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The Journal of Immunology, 2004, 172: 5512-5521.
Copyright © 2004 by The American Association of Immunologists

Human CD1D Gene Has TATA Boxless Dual Promoters: An SP1-Binding Element Determines the Function of the Proximal Promoter1

Qiao-Yi Chen2,*,{dagger} and Natalie Jackson*

* Research Institute for Children, Children’s Hospital, New Orleans, LA 70118; and {dagger} Department of Pediatrics, Louisiana State University Health Science Center, New Orleans, LA 70112


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD1d presents lipid Ags to a specific population of NK T cells, which are involved in the host immune defense, suppression of autoimmunity, and the rejection of tumor cells. The transcriptional control mechanism that determines the regulation and the tissue distribution of CD1d remains largely unknown. After investigating 3.7 kb 5' upstream of the coding region, we found that human gene encoding CD1d molecule (CD1D) has TATA boxless dual promoters with multiple transcription initiation sites. The proximal promoter is located within the region of –106 to +24, and the distal promoter is located within the region of –665 to –202 with the A of the translational start codon defined as +1. The longest 5'-untranslated region derived from 5'-RACE and apparently generated by the distal promoter has 272 bp in length covering the genomic sequence of the proximal promoter. The region covering the proximal promoter gave a much higher luciferase activity in Jurkat cells than in K562 cells, whereas it was in reverse for the region covering the distal promoter, indicating a cell type sp. act. of the two promoters. Transcription factor SP1 plays a crucial role in the function of the proximal promoter. The analysis of the CD1D promoter region indicates that IFN-{gamma}, NF-IL-6, and T cell factor 1/lymphoid enhancer-binding factor 1 are most likely involved in the regulation of CD1d expression. The illustration of the dual CD1D gene promoters will help to reveal the regulatory factors that control CD1d expression and its tissue distribution for a better understanding of the cross-regulation between CD1d and NK T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD1d molecules are noncovalently associated with {beta}2-microglobulin to form a heterodimeric three-dimensional structure that is similar to the MHC class I molecules (1). The gene encoding CD1d molecule (CD1D)3 gene belongs to the group II CD1 gene family in human. The group I CD1 genes include CD1A, CD1B, CD1C, and CD1E based on their sequence homology (2, 3). The CD1 genes are located in chromosome 1 q22–23 in the human (4, 5). CD1D gene has no significant homology in the 5'-untranslated region (UTR) with the group I genes, whereas the latter genes share significant homology in this region (6). Unlike MHC class I and II genes, CD1 genes are not polymorphic (1, 3). The lack of polymorphism in the CD1 genes has led to the assumption that CD1 molecules may be associated with a conserved and essential Ag presentation in immune regulation (7). Among the CD1 molecules, CD1d is highly conserved across species and is the only group CD1 molecule functionally present in mice and rats (8, 9, 10, 11). Both human and mouse CD1d can present {alpha}-galactosylceramide to NK T cells, which express a restricted range of TCRs bearing a single invariant V{alpha} chain (V{alpha}14J{alpha}281 in mice and V{alpha}24inv in human) to stimulate specific immune responses (12, 13).

CD1d is expressed in various types of human tissues, including T cells, B cells, monocytes, and epithelial cells (14, 15, 16). CD1d is also expressed on immature cortical thymocytes and down-regulated on mature thymocytes in parallel with the expression of group I CD1 molecules (17, 18, 19). However, the transcriptional regulatory mechanism that determines the CD1d expression and tissue distribution remains largely unknown. Human CD1d expression can be up-regulated on intestinal epithelial cells and keratinocytes by IFN-{gamma} (20, 21) or on peripheral blood T cells by mitogen stimulation (15, 22). Overexpression of CD1d has been seen in patients with psoriasis on keratinocytes (21), in patients with allergic reactions to cow’s milk in the duodenal lamina propria (23), and in patients with primary biliary cirrhosis on the epithelial cells of the small bile ducts (24). The levels of CD1d expression can vary significantly between different individuals on T lymphocytes (15), monocytes, or monocyte-derived dendritic cells (25). It is unclear whether the difference in CD1d expression between individuals is due to their genetic variation or an environmental stimulation or both.

Understanding the transcriptional control mechanism would help to reveal how the CD1d expression is regulated in NK T cell-associated immune responses. The CD1D promoter structure and the associated cis-acting regulatory elements have not been illustrated. Thymus and trophoblast cells have multiple alternatively spliced CD1D mRNA transcripts (26, 27), which could be modulated by the promoter structure (28). In this study, we investigated the 5' upstream region of the human CD1D gene and discovered that the CD1D gene has multiple transcription initiation sites and has dual promoters that are located within 700 bp 5' upstream of the coding region. The core domain of the proximal promoter is located within 106 bp 5' upstream of the coding region, and its promoter function is determined by a transcription factor SP1-binding element.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and tissue culture conditions

Jurkat cells and K562 cells purchased from the American Type Culture Collection (Manassas, VA) were used in this study. The cells were cultured in IMDM (Invitrogen, Carlsbad, CA) in the presence of 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). The cells were cultured at 37°C in a humidified atmosphere with 5% CO2.

RT-PCR

The expression of CD1D mRNA in Jurkat cells was detected using RT-PCR. RNA was prepared from the cells using the TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. The isolated RNA was digested with DNase I (Promega, Madison, WI) and precipitated using isopropanol. A total of 2 µg of the isolated total RNA was processed for reverse transcription using a cDNA synthesis kit (New England Biolabs, Beverly, MA), according to the manufacturer’s instructions. The generated first strand cDNA was used as the template for CD1D-specific amplification by PCR using condition as previously described (29). The primers used for CD1D cDNA amplification are 5'-CGC GCA GCG GCG CTC CGC G-3' located in exon 1 and 5'-GGA CCA AGG CTT CAG AGA G-3' located in exon 2. The primers used for the amplification of the housekeeping gene GAPDH were 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3' and 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3'. The PCR products were separated in 2% agarose gel.

Flow cytometry to detect CD1d expression

Jurkat cells were analyzed for surface CD1d expression using a mAb specific for human CD1d (clone CD1d42) conjugated with PE and the corresponding isotype control (BD Biosciences, Franklin Lakes, NJ). The cells (1 x 106) were exposed to 20 µl of the Ab in PBS, pH 7.4, containing 0.1% BSA, in a total volume of 100 µl at 4°C for 30 min. After the reaction, the cells were washed with 1 ml of PBS and suspended in 0.5 ml of PBS containing 0.05% sodium azide and 1% formaldehyde. The cells were analyzed for CD1d expression using FACSVantage (BD Biosciences).

5'-RACE

The 5' end of the cDNA was amplified using the GeneRacer kit (Invitrogen), according to the manufacturer’s instructions. In brief, RNA was isolated from Jurkat T cells, as described above. Two micrograms of the total RNA were used to generate cDNA with 5' cap using the kit according to the manufacturer’s instruction. The generated cDNA containing a potential full-length 5' end was used as a template in a nested PCR. The primers used for the initial PCR were GeneRacer 5' primer: 5'-CGA CTG GAG CAC GAG GAC ACT GA-3', which was derived from the ligated oligo, and a gene-specific primer: 5'-TGC TAT TGG CGA AGG ACG AGA TCT-3', which is located in exon 2 of the CD1D gene. The PCR amplifications were performed in 25 µl vol containing 1 U of TaqDNA polymerase (Invitrogen), using the buffer provided by the company, but with the addition of 8% DMSO (Sigma-Aldrich, St. Louis, MO). The PCR were subjected to a touchdown reaction with an annealing temperature of 68°C for 3 cycles, 66°C for 3 cycles, 64°C for 3 cycles, and 62°C for 25 cycles using an automated thermal cycler (9700; Applied Biosystems, Foster City, CA). The PCR product was separated in a 2% gel. Because the intensity of the PCR product under UV was weak, a nested primer pair was used to reamplify the PCR product. The nested primer pair was GeneRacer 5' nested primer, 5'-GGA CAC TGA CAT GGA CTG AAG GAG TA-3', provided by the company, and a gene-specific primer, 5'-GGC AGC GGA GGG GGA AAA GCC TTT-3', located in the 5' upstream of the exon 2. The nested PCR product was separated in a 2.5% gel. The DNA in the gel was isolated using a kit (QBiogene, Carlsbad, CA) and cloned into a TA cloning vector (Invitrogen). Plasmid DNAs were purified using a commercial kit (Promega) and sequenced using the ABI PRISM 377 DNA Sequencer (Applied Biosystems), according to the manufacturer’s instructions.

Primer extension analysis

Primer extension was performed to confirm the major transcription initiation sites for human CD1D gene. Human thymus RNA purchased from Clontech Laboratories (Palo Alto, CA) was used in the reaction. The primers used in the reaction included 5'-CAG CAG AAA CAG CAG GCA-3' (+7 to + 24), 5'-GCT CAG CTC ACG TCC CTT-3' (–91 to –74), and CAC CTC CCT CTC CCT CAC T-3' (–990 to –1009). The nucleotide +1 was defined as the A of the ATG-translation initiation codon, and the nucleotide 5' to +1 is numbered –1 (30). The sequence of the published cDNA (2) and a genomic DNA sequence obtained from GenBank (accession AL138899) were used as the reference throughout the work. Each (10 pmol) of the primers was labeled with [32P]ATP (PerkinElmer Life and Analytical Sciences, Boston, MA) using T4 polynucleotide kinase (Promega), according to the manufacturer’s instructions. Approximately 10 µg of the total RNA was reacted with 2 pmol of the labeled primer using the first strand cDNA synthesis kit (New England Biolabs), according to the manufacturer’s instructions. The labeled primer was also used in a sequence reaction using the fmol DNA cycle sequencing system (Promega), according to the manufacturer’s instruction, to generate the size marker for the primer extension analysis. A plasmid DNA covering the genomic region of –3478 to +508 was used as the template for the sequencing reaction. The generated DNA products were separated in denaturing polyacrylamide gel containing 8% acrylamide (19:1 acrylamide:bis) and 7 M urea. The signals were detected by autoradiograph.

PCR to amplify genomic DNA

The human CD1D genomic region covering –3478 to +508 or –1738 to +24 was amplified using the primer pair of 5'-TGG CTC TGG TAA GTC TGG AG-3' and 5'-GGA CCA AGG CTT CAG AGA G-3', or 5'-CTA GAG TGT GGT GCA GTA GC-3' and 5'-CAG CAG AAA CAG CAG GCA-3'. The PCR amplification was performed on 50 ng of genomic DNA in 25 µl vol containing 1 U of Platinum Pfx DNA polymerase (Invitrogen), as previously described (29). The amplified PCR product was reacted with 1 U of Taq polymerase at 72°C for 10 min to add a single deoxyadenosine to the 3' ends, then processed for TA cloning (Invitrogen), according to the manufacturer’s instructions. Plasmid DNA was isolated using a commercial kit (Promega) and verified by sequencing using multiple primers. The primers used in the sequencing reactions were M13-F, M13-R, provided by the manufacturer (Invitrogen), and the gene-specific primers, which include 5'-TGG CTC TGG TAA GTC TGG AG-3'; 5'-AGT CGC TGG A GT TTT GCT-3'; 5'-GAA TTC TGA ACT GAA ACC AAG TAA-3'; 5'-AAC ATT CTT GCA CAC TT-3'; 5'-GCT ACT GCA CCA CAC TCT AG-3'; 5'-CTA GAG TGT GGT GCA GTA GC-3'; 5'-TTA GTG GAA ATA ACT AGG CA-3'; 5'-AGT GAG GGA GAG GGA GGT GT-3'; 5'-ATT TAT GTT TTG GAA GCA GG-3'; 5'-CAG CAG AAA CAG CAG GCA-3'; 5'-GAC CAA GGC TTC AGA GAG-3'.

Construction of luciferase reporter gene vectors

To locate the CD1D promoter region, four DNA segments (–3478 to +508, –1804 to +508, –1738 to +24, and –985 to +24) containing the translation initiation codon were subcloned into the pGL3 basic vector (Promega). In brief, the two verified clones (–3478 to +508 and –1738 to +24) in the TOPO PCR2.1 vector with a forward orientation were subcloned into the pGL3 basic vector at the KpnI and XhoI sites. The pGL3 basic vector with the insert of –3478 to +508 was digested with HindIII to generate a 2.312-kb product (–1804 to +508) and subcloned into the HindIII site in the pGL3 basic vector. The clone with the insert of –1738 to +24 was digested with PstI, and a 1.009-kb region (–985 to +24) was subcloned into the pGL3 basic vector at the PstI site.

Four additional DNA segments (–2579 to –980, –1738 to –980, –1460 to –980, and –1393 to –980) containing a putative transcriptional initiation site based on a recent released cDNA (BC027926) were also subcloned into the pGL3 basic vector. The pGL3 basic vector with the insert of –3478 to +508 was digested with EcoRI/PstI to generate a 1.594-kb product (–2579 to –980) and subcloned into the pGL3 basic vector at the respective sites. The clone with the insert of –1738 to +24 was digested with PstI to remove a 1.009-kb region (–985 to +24) and religated to generate a construct with the insert of –1738 to –980. The clone with the insert of –1738 to –980 was digested with SpeI/AvrII and religated to generate an additional clone with the insert of –1460 to –980. The fourth construct covering the region of –1393 to –980 was generated by PCR using the conditions described above with the verified clone as the template. The forward primer used in the reaction was 5'-CAT AGA GCT CTT AGT GGA AAT AAC TAG GCA-3', and the reverse primer was 5'-CTT TAT GTT TTT GGC GTC TTC CA-3' located in the pGL3 basic vector. The PCR product was then digested with SacI/XhoI and cloned into the pGL3 basic vector.

In addition, multiple sequentially deleted segments in the region of –985 to +24 were amplified by PCR and subcloned into the SacI and NheI sites in pGL3 basic vector using conditions as described above. The forward primers that were used to amplify the sequentially deleted 5' end segments are listed in Table I (1, 2, 3, 4, 5, 6, 7, 8, 9), and the common reverse primer for the PCR is 5'-CAG TGC TAG CAG CAG AAA CAG CAG GCA-3'. The reverse primers that were used to amplify the sequentially deleted 3' end segments are also listed in Table I (10, 11, 12). The common forward primer for these segments was 5'-CTA AGA GCT CAG GAG GAA AGA GAG GCT-3'. The PCR products were subcloned into the SacI and NheI sites in pGL3 basic vector. Each of the above constructs was verified using direct DNA sequencing. The plasmid DNA from the above constructs, the pGL3 basic vector, and the Renilla luciferase reporter vector (Promega) were prepared quantitatively using the S.N.A.P. Midiprep kit (Invitrogen), according to the manufacturer’s instructions.


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  		Table I. Partial primers used in the PCR

 
Site-directed mutagenesis analysis

The GeneTailor Site-Directed Mutagenesis System (Invitrogen) was used to generate constructs with the deletion of SP1-binding elements (see Results) following the manufacturer’s instructions. The primers used for the deletion of the predicted SP1 element GGCGGG (–73 to –68) were 5'-GGA CGT GAG CTG AGC GGA GAA GAG TGC GCA G-3' and 5'-GCT CAG CTC ACG TCC CTT-3'. The primers used for the deletion of a second predicted SP1-binding element GGGCGG (–40 to –45) were 5'-GAG TGC GCA GGT CAG ACG CGC AGC GGC GCT CC-3' and 5'-TCT GAC CTG CGC ACT CTT-3'. Five additional predicted cis-acting elements located in the distal promoter region (see Results) were mutated using the above mutagenesis analysis. These five elements are an E-box (–607 to –612) mutated from CAGTTG to CGGCTG, a CAAT box (–570 to –573) from CAAT to CCAG, a second E-box (–508 to –513) from CACTTG to CCCGTG, a CATAAA box (–335 to –340) from CATAAA to CACAGA, and a lymphoid enhancer-binding factor 1 (LEF-1) element (–294 to –288) from CTTTGAA to CTCTGTA. The primer pairs used for these five elements are 5'-AGG AAT CCT GGG ATA TGA CGG CTG TAA AGA ATT G-3' and 5'-GTC ATA TCC CAG GAT TCC TCC ACC TCA GT-3' for E-box (–607 to –612); 5'-CCT AAT CCA GTT CTC TCC AGG TGC AAC TGA GG-3' and 5'-GAG AGA ACT GGA TTA GGT TGG CTA C-3' for CAAT box (–570 to –573); 5'-ATT GTA AAT GTG GTA CTT GCC CGT GAG AAA TTT TG-3' and 5'-GCA AGT ACC ACA TTT ACA ATA AAG-3' for a second E-box (–508 to –513); 5'-TAT GTC CTG CTT CCA AAA CAC AGA TAA TGG TTG-3' and 5'-TGT TTT GGA AGC AGG ACA TAA GGT TG-3' for CATA box (–335 to –340); and 5'-ACG TTG ACC CAA AGT CTC CTC TGTAAC AGG AAA TTG A-3' and 5'-AGG AGA CTT TGG GTC AAC GTG TGG-3' for LEF-1 element (–294 to –288). The underlined letters are the mutated nucleotides for the respective elements. The templates used in the reactions were the pGL3 basic vector constructs inserted with the CD1D genomic region of –213 to +24, –82 to +24, –665 to +24, or –665 to –202. The generated constructs with the deleted or mutated sequences were verified using direct DNA sequencing.

Transfection and luciferase assay

The promoter activity of the CD1D genomic DNA was detected using the luciferase reporter gene assay. The cells were seeded at 5 x 105 per well in a 24-well plate and cotransfected with 0.8 µg of pGL3 basic vector with the insert of interest and 30 ng of pRL-CMV vector (Promega) using DMRIE-C transfection reagent (Invitrogen), according to the manufacturer’s instructions. pRL-CMV expressing Renilla luciferase was used to normalize the transfection. The transfected cells were added with 1 µg/ml PHA (Sigma-Aldrich) and 50 ng/ml PMA (Sigma-Aldrich). Forty-eight hours after the transfection, the cells were harvested and tested using the Dual-Glo luciferase assay system (Promega), according to the manufacturer’s instructions. The luminescence was measured using the Packard NYT Top Counter NXT (Packard Instrument, Meriden, CT). The results were expressed as fold over the luminescence activity derived from the pGL3 basic vector. An error bar was used to show the SD derived from three or more separate experiments for each of the tested samples.

EMSA

EMSA was used to test whether the predicted SP1-binding elements in the proximal promoter region (see Results) are functional. Sense and the antisense synthesized oligonucleotides were annealed to form double-stranded oligonucleotides, which (1.75 pmol) were end labeled with 5 µCi of [{gamma}-32P]ATP (PerkinElmer Life and Analytical Sciences) using T4 polynucleotide kinase (Promega). The labeled oligonucleotides were separated from the free [{gamma}-32P]ATP using a column (Bio-Rad, Hercules, CA), according to the manufacturer’s instructions. A total of 10 µg of nuclear extract derived from Jurkat cells (Active Motif, Carlsbad, CA) was incubated with 1.75 pmol (~100x) of cold oligo or cold oligo with the deletion of the predicted SP1 element, or 2 µl of Ab to SP1 or isotype control (Active Motif) in EMSA-binding buffer (Promega) for 10 min at room temperature, then the [{gamma}-32P]ATP-labeled oligo (20,000 cpm) was added and incubated for additional 20 min. The mixtures were electrophoresed in prerun, nondenaturing 4% polyacrylamide gels. The signals were revealed by autoradiography. The sense sequences for the ologonucleotides tested in the EMSA were 5'-GGT CAG AGG GCG GCG CGC AGC GG-3' (–52 to –30) and 5'-AGC TGA GCG GCG GGG GAG AAG A-3' (–82 to –60). The sense sequences for the two oligonucleotides with the deletion of the respective putative SP1 element were 5'-GAG TGC GCA GGT CAG ACG CGC AGC GGC GCT CC-3' and 5'-GGA CGT GAG CTG AGC GGA GAA GAG TGC GCA G-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of the 5' flanking sequence of the human CD1D gene

We initially searched GenBank for available CD1D cDNA or an expressed sequence tag (EST) with a potential full-length 5'-UTR. A CD1D cDNA (AC: BC027926) and an EST (AU099295) were identified. The cDNA (BC027926) has the longest 5'-UTR with a transcribed exon 1a. The EST (AU099295) does not contain the transcribed exon 1a, but has a 5'-UTR longer than other published cDNAs or ESTs. The EST AU099295 was cloned for 5' ends of human mRNAs using a full-length enriched and 5' end-enriched cDNA library constructed by the oligo-capping method (31). The cDNA (BC027926) was derived from adult pancreas and spleen, whereas the EST (AU099295) was derived from B cells of Burkitt lymphoma. If the EST clone was derived from a CD1D cDNA with a full-length 5' end, the search results would suggest that the CD1D gene has two transcription initiation sites.

To confirm that human CD1D gene has two transcription initiation sites, we analyzed the 5' flanking sequence of the human CD1D gene using 5'-RACE kit. For the 36 clones sequenced, the 5' ends were distributed in two separate regions. One region covers –12 to –62 with the majority located at –12A. The second region covers –201 to –272 (Fig. 1A). The 5' end of the clone with the longest 5'-UTR was –272A. One of the clones shared the same 5' end (–213C) with the EST (AU099295) (Fig. 1B). We were unable to obtain a clone with the 5' flanking sequence like that of the cDNA (BC027926). The results indicate that the human CD1D gene may have multiple transcription initiation sites, which are located in two separate regions. However, these clones with multiple 5' ends might be derived from the truncated mRNA. We then used the method of primer extension to confirm our results. We identified a major band at –12A using the primer located at +7 to +24 (Fig. 2A), and four bands corresponding to –278T, –235C, –213C, and –196A using the primer located at –91 to –74 (Fig. 2B). However, we could not obtain a signal using the primer located at the position of –990 to –1009 for the detection of the putative transcription initiation site based on the cDNA clone of BC027926. Accordingly, our results have shown that the human CD1D gene has multiple transcription initiation sites, which are located in two separate regions.



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  		FIGURE 1. Analysis of the 5' flanking sequence of CD1D gene. A, Thirty-six sequenced clones with 5' cap were identified to be distributed in two separate regions. The numbers over the short lines are the position of the 5' end, and the numbers underneath the short lines are the detected number of the clones with a same 5' end. B, A representative clone with the transcription initiation site –213C. The vertical arrow separates the ligated oligo from the CD1D 5'-UTR.

 


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  		FIGURE 2. Primer extension to determine the transcription initiation sites. A, A major transcription initiation site corresponding to –12A, as indicated by the arrow, was identified using the primer located at +7 to +24. B, Four transcription initiation sites corresponding to the nucleotides as marked were identified using the primer located at –91 to –74.

 
Location of the CD1D promoter region

Four constructs (–3478 to +508, –1804 to +508, –1738 to +24, and –985 to +24) were tested for luciferase activity to locate the CD1D promoter in Jurkat cells, which express CD1d (Fig. 3). The luciferase activity was not detectable for the region of –3478 to +508. However, the luciferase activity increased slightly with the deletion of the region of –3478 to –1803. The deletion of the region between +23 and +508 or between –1738 and –984 also increased the luciferase activity (Fig. 4A). The results indicated that the CD1D promoter is potentially located within the region of –985 to +24, corresponding to the transcription initiation site according to the analysis of 5' flanking sequence, because a functional promoter requires the presence of a transcription initiation site.



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  		FIGURE 3. Detection of the expression of CD1d in Jurkat T cells. A, Detection of CD1D transcripts by RT-PCR. B, Detection of CD1d protein on Jurkat cells by flow cytometry.

 


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  		FIGURE 4. Location of CD1D promoter region. A, The functional region was located within the region of –985 to +24. B, The luciferase activity derived from the four constructs showed that the region between –2578 and –980 appears not to harbor a functional promoter.

 
We then tested whether a distal CD1D promoter is present in the region of 1738 to –980, which may cover a putative distal promoter according to the cDNA BC027926. Fig. 4B showed that the four constructs with sequential deletion gave no obvious luciferase activity, suggesting that this region may not contain a functional CD1D promoter. Similar results were also obtained in five other cell lines, including K562, CCRF-CEM, Daudi, HT29, and Hela cells (data not shown).

Identification of dual human CD1D promoters

We investigated the region between –985 and +24 to define the core domain of the CD1D promoter using sequentially deleted constructs. Fig. 5A showed that the luciferase activity increased in the deleted constructs and peaked at the region of –213 to +24. The luciferase activity was not detectable using the construct with the insert of –39 to +24. The removal of the region between –985 and –825 increased the luciferase activity, suggesting that this region may contain a cis-suppressing element(s). In contrast, the region of –106 to –82 may contain a cis-enhancing element because the removal of this region dramatically decreased the luciferase activity. The results suggest that a functional CD1D promoter is located between –106 and +24, with its core promoter domain located within the region of –65 to –40. This promoter could be responsible for driving the transcripts with the major start site of –12A based on the results of 5'-RACE and the primer extension. However, the genomic DNA region between –106 and +24 is within the exon 1 of the CD1D mRNA transcripts according to the 5'-RACE results, because the CD1D mRNA covers this region (Figs. 1 and 2). It suggests that CD1D gene must have a second functional promoter responsible for the transcription of the mRNA with a 5'-UTR up to 278 bp according to the results of primer extension shown in Fig. 2.



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  		FIGURE 5. Identification of CD1D dual promoters. A, Sequential deletion to narrow down the promoter region. The core promoter domain was narrowed down to the region of –65 to +24. This region is within the genomic sequence of exon 1. The CD1D 5'-UTR with the longest 5' end was marked as light gray before the region encoding the leader sequence (L) of CD1d. B, Identification of an independent CD1D distal promoter. The region between –665 and –202 had an independent promoter function to drive the expression of luciferase.

 
To test whether CD1D has an independent distal promoter region, three additional constructs covering the region of –665 to –314, –665 to –246, or –665 to –202 were tested for luciferase activity. The results showed that the region between –665 and –202 could drive the expression of luciferase, indicating that this region contains an independent functional promoter (Fig. 5B). The luciferase activity was 10.5 (±1.6) tested for the proximal promoter region (–106 to + 24) and 3.5 (±0.5) for the distal promoter region (–665 to –202), suggesting that the proximal promoter can be more efficient than the distal promoter to generate CD1D transcripts in Jurkat cells. In addition, the luciferase activity was reduced with the removal of the regions covering the major transcription initiation sites detected by 5'-RACE or primer extension. The luciferase activity was abolished when the entire region containing all of the transcription initiation sites was removed (Fig. 5B) in concordance with the known fact that a functional promoter requires the inclusion of a transcription initiation site (32).

Analysis of the genomic DNA sequence covering the dual CD1D promoters

The region covering the proximal and the distal promoter was analyzed for putative cis-acting regulatory elements using the SIGSCAN version 4.05 (33). A putative signal element is defined as any short DNA sequence that has a homology with the published transcriptional elements (33). Fig. 6 showed the representative putative elements that are essential to a TATA-boxless promoter and the representative putative elements that are potentially associated with CD1D gene regulation in immune related cells. The region between –985 and +24 lacks a typical TATA box, which is usually located between 25 and 35 bp 5' upstream of a transcription initiation site (34). The proximal promoter region contains two putative SP1-binding elements, one AP-2-binding element, and one CCAAT-binding element. The distal promoter region contains putative elements of SP-1, AP-2, E-box, Gtx box, and T-Ag, etc. (Fig. 6). These properties contain promoter features associated with housekeeping genes (35, 36, 37). There are other putative transcription factor-binding elements either in a forward or reverse binding orientation in the region between –985 and +24. Some of the interesting putative sites include IFN-{gamma} response element consensus sequence ({gamma}-IRE_CS) (9 sites), NF-IL-6 (5 sites), T cell factor 1 (TCF-1) (15 sites), and LEF-1 (1 site).



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  		FIGURE 6. Representative putative cis-acting regulatory elements were marked for the dual promoter region of human CD1D gene. Three major transcription initiation sites (–235C, –213C, and –12A) were indicated with three arrows.

 
An SP1-binding element determines the function of the human CD1D-proximal promoter

Two putative SP1-binding elements, GGGCGG (–40 to –45) and GGCGGG (–73 to –68), are present in the human proximal promoter region based on DNA sequence analysis. To test whether these two putative SP1-binding elements are functional, two oligos (–52 to –30 and –82 to –60) covering the elements were tested for SP1-binding activity using EMSA. Fig. 7A shows that the nuclear proteins derived from Jurkat cells bound to the oligo –52 to –30 and gave rise to three bands (Fig. 7A, lane 2, a–c), suggesting that at least three separate nuclear proteins bound to the oligo. Band a was present as a smear. This may indicate that a protein with a similar size to SP1 might bind to the oligo. The binding activity of the nuclear proteins to the oligo was blocked with a 100x molar excess of the cold oligo –52 to –30 (Fig. 7, lane 3). The intensity of band a reduced when the cold oligo that had the deletion of GGGCGG (–40 to –45) was present in the above reaction. It is possible that this oligo did not block the binding of the SP1 protein to the element GGGCGG, but blocked the binding of the protein with a similar size to SP1 to the oligo in a position other than GGGCGG. However, this cold oligo with the deletion of SP1 element blocked band c, indicating that the nuclear protein present in band c did not bound to the region associated with the putative SP1 element (Fig. 7, lane 4).



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  		FIGURE 7. Identification of an SP1-binding element in the proximal promoter region of CD1D gene. A, The 32P-labeled oligo (–52 to –30) (lane 1) was reacted with a nuclear extract sample derived from Jurkat cells (10 µg/lane). Three bands (arrows a–c) were detected (lane 2), and were blocked with 100x molar excess of the cold same oligo (lane 3). Only band c was blocked with the cold oligo that had the element GGGCGG deleted (lane 4) or with the cold oligo –82 to –60 (lane 5). Ab specific to SP1 supershifted a signal (lane 6, arrow d) compared with the isotype control (lane 7). B, Functional effects of the putative SP1-binding elements in the proximal promoter region. The position for the predicted SP1 element in the region was marked as a for GGCGGG (–73 to –68) or b for GGGCGG (–40 to –45). The deleted element was marked as filled oval, and the nondeleted as open oval.

 
To understand whether the two putative SP1 elements, GGGCGG (–40 to –45) and GGCGGG (–73 to –68), could be competitive for SP1 protein to bind, we did a cross inhibition using the two oligos (–52 to –30 and –82 to –60) in EMSA. Fig. 7, lane 5, showed that the nuclear proteins bound to the 32P-labeled oligo –52 to –30 shown in bands a and b, which were associated with the nuclear proteins that bound to the element GGGCGG (–40 to –45), were not blocked with a 100x molar excess of the cold oligo –82 to –60 that contains the element GGCGGG (–73 to –68). This indicates that the two elements had different properties for proteins to bind. However, band c was blocked with the cold oligo –82 to –60 presumably due to the presence of a shared sequence AGCGG between these two oligos. The sequence AGCGG was also present in the oligo that had the deletion of SP1 element (–40 to –45) shown in lane 4.

To reveal whether the GGGCGG (–40 to –45)-associated signals in bands a and b were SP1 specific, an Ab specific to SP1 protein was used in the EMSA. A supershifted signal was detected as indicated with arrow d (lane 6), suggesting that SP1 protein bound specifically to the oligo –52 to –30. Some signals left in band a and the signals in bands b and c appeared similar to those detected using an isotype control (lane 7), indicating that unknown proteins other than SP1 also bound to the oligo –52 to –30. A putative transcriptional factor AP-2 element was also present in the oligo –52 to –30 according to DNA sequence analysis. The presence of an Ab to AP-2 did not give a supershifted signal (data not shown), suggesting that the nuclear proteins present in the identified three bands did not include AP-2. SP1 protein did not specifically bind to the second oligo (–82 to –60), because Ab to SP1 did not give a supershifted signal in EMSA (data not shown). However, binding signals including a band corresponding to the size of band c described above did bind to this oligo and could be partially blocked with the cold oligo –52 to –30 (data not shown).

To test the functional role of GGGCGG (–40 to –45) and GGCGGG (–73 to –68) in the human CD1D-proximal promoter, the two elements were deleted either separately or in combination in the construct inserted with the region of –213 to +24 or –82 to +24. The luciferase activity was abolished when the SP1-binding element GGGCGG (–40 to –45) was deleted either in the construct of –213 to +24 or –82 to +24 (Fig. 7B). The luciferase activity reduced from 12.2 (±2.5)-fold to 3.5 (±1.27)-fold when the element GGCGGG (–73 to –68) was deleted in the construct of –213 to +24. This indicates that the SP1-binding element GGGCGG (–40 to –45) is crucial to the function of the proximal promoter, and the element GGCGGG (–73 to –68) is associated with an enhancer in the proximal promoter.

Cell-type sp. act. of the dual promoters

To reveal whether the dual CD1D promoters have a cell-type sp. act., the pGL3 basic constructs inserted with the CD1D genomic region of –213 to +24, –665 to +24, or –665 to –202 were comparatively tested in Jurkat and K562 cells. The region (–213 to +24) covering the proximal promoter gave a much higher luciferase activity in Jurkat cells than in K562 cells, whereas it was in reverse for the region (–665 to –202) covering the distal promoter (Fig. 8). The luciferase activity tested in Jurkat cells for the region (–665 to +24) covering both of the promoters was not as high as that of the region covering the proximal promoter, indicating that the activity for the two promoters may be not simply added up in the cells. The data suggest that the two promoters could have a cell-type sp. act. in driving CD1D gene expression.



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  		FIGURE 8. Cell-type sp. act. of the dual CD1D promoters. The luciferase activity for the genomic region of –213 to +24 covering the proximal promoter or –665 to –202 covering the distal promoter differs between Jurkat cells and K562 cells. A predicted LEF-1 element (open oval) was mutated (gray oval) and tested in Jurkat and K562 cells. The mutation of the LEF-1 element enhanced the luciferase activity.

 
To support the cell-type sp. act. of the dual promoters, five putative cis-acting regulatory elements located in the distal promoter region were mutated in the constructs of pGL3 basic vector inserted with the region of –665 to +24 or –665 to –202. These five putative elements include an E-box (–607 to –612), a CAAT box (–570 to –573), a second E-box (–508 to –513), a CATAAA box (–335 to –340), and an LEF-1 element (–294 to –288), as marked in Fig. 6. The introduction of the mutations in the two putative E-boxes, the CAAT box, and the CATAAA box did not apparently affect the luciferase activity in either of the two constructs (data not shown). However, the removal of the putative LEF-1 element by mutation enhanced the luciferase activity especially in K562 cells for both of the constructs (Fig. 8). The results support the cell-type sp. act. of the dual promoters and indicate that the putative LEF-1 element plays a negative regulatory role in CD1D gene expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results provide the first description about the characteristics of the human CD1D promoter. We demonstrated that human CD1D gene has two functional TATA boxless promoters located within the region of –985 to +24. The distal promoter is located within the region of –665 to –202, and the proximal promoter is located within the region of –106 to +24 with the nucleotide A of the translational initiation codon defined as +1. It is unusual for the proximal CD1D promoter to locate at the genomic region corresponding to the 5'-UTR of the CD1D transcripts. However, we presented four lines of evidence to support the presence of these two TATA boxless promoters. We showed that human CD1D gene has multiple transcription initiation sites located in two separate regions using the method of 5'-RACE and primer extension. Three of the identified 5' ends (–235C, –213C, and –12A) of CD1D transcripts were detected to be identical using the method of 5'-RACE and the primer extension. These three ends were the major transcription initiation sites for CD1D gene. The clones with different 5' ends derived using 5'-RACE may include those derived from truncated mRNA of CD1D transcripts. We then showed that two separate genomic regions (–665 to –202 and –106 to +24) containing the respective major transcription initiation site (–213C or –12A) had the promoter activity to drive the expression of luciferase in a promoterless reporter gene vector system. We found that a functional SP1 element (GGGCGG) plays a crucial role in the proximal promoter region, and the mutation of a putative LEF-1 element in the distal promoter region enhanced the distal promoter activity. Finally, we showed that the two promoters apparently have a cell-type sp. act. tested in Jurkat cells and K562 cells.

SP1-binding element plays an important role in the TATA boxless CD1D-proximal promoter. The proximal CD1D promoter is located within the region of –106 to +24, with its core domain located within the region of –65 to –40. This region does not contain a typical TATA box, which is usually located between 25 and 35 bp 5' upstream of transcription initiation site (34). SP1 plays an important role for RNA polymerase II to bind to the transcription initiation site in TATA boxless promoters and is associated with multiple transcription initiation sites (35, 36, 37). The functional SP1-binding element GGGCGG (–40 to –45) is located 27 bp 5' upstream of the major transcription initiation site (–12A) of the CD1D gene. Deletion of this SP1-binding element abolished the proximal promoter activity, indicating that this SP1-binding element is crucial to the proximal promoter. The core domain of the proximal promoter is located within the region of –65 to –40, which contains the functional SP1-binding element, suggesting that SP1 protein binding to this element appears sufficient for the basal proximal promoter activation. The second predicted SP1-binding element GGCGGG (–73 to –68) is not functional, but is associated with an enhancing element for the proximal promoter because the deletion of this element reduced the proximal promoter activity.

There is no TATA box nor SP1-binding element located at 25–35 bp 5' upstream of the transcription initiation sites (–278T, –235C, –213C, and –196A) in the distal promoter. A TATA boxlike element CATAAA (–340 to –335) was tested to be not functional using mutagenesis analysis. Unknown initiators that can carry out a similar function as TATA box and are usually contained between nucleotides –3 and +5 relative to the initiation site (38) may be present in the distal promoter. These elements remain to be elucidated to understand how the distal promoter uses the multiple transcription initiation sites to drive the transcripts of human CD1D gene with different lengths of 5'-UTR.

The two CD1D promoters apparently have a cell-type sp. act. because the proximal promoter was more active in Jurkat cells than in K562 cells, whereas the distal promoter was in reserve in these two cell lines. Jurkat is a T cell leukemia cell line and K562 is an erythroid leukemia cell line, which has detectable CD1D mRNA expression (data not shown). It is expected that they would have different cellular factors in driving CD1D gene expression. Study of these two cell lines may help to understand the cell-type-specific regulatory factors controlling CD1D gene expression. We examined two predicted E-boxes, a CATA box, and a CATAAA box in the distal promoter region by mutagenesis analysis and tested in these two cell lines. However, the removal of these four putative elements by mutation did not affect the promoter function of the distal promoter. It suggests that these four predicted elements are not functional in the regulation of CD1D distal promoter or require the presence of an unknown cellular condition.

The sequence of the published cDNA (BC027926) indicates the presence of a putative distal transcription initiation site at the genomic DNA position of –1155. This cDNA has the genomic region of –940 to –283 spliced out as intron 1a. However, we could not demonstrate the presence of a functional promoter for this transcript after having tested the region of –2579 to –980 in Jurkat cells and five other cell lines with or without the surface CD1d expression. The activation of this putative promoter may require a specific cellular condition (39, 40) if such a promoter exists.

Both the distal and the proximal CD1D promoter apparently have the cis-acting suppressing and/or enhancing elements. The region between –985 and –825 can contain a cis-suppressing element, because the removal of this region increased the luciferase activity from ~4- to 10-fold over the background activity derived from the pGL3 basic vector. It remains to be defined whether the suppressor in this region is functionally associated with the distal and/or the proximal promoter. By contrast, the region between –82 and –106 may contain an enhancing element because the removal of this region decreased the proximal promoter activity. In addition, an unknown nuclear protein derived from Jurkat cells may bind to the sequence AGCGG, which is present in both the oligo –52 to –30 and the oligo –82 to –60 tested in EMSA. This presumption was based on the evidence that a band (c in Fig. 7A) that is not associated with the putative SP1 elements in the above two oligos was detected in EMSA. This band detected in the reaction between the nuclear protein and the 32P-labeled oligo –52 to –30 was completely blocked with the cold oligo –82 to –60 (100x molar excess). The same amount of the cold oligo –52 to –30 partially blocked the corresponding size of a band detected in the reaction between the nuclear protein and the 32P-labeled oligo –82 to –60 (data not shown). The partial blockage could be due to a different binding affinity between the unknown protein and the sequence AGCGG in these two oligos. This is because the sequence AGCGG is located at the end of the oligo –52 to –30, but in the middle of the oligo –82 to –60. This could result in a comparative lower affinity for the protein to bind to the oligo –52 to –30 than to the oligo –82 to –60. We are in the process of confirming the above presumption to define the functional role of the element AGCGG and its binding nuclear protein in CD1D gene regulation.

The analysis of the characteristics of the CD1D promoter will help to reveal the regulatory elements involved in the CD1d expression and/or tissue distribution. Multiple putative TCF-1 and/or LEF-1 elements are present in the CD1D promoter region, suggesting that TCF-1 and/or LEF-1 could be involved in the CD1D gene regulation. This presumption was supported by the evidence that mutation of a putative LEF-1 element located in the distal promoter region enhanced the luciferase activity. It indicates that this putative LEF-1 element plays a negative regulatory role in controlling CD1D promoter. TCF-1 is expressed primarily in the T-lineage lymphocytes, and LEF-1 is expressed primarily in mature and immature T cells and in immature B cells in mice (41). Activated peripheral blood T cells express CD1d (15, 22). We are in the process of confirming whether LEF-1 could bind to this putative element and revealing the precise role of TCF-1/LEF-1 in CD1D gene expression. The results would help to understand their functional effects in CD1D gene regulation. Other cellular factors such as IFN-{gamma} and NF-IL-6 may also be involved in the CD1D gene regulation. Multiple copies of putative {gamma}-IRE_CS are present in the CD1D promoter region. It is known that IFN-{gamma} can regulate the CD1D gene expression (15, 20, 21, 22), suggesting that functional {gamma}-IRE_CS element(s) may play a role in the CD1D gene regulation. There are five putative NF-IL-6 response elements in the CD1D promoter region. NF-IL-6 is a nuclear protein, which contributes to the activation of IL-6 in response to IL-1 (42). NF-IL-6 has been shown to be an important nuclear target in the IL-6 signaling pathway in rodents (43). IL-1 can up-regulate CD1a expression in human monocytes (44, 45), but it is unclear regarding CD1d expression. The recent identification of a subset of B cells expressing CD1d in association with an up-regulation of IL-1 in mice (46) suggests that CD1d may be induced by NF-IL-6 through the up-regulated IL-1. However, this postulation needs to be confirmed experimentally.

Our study revealed that human CD1D gene is controlled by at least two cell-type-specific promoters, which use multiple transcription initiation sites. The proximal promoter is located within a region of 213 bp 5' upstream of the CD1D coding region. An SP1-binding element is crucial to the function of the proximal promoter, and a putative LEF-1 element plays a negative regulatory role in the distal promoter. The analysis on the CD1D promoter region suggests that IFN-{gamma} and NF-IL-6 may be involved also in the regulation of CD1D gene expression. Our results would help to elucidate the regulatory factors that control CD1d expression and tissue distribution to understand the cross-regulation between CD1d and NK T cells in host immune defense, suppression of autoimmunity, and antitumor immunity.


   Acknowledgments
 
We thank Drs. Seth Pincus and Michael Lan for their useful suggestions and discussions, and Dr. Mary Breslin for critical reading of the manuscript. We also thank David Ricks for partial technical assistance.


   Footnotes
 
1 This study was supported by intramural research grants to Q.-Y.C. from the Children’s Hospital of New Orleans and Department of Pediatrics, Louisiana State University Health Science Center. Back

2 Address correspondence and reprint requests to Dr. Qiao-Yi Chen, Research Institute for Children, 200 Henry Clay Avenue, New Orleans, LA 70118. E-mail address: qchen{at}chnola-research.org Back

3 Abbreviations used in this paper: CD1D, gene encoding CD1d molecule; EST, expressed sequence tag; {gamma}-IRE_CS, IFN-{gamma} response element consensus sequence; LEF-1, lymphoid enhancer-binding factor 1; TCF-1, T cell factor 1; UTR, untranslated region. Back

Received for publication August 14, 2003. Accepted for publication February 13, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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From The Cover: Two CD1 genes map to the chicken MHC, indicating that CD1 genes are ancient and likely to have been present in the primordial MHC
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