shows striking homology to the initiator (Inr) element consensus sequence (5'-PyPyA⁽⁺¹⁾N(A/T)PyPy-3') (28) (Fig. 2). Analysis of the 5'-flanking region of NKR-P1C highlights multiple Ets-1 consensus sequences at positions –94, –98, –102, –104, –108, –117, and –123 relative the to major transcriptional start site (Fig. 2; large right arrow). Several Ikaros binding sites are evident at positions –57, –111, –183, and –328. In addition, unique binding sites are observed for the following transcription factors with more complex sequence elements: GATA, position –203; TCF-1, position –73; Sp1, position –18; NFAT, position –105; Oct-1, position –237 (Fig. 2). It is likely that at least some of these binding sites play a significant role in recruiting transcription factors to mediate lineage-specific expression of NKR-P1C.

Detection of NKR-P1C mRNAs-containing Novel 5'-Exons— Both 5'-RACE (Fig. 1C) and primer extension (Fig. 3A) analyses revealed the presence of a novel exon, designated exon 1.1 (E1.1), located upstream of the originally described exon 1 (E1) (7). Fig. 2 shows the transcriptional initiation site for this novel exon (small thin right arrow) and the location of the novel splice donor/acceptor sites (N|gt...ag|N) corresponding to exons 1.1/2.1. To confirm the physiological existence of exon 1.1, we performed RT-PCR analysis with specific primers (F1B and F1C) in Fig. 2. The results demonstrate that exon 1.1 is transcribed significantly, albeit to a lesser extent than exon 1 (Fig. 3B). Interestingly, transcripts containing exon 1.1 could be detected among adult tissues (CTLL-2 cells, splenic NK cells), but not during fetal ontogeny (day 15 fetal liver and thymus are shown) (Fig. 3B). Moreover, primer extension analysis revealed that NKR-P1C transcripts initiated from the upstream site are not very abundant (Fig. 3A). Although exon 1.1 contains one in-frame ATG start codon (Fig. 2, italicized), no easily discernable structural motifs are present within this sequence.

View larger version (41K): [in this window] [in a new window]   FIG. 3. Identification of exon 1.1 (E1.1) by primer extension and RT-PCR. A, primer extension products were size-fractionated on a denaturing polyacrylamide gel alongside a Sanger dideoxynucleotide sequencing ladder primed on an NKR-P1C genomic DNA subclone. The G nucleotide corresponds to position –540 relative to the major transcriptional initiation site (large right arrow in Fig. 2). B, RT-PCR of various tissues showing expression levels of NKR-P1C transcripts corresponding to exon 1, exon 1.1, or -actin as a control. cDNA was generated from total RNA isolated from C57BL/6 (B6) splenic NK cells, CTLL-2 cells, and B6 day 15 fetal thymus and fetal liver. PCR was performed using 5'-primers that correspond to either exon 1.1 (RT-PCR oligo F1B; Fig. 2) or exon 1 (RT-PCR oligo F1C; Fig. 2), and a common 3'-primer (RT-PCR oligo R1B/C; Fig. 2).

To investigate whether a transcript containing exon 1.1 could encode for a protein, we generated plasmids containing either transcript 2 cDNA or the native NKR-P1C cDNA (transcript 1) (Fig. 4A). Transfection of these vectors into Jurkat cells (human T cell line) yielded detectable surface expression (as determined by NK1.1 staining) only with the original NKR-P1C exon 1-containing cDNA (Fig. 4B). Notably, even the addition of a proteasome inhibitor (MG-132, Calbiochem, San Diego, CA) could not allow for the detection of a product from the novel exon 1.1-containing cDNA, either on the surface or intracellularly (data not shown). The lack of detectable surface or intracellular staining suggests that the newly identified exon 1.1 is non-coding. In this regard, it is interesting to note that although exon 1.1 possesses an ATG (Fig. 2, italicized) that is in-frame with the rest of the gene, several other start sites, which are followed by multiple in-frame stop codons, precede the in-frame ATG in exon 1.1 (Fig. 4A). The presence of multiple stop codons downstream of the first ATG in exon 1.1 is likely to contribute to the failure to detect NK1.1 expression encoded by transcripts containing exon 1.1.

View larger version (24K): [in this window] [in a new window]   FIG. 4. Lack of NK1.1 expression following transfection with a construct containing exon 1.1 of NKR-P1C. A, genomic fragment of the NKR-P1C upstream region showing exons 1.1, 1, and 2. Large arrow represents the major transcription initiation site, while the small arrow represents the upstream transcription initiation site. Schematic representation of the E1.1- and E1-containing transcripts (transcript 2 and transcript 1, respectively) and the corresponding protein regions are indicated (* represents ATG codons and # represents stop codons). B, flow cytometric analysis of NK1.1 expression on Jurkat cells transiently transfected with plasmids corresponding to full length NKR-P1C cDNAs containing either exon 1 (transcript 1) or exon 1.1 (transcript 2). Cells were co-transfected with GFP expression plasmid (pEGFP), and surface expression of NK1.1 was analyzed on GFP+ cells (R1-gated). RCN, relative cell number; FSC, forward light scatter.

Promoter/Enhancer Activity of the NKR-P1C 5'-Flanking Region—The functional promoter activity of the NKR-P1C upstream region was assessed by transient expression of luciferase reporter constructs (Fig 5A). Initially, a genomic fragment containing a full 9.76 kb of the NKR-P1C upstream region was cloned into the pGL-2 vector (pGL-2/9.76kb). To assess lineage-specific expression, this construct was introduced into MNK-1 cells, and luciferase activity in cell extracts was measured. As a non-NK lineage control, the construct was also transfected into SL-12 cells (an NK1.1^– mouse pre-T cell line) (29). To determine the minimal promoter function, a shorter 600 bp NKR-P1C upstream flanking sequence was also inserted into the pGL-2 vector (pGL-2/600bp). In addition, a pGL-2/SV40 construct (luciferase driven by the SV40 promoter/enhancer) was used as a positive control for luciferase activity, while a CMV--galactosidase vector (CMV promoter/enhancer driving -galactosidase) was used to normalize transfection efficiency.

Fig. 5 shows the results of the transfections, normalized for transfection efficiency and fold induction of luciferase activity relative to the promoterless/enhancerless pGL-2/Basic vector. As expected, the control pGL-2/SV40 vector produced a large induction in luciferase activity in both cell lines (Fig. 5A). Importantly, the pGL-2/600bp NKR-P1C promoter region alone also yielded a significant increase in luciferase activity in both cell lines (Fig. 5A). However, when the activity of the large genomic fragment containing the 9.76-kb NKR-P1C upstream region was assessed, a 30-fold higher luciferase activity was observed in MNK-1 cells relative to that of SL-12 cells (Fig. 5A). Strikingly, the 9.76-kb insert drove expression of luciferase in MNK-1 cells almost equal to that of the SV40 construct. In contrast, the activity of the 9.76-kb upstream region was actually reduced 3-fold in SL-12 cells relative to the activity of the shorter 600-bp fragment. Collectively, these results suggest that the 9.76-kb upstream region contains elements necessary and sufficient for lineage-specific expression of the NKR-P1C gene, and that the 600-bp element has core promoter activity.

To further refine the elements in the upstream region responsible for lineage-specific expression, we explored the minimal DNA fragment size of the 5'-region that is necessary for full tissue-specific transcriptional regulation of the NKR-P1C gene. We achieved this by performing deletion analysis of the cloned 9.76-kb 5'-region, and subcloning these fragments into the pGL-2/Basic vector. Fig. 5B shows functional analysis of the 5'-region broken down into 10 different reporter constructs. As noted in Fig. 5A, the 600-bp construct was able to induce luciferase activity in both cell types, thus defining a core promoter region containing the cis-acting regulatory elements responsible for directing NKR-P1C expression. In addition, transfection of reporter constructs ranging from 600 bp to the full 9.76 kb revealed a pattern of enhanced activities in MNK-1 cells, yet suppressed activities in SL-12 cells. This suggests that additional binding sites for regulatory proteins exist in the 5'-regions of the NKR-P1C gene. Significantly, constructs containing a minimum of the first 1 kb of flanking sequence enhanced lineage-specific expression (augmented levels in MNK-1 SL-12), while fragments 3 kb and larger silenced ectopic expression (depressed levels in SL-12 MNK-1). In general, larger fragments further augmented the difference between MNK-1 and SL-12 activities. This pattern suggests the existence of both enhancer and silencer elements upstream of NKR-P1C.

The identification of a longer NKR-P1C transcript containing novel 5'-untranslated exons suggested that an additional promoter existed. To survey cell lines for the activity of this putative new promoter, termed promoter 2, we designed a reporter luciferase construct (Fig. 5C, 500bp-P2) containing the putative promoter 2 in the absence of the 600 bp NKR-P1C minimal promoter (600 bp promoter 1). As can be observed from Fig. 5D, the promoter 2 (500bp-P2) failed to induce significant luciferase activity, and thus appears to be inactive in MNK-1 cells. This observation helps explain our inability to detect a product in the primer extension analysis of exon 1.1 in MNK-1 cells (data not shown). Interestingly, the 500 bp promoter 2 construct displayed low activity in the mature NK1.1⁺ T cell line, CTLL-2 (Fig. 5D), suggesting that it is a very weak promoter and explaining the faint signal obtained from primer extension experiments using CTLL-2 cells.

Identification of a DNaseI Hypersensitive Site (HS1) in the Upstream NKR-P1C Region That Exhibits Enhancer/Promoter Activity—In order to identify 5' DNA elements that functionally bind regulatory proteins, we employed a DNaseI hypersensitivity assay. To facilitate this, the full 9.76-kb NKR-P1C upstream region was cloned in front of GFP as a reporter, and this vector was used to generate stable transfectants of MNK-1 cells. Flow cytometric analysis reveals that more than 90% of these cells were GFP⁺ (Fig. 6A), demonstrating that the 9.76-kb fragment drives high level GFP expression in stable MNK-1 transfectants. Similarly transfected SL-12 cells failed to show any expression of GFP (data not shown). To analyze the binding of MNK-1 transcription factors to the 9.76-kb upstream DNA, nuclei from these cells were prepared and treated with different amounts of DNaseI. Recovered DNA was digested with NotI and assayed for DNaseI hypersensitive sites by Southern blotting using a GFP-specific probe. These experiments revealed a single hypersensitive site 9-kb upstream of the starting ATG codon (Fig. 6B). The region of DNaseI hypersensitivity appeared to span about 500 bp of upstream region and was termed HS1. Computational analysis of HS1 for DNA binding sites revealed multiple high scoring GATA, Ikaros, and AP-1 sites (core similarity = 1.0, matrix similarity = 0.95) (Fig. 7). This suggested that HS1 might exhibit functional enhancer activity.

To test whether HS1 exhibits enhancer activity, this 500 bp genomic region was cloned into a luciferase backbone containing a minimal promoter (TATA box only). This construct was transfected into MNK-1 and BW5417 cells (Fig. 8A). The HS1 induced 7.5-fold increase in luciferase activity when transfected into MNK-1 cells compared with the TATA box promoter alone (Fig. 8A), while only a moderate 2-fold increase was observed in BW5417 cells (Fig. 8A).

View larger version (34K): [in this window] [in a new window]   FIG. 8. Functional analysis of the NKR-P1C HS1 DNA element. A, a 500-bp HS1 fragment was cloned in front of the minimal promoter (TATA box)-containing luciferase backbone plasmid. Constructs were transiently transfected into MNK-1 and BW5147 cells and assayed for luciferase activity. Bar graphs represent fold induction of luciferase activity relative to that obtained from cells transfected with the TATA construct alone. The mean values (±S.D.) of two experiments are shown. B, the 500-bp HS1 DNA fragment was cloned in front of the 600-bp minimal NKR-P1C promoter driving a luciferase reporter gene (pGL-2/600bp). Constructs were transiently transfected into MNK-1, SL-12, and BW5147 cells and assayed as in A. Bar graphs represent the relative luciferase activities for each cell line. The values (±S.E.) of three independent experiments are shown. C, relative luciferase values from B were plotted as fold induction relative to those of the pGL-2/600bp construct.

Since the HS1 enhanced the expression of luciferase from a heterologous promoter, we decided to test its ability to increase luciferase expression from the NKR-P1C minimal functional promoter. Thus, the HS1 fragment was cloned into pGL-2 directly in front of the 600 bp NKR-P1C upstream core promoter region. This construct (pGL-2/HS1/600bp) was transfected into the MNK-1, SL-12, and BW5417 cell lines (Fig. 8B). The parent vector without HS1 (pGL-2/600bp) was used as a promoter-alone control. Interestingly, the HS1 construct induced a 4-fold increase in luciferase activity when transfected into MNK-1 cells, compared with the promoter alone (Fig. 8C). In contrast, a more moderate 1.5–2-fold increase was seen in SL-12 and BW5417 cells, respectively (Fig. 8C). This suggests that the 500-bp HS1 element of the NKR-P1C gene contains enhancer-like activity, which is at least partially cell-type specific. Thus, although other sites not identified in this study may be responsible for further lineage-specific control of NKR-P1C expression, these experiments collectively outline a minimal functional NKR-P1C promoter and a unique enhancer element, HS1.

To further characterize HS1, this element was cloned into an empty luciferase backbone (HS1 construct). This construct and the construct containing the NKR-P1C minimal promoter (600 bp) were transfected into MNK-1 and CTLL-2 cells (Fig. 9A). Although the 600-bp minimal promoter is capable of driving luciferase expression in both lines, the HS1 construct was capable of inducing luciferase activity only in MNK-1 cells. Nevertheless, the observed luciferase activity induced in MNK-1 cells by HS1 was significantly lower than that induced by the 600-bp NKR-P1C promoter (Fig. 9A). This level of luciferase expression in MNK-1 cells could be due to the presence of multiple NK cell-specific elements (Fig. 7) that might be responsible for inducing luciferase expression in MNK-1 cells even in the absence of a defined promoter, as has been reported for other cell lines (30), or perhaps the HS1 element contained a novel early NK cell-specific promoter. To address the possibility that HS1 could serve as promoter, we examined whether we could detect transcripts initiated by this element. Fig. 9B shows an RT-PCR analysis using a forward primer positioned downstream of the potential initiator consensus sites (Fig. 7, underlined, bold) and a reverse primer located within exon 2 (Fig. 2, GSP nested). Using this approach, a barely-detectable transcript derived from HS1 was observed only in sorted NK1.1⁺ cells from the fetal thymus (Fig. 9B), suggesting that HS1 contained a novel promoter (promoter 3). The PCR product was cloned and sequenced to verify that the observed band resulted from HS1/promoter-3 activity. The sequence analysis revealed a novel exon (E1.2) that spliced to exon 1, creating a novel exon 2.2 (splice donor and acceptor sites are shown in Fig. 7 and Fig. 2, respectively, as dotted vertical lines). Similarly to exon 1.1 from promoter 2, the initial ATG codon in E1.2 was followed by several in-frame stop codons, suggesting that this exon was also noncoding.

View larger version (27K): [in this window] [in a new window]   FIG. 9. Characterization of promoter 3 function and detection of promoter 3-derived transcripts. A, the 500-bp HS1 fragment was cloned into an empty luciferase backbone (HS1 construct). HS1 and 600 bp minimal promoter construct were transiently transfected into MNK-1 and CTLL-2 cells and assayed for luciferase activity. Bar graphs represent fold induction of luciferase activity relative to that obtained from cells transfected with an empty luciferase backbone (pGL-2/Basic). The mean values (±S.D.) of three experiments are shown. B, RT-PCR of various tissues showing expression levels of NKR-P1C transcript 3-containing exons 1.2 (E1.2) and 2.2 (E2.2), or -actin as a control. cDNAs were generated from total RNA isolated from C57BL/6 day 15 fetal thymus, NK1.1+ sorted day 15 fetal thymus, CTLL-2 cells, and MNK-1 cells. PCR was performed using 5'-primer F1A (Fig. 7), and 3'-primer GSP nested (Fig. 2). C, genomic fragment of the NKR-P1C upstream region showing exons 1.2, 2.2, and 2. Large arrow represents the major transcription initiation site, while the small arrow represents the upstream transcription initiation site. Schematic representation of the E1.2- and E1-containing transcripts (transcript 3 and transcript 1, respectively) and the corresponding protein regions are indicated. * represents ATG codons and # represents stop codons.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we have cloned and sequenced about 10 kb of the upstream region of the mouse NKR-P1C gene. We have defined the transcriptional initiation sites, a minimal functional promoter, and an enhancer region responsible for regulating expression of NKR-P1C in a lineage-specific manner. Using a combination of primer extension and 5'-RACE methodologies, a single major transcriptional initiation site was identified 156 bp upstream of the ATG coding the N-terminal methionine of NKR-P1C. This initiation site contains the TCA⁽⁺¹⁾GTCA sequence, which shows remarkable homology to the initiator (Inr) consensus. There is no canonical TATA box within normally expected distances from the transcriptional initiation site of the mouse NKR-P1C. Although the majority of TATA-less genes are generally thought to be constitutively active "housekeeping" genes, some can be expressed in a developmentally specific and tissue-specific manner (31). Two other NK cell-specific genes, human NKG2A (32) and murine 2B4 (33) both have tissue-restricted expression without TATA sequences in their 5' promoter regions. Several other lymphocyte-specific genes, such as Lck (34), Tdt (28), CD19 (35) RAG1, and RAG2 (36) also lack a TATA box.

The biological significance of the absence of a TATA box among such lymphocyte specific genes is not yet understood. However, studies of numerous TATA-less genes have demonstrated that the Inr element constitutes the simplest functional promoter identified and provides one explanation for how TATA-less promoters direct transcriptional initiation (28). About 30-bp downstream of the NKR-P1C transcriptional start site, we observed the sequence 5'-AGACAGG-3', which shows striking homology to the downstream promoter element (DPE) (37). Both Inr and DPE are required for sequence-specific binding of TFIID in TATA-less promoters (37). Furthermore, at positions –30 to –35, there is an A/T-rich sequence (5'-TTAAGT-3'), which, in addition to Inr and DPE, may contribute to the assembly of the transcription preinitiation complex.

Examination of the nucleotide composition of the NKR-P1C 5'-flanking region reveals the region to be depleted in CpG dinucleotides. This is a characteristic of many differentiation-associated and developmentally regulated genes with TATA-less promoters (28). There are no CpG dinucleotides in any of the putative cis-elements in the 5'-flanking region of mouse NKR-P1C. The NKR-P1C promoter region contains multiple consensus binding sites for the Ets-1, Ikaros, GATA, and TCF-1 factors important in regulating expression of many lymphocyte-specific genes. It is not surprising to find several (at least 5) Ets-1 binding sites in the 100-bp region immediately upstream of the transcriptional initiation site, as mice with a disrupted Ets-1 gene lack NK cells due to impaired survival and/or development of NK cells (38, 39).

In expression experiments, the NKR-P1C 5'-flanking region confers significant reporter activity when introduced into NK lineage cells. The proximal promoter region (600-bp construct) drove transcription in both NK lineage (MNK-1) and non-NK (SL-12 and BW5147) cell lines. The observation that this core promoter activity was obtained in a non-NK cell line, in which NKR-P1C is not expressed, indicates that the tissue-specific expression of NKR-P1C involves other factors, such as enhancer or silencer cis-elements, that may act in concert with the promoter region. Consistent with this, constructs containing the upstream NKR-P1C region, including the HS1 DNaseI hypersensitive site alone, impart a cell-type specific expression pattern. Further biochemical and functional studies are currently in progress to identify the precise cis-elements contributing to the observed enhancer/silencer functions of the 9.76-kb upstream fragment.

We provide evidence for the existence of several novel promoters (promoters 1–3), with the 600 bp 5'-flanking region serving as the principal promoter (promoter 1) active in all NK1.1⁺ cells (Fig. 10A). The additional upstream promoters (2 and 3) appeared to be developmentally regulated, with promoter 2 active in mature NK cells and promoter 3, which is located within the HS1 site, active in fetal-derived NK cells. Additionally, transcripts from both of these promoters contained noncoding exons, due to the presence of multiple stop codons following the first few ATGs. The above findings suggest that mouse NKR-P1C gene shares many similar features with the genes encoding the other NKC-encoded lectin-like proteins, such as the Ly49 gene family (30, 40–42). Indeed, it has recently been shown that in addition to the Ly49 promoter used by mature NK cells (Pro-2 or Pro-3) there is also a fairly upstream Ly49 promoter (Pro-1) that is primarily active in tissues where Ly49 gene expression is first detected during ontogeny (40) (Fig. 10A). Moreover, as shown for NKR-P1C, the first exon of Ly49a is non-coding, followed by the cytoplasmic (second exon), transmembrane (third exon), stalk (fourth exon), and putative carbohydrate recognition domains (CRD; fifth to seventh exons), respectively (Fig. 10B) (41). These findings suggest a clear commonality in the structure and transcriptional regulation of these NK cell related genes that may have been conserved throughout evolution in order to maintain important features of NK cell function.

View larger version (22K): [in this window] [in a new window]   FIG. 10. Comparison of the transcriptional regulation, and exon/intron structures and functional domain organization of NKR-P1C and Ly49a. A, schematic representation showing the locations of exons 1.2, 1.1, and 1, and promoters 1, 2, and 3 in the NKR-P1C upstream region. The locations of Ly49 exons and promoters are also indicated (adapted from Saleh et al., Ref. 40). B, the organization of NKR-P1C exons 1 and 1.1/1.2 (identified in this study) bear striking similarity to the translated exon 2 and untranslated exon 1 of Ly49A, as do the remaining exons.

In conclusion, the experiments outlined in this study provide an insight into the molecular basis of NKR-P1C expression in NK cells, NK1.1⁺ T cells, and progenitor thymocytes. We have shown that the 600 bp 5'-flanking region of the NKR-P1C gene contains cis-elements required for basal transcription or core promoter function. In addition, we have identified a DNA element (HS1) 9-kb upstream that functions to enhance transcription in a lineage-specific manner and may also serve as a developmentally-regulated promoter. Studies are currently underway to define tissue- and stage-specific elements regulating NKR-P1C expression in vivo.

	FOOTNOTES

 
^* This work was supported by funds from the Canadian Institute for Health Research (CIHR). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by an Ontario Graduate Scholarship Award.

^|| Supported by a Long Term Fellowship from the Human Frontier Science Program.

^** Supported by an Investigator Award from the CIHR. To whom correspondence should be addressed: Dept. of Immunology, University of Toronto, Sunnybrook and Women's Health Sciences Centre, Room A-331, 2075 Bayview Ave., Toronto, Ontario M4N 3M5, Canada. Tel.: 416-480-6112; Fax: 416-480-4375; E-mail: jc.zuniga.pflucker{at}utoronto.ca.

¹ The abbreviations used are: NKC, natural killer gene complex; mAb, monoclonal antibody; HS, DNaseI hypersensitive site; RACE, rapid amplification of cDNA ends; GFP, green fluorescent protein.

	ACKNOWLEDGMENTS

 
We thank Quyen Fong for help with cloning and sequencing of NKR-P1C flanking region.

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