This information is current as
of July 28, 2017.
Human CD1D Gene Has TATA Boxless Dual
Promoters: An SP1-Binding Element
Determines the Function of the Proximal
Promoter
Qiao-Yi Chen and Natalie Jackson
J Immunol 2004; 172:5512-5521; ;
doi: 10.4049/jimmunol.172.9.5512
http://www.jimmunol.org/content/172/9/5512
Subscription
Permissions
Email Alerts
This article cites 46 articles, 16 of which you can access for free at:
http://www.jimmunol.org/content/172/9/5512.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
References
The Journal of Immunology
Human CD1D Gene Has TATA Boxless Dual Promoters: An
SP1-Binding Element Determines the Function of the Proximal
Promoter1
Qiao-Yi Chen2*† and Natalie Jackson*
D1d molecules are noncovalently associated with ␤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 –11). Both human and mouse CD1d can present
C
*Research Institute for Children, Children’s Hospital, New Orleans, LA 70118; and
†
Department of Pediatrics, Louisiana State University Health Science Center, New
Orleans, LA 70112
Received for publication August 14, 2003. Accepted for publication February
13, 2004.
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.
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.
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:
[email protected]
3
Abbreviations used in this paper: CD1D, gene encoding CD1d molecule; EST,
expressed sequence tag; ␥-IRE_CS, IFN-␥ response element consensus sequence;
LEF-1, lymphoid enhancer-binding factor 1; TCF-1, T cell factor 1; UTR, untranslated region.
Copyright © 2004 by The American Association of Immunologists, Inc.
␣-galactosylceramide to NK T cells, which express a restricted
range of TCRs bearing a single invariant V␣ chain (V␣14J␣281 in
mice and V␣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 –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 (17–19). 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-␥ (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
0022-1767/04/$02.00
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
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 5ⴕ 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 ⴚ106 to ⴙ24, and the distal promoter is located within the region of ⴚ665 to
ⴚ202 with the A of the translational start codon defined as ⴙ1. The longest 5ⴕ-untranslated region derived from 5ⴕ-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-␥,
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. The Journal of Immunology,
2004, 172: 5512–5521.
The Journal of Immunology
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
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 ␮g/ml streptomycin (Invitrogen). The cells were
cultured at 37°C in a humidified atmosphere with 5% CO2.
RT-PCR
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 ⫻ 106) were exposed to 20 ␮l of the Ab in PBS, pH 7.4, containing 0.1%
BSA, in a total volume of 100 ␮l 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 ␮l 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 ␮g
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 ␮l 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 (1–9), and the common reverse primer for the
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
The expression of CD1D mRNA in Jurkat cells was detected using RTPCR. 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 ␮g 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.
5513
5514
DUAL HUMAN CD1D PROMOTERS
Table I. Partial primers used in the PCR
N
1
2
3
4
5
6
7
8
9
10
11
12
Primers
5⬘-CTA
5⬘-CTA
5⬘-CTA
5⬘-CTA
5⬘-CTA
5⬘-CTA
5⬘-CTA
5⬘-CTA
5⬘-CTA
5⬘-CAT
5⬘-CAG
5⬘-CAT
AGA
AGA
AGA
AGA
AGA
AGA
AGA
AGA
AGA
GGC
TGC
GGC
GCT
GCT
GCT
GCT
GCT
GCT
GCT
GCT
GCT
TAG
TAG
TAG
CAG
CAG
CCT
CTC
CAG
CTG
CGA
CAG
CCG
CTG
CAC
CTG
Position
AG GCG CAT ATT TGG A-3⬘
GAG GAA AGA GAG GCT-3⬘
GCT TCC AAA ACA TAA-3⬘
TTT GCA GCT CGC ACA-3⬘
CAA ACC GCC GGC AAG CC-3⬘
GCA CCC AGC GGA AAG G-3⬘
GCT GAG CGG CGG GG-3⬘
AAG AGT GCG CAG GTC A-3⬘
CGC AGC GGC GCT CCG-3⬘
GGA CGC TTT ACA ACC ATT-3⬘
TTC AGT AGG TTT CAC-3⬘
CAA AGA GGT CGG G-3⬘
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 ⫻ 105 per well
in a 24-well plate and cotransfected with 0.8 ␮g 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 ␮g/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 doublestranded oligonucleotides, which (1.75 pmol) were end labeled with 5 ␮Ci
of [␥-32P]ATP (PerkinElmer Life and Analytical Sciences) using T4
polynucleotide kinase (Promega). The labeled oligonucleotides were separated from the free [␥-32P]ATP using a column (Bio-Rad, Hercules, CA),
according to the manufacturer’s instructions. A total of 10 ␮g of nuclear
extract derived from Jurkat cells (Active Motif, Carlsbad, CA) was incubated with 1.75 pmol (⬃100⫻) of cold oligo or cold oligo with the deletion
of the predicted SP1 element, or 2 ␮l of Ab to SP1 or isotype control
(Active Motif) in EMSA-binding buffer (Promega) for 10 min at room
temperature, then the [␥-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
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⬘
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
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 –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.
⫺825 to ⫺808
⫺665 to ⫺648
⫺354 to ⫺336
⫺213 to ⫺196
⫺165 to ⫺146
⫺106 to ⫺88
⫺82 to ⫺67
⫺65 to ⫺48
⫺39 to ⫺23
⫺314 to ⫺333
⫺246 to ⫺263
⫺202 to ⫺220
The Journal of Immunology
5515
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.
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.
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.
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.
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
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.
5516
DUAL HUMAN CD1D PROMOTERS
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).
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.
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
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
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.
The Journal of Immunology
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 (35–37). 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-␥ response element consensus sequence (␥-IRE_CS) (9 sites), NF-IL-6 (5 sites), T cell
factor 1 (TCF-1) (15 sites), and LEF-1 (1 site).
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
100⫻ 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).
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.
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
⫺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.
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).
5517
5518
DUAL HUMAN CD1D PROMOTERS
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 100⫻ 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).
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.
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
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 ␮g/lane). Three bands (arrows a– c) were detected (lane 2), and were blocked with
100⫻ 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.
The Journal of Immunology
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
Discussion
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 (35–37). 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 SP1binding 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.
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
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.
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.
5519
5520
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-␥ 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.
References
1. Porcelli, S. A., and R. L. Modlin. 1999. The CD1 system: antigen-presenting
molecules for T cell recognition of lipids and glycolipids. Annu. Rev. Immunol.
17:297.
2. Calabi, F., J. M. Jarvis, L. Martin, and C. Milstein. 1989. Two classes of CD1
genes. Eur. J. Immunol. 19:285.
3. Hughes, A. L. 1991. Evolutionary origin and diversification of the mammalian
CD1 antigen genes. Mol. Biol. Evol. 8:185.
4. Yu, C. Y., and C. Milstein. 1989. A physical map linking the five CD1 human
thymocyte differentiation antigen genes. EMBO J. 8:3727.
5. Albertson, D. G., R. Fishpool, P. Sherrington, E. Nacheva, and C. Milstein. 1988.
Sensitive and high resolution in situ hybridization to human chromosomes using
biotin labelled probes: assignment of the human thymocyte CD1 antigen genes to
chromosome 1. EMBO J. 7:2801.
6. Balk, S. P., P. A. Bleicher, and C. Terhorst. 1989. Isolation and characterization
of a cDNA and gene coding for a fourth CD1 molecule. Proc. Natl. Acad. Sci.
USA 86:252.
7. Brossay, L., and M. Kronenberg. 1999. Highly conserved antigen-presenting
function of CD1d molecules. Immunogenetics 50:146.
8. Bradbury, A., K. T. Belt, T. M. Neri, C. Milstein, and F. Calabi. 1988. Mouse
CD1 is distinct from and co-exists with TL in the same thymus. EMBO J. 7:3081.
9. Bradbury, A., F. Calabi, and C. Milstein. 1990. Expression of CD1 in the mouse
thymus. Eur. J. Immunol. 20:1831.
10. Balk, S. P., P. A. Bleicher, and C. Terhorst. 1991. Isolation and expression of
cDNA encoding the murine homologues of CD1. J. Immunol. 146:768.
11. Porcelli, S. A. 1995. The CD1 family: a third lineage of antigen-presenting molecules. Adv. Immunol. 59:1.
12. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno,
R. Nakagawa, H. Sato, E. Kondo, et al. 1997. CD1d-restricted and TCR-mediated
activation of V␣14 NKT cells by glycosylceramides. Science 278:1626.
13. Joyce, S. 2001. CD1d and natural T cells: how their properties jump-start the
immune system. Cell. Mol. Life Sci. 58:442.
14. Canchis, P. W., A. K. Bhan, S. B. Landau, L. Yang, S. P. Balk, and
R. S. Blumberg. 1993. Tissue distribution of the non-polymorphic major histocompatibility complex class I-like molecule, CD1d. Immunology 80:561.
15. Exley, M., J. Garcia, S. B. Wilson, F. Spada, D. Gerdes, S. M. Tahir, K. T. Patton,
R. S. Blumberg, S. Porcelli, A. Chott, and S. P. Balk. 2000. CD1d structure and
regulation on human thymocytes, peripheral blood T cells, B cells and monocytes. Immunology 100:37.
16. Blumberg, R. S., C. Terhorst, P. Bleicher, F. V. McDermott, C. H. Allan,
S. B. Landau, J. S. Trier, and S. P. Balk. 1991. Expression of a nonpolymorphic
MHC class I-like molecule, CD1D, by human intestinal epithelial cells. J. Immunol. 147:2518.
17. McMichael, A. J., J. R. Pilch, G. Galfre, D. Y. Mason, J. W. Fabre, and
C. Milstein. 1979. A human thymocyte antigen defined by a hybrid myeloma
monoclonal antibody. Eur. J. Immunol. 9:205.
18. Amiot, M., A. Bernard, B. Raynal, W. Knapp, C. Deschildre, and L. Boumsell.
1986. Heterogeneity of the first cluster of differentiation: characterization and
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
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
(100⫻ 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-␥ and NF-IL-6 may also be involved in the CD1D
gene regulation. Multiple copies of putative ␥-IRE_CS are present
in the CD1D promoter region. It is known that IFN-␥ can regulate
the CD1D gene expression (15, 20 –22), suggesting that functional
␥-IRE_CS element(s) may play a role in the CD1D gene regulation. There are five putative NF-IL-6 response elements in the
DUAL HUMAN CD1D PROMOTERS
The Journal of Immunology
19.
20.
21.
22.
23.
24.
25.
27.
28.
29.
30.
31.
32. Hampsey, M. 1998. Molecular genetics of the RNA polymerase II general transcriptional machinery. Microbiol. Mol. Biol. Rev. 62:465.
33. Prestridge, D. S. 1991. SIGNAL SCAN: a computer program that scans DNA
sequences for eukaryotic transcriptional elements. Comput. Appl. Biosci. 7:203.
34. McKnight, S. L., and R. Kingsbury. 1982. Transcriptional control signals of a
eukaryotic protein-coding gene. Science 217:316.
35. Melton, D. W., D. S. Konecki, J. Brennand, and C. T. Caskey. 1984. Structure,
expression, and mutation of the hypoxanthine phosphoribosyltransferase gene.
Proc. Natl. Acad. Sci. USA 81:2147.
36. Reynolds, G. A., S. K. Basu, T. F. Osborne, D. J. Chin, G. Gil, M. S. Brown,
J. L. Goldstein, and K. L. Luskey. 1984. HMG CoA reductase: a negatively
regulated gene with unusual promoter and 5⬘ untranslated regions. Cell 38:275.
37. Valerio, D., M. G. Duyvesteyn, B. M. Dekker, G. Weeda, T. M. Berkvens,
L. van der Voorn, H. van Ormondt, and A. J. van der Eb. 1985. Adenosine
deaminase: characterization and expression of a gene with a remarkable promoter. EMBO J. 4:437.
38. Smale, S. T., M. C. Schmidt, A. J. Berk, and D. Baltimore. 1990. Transcriptional
activation by Sp1 as directed through TATA or initiator: specific requirement for
mammalian transcription factor IID. Proc. Natl. Acad. Sci. USA 87:4509.
39. Lauring, J., and M. S. Schlissel. 1999. Distinct factors regulate the murine RAG-2
promoter in B- and T-cell lines. Mol. Cell. Biol. 19:2601.
40. Chiu, I. M., K. Touhalisky, and C. Baran. 2001. Multiple controlling mechanisms
of FGF1 gene expression through multiple tissue-specific promoters. Prog. Nucleic Acid Res. Mol. Biol. 70:155.
41. Waterman, M. L., and K. A. Jones. 1990. Purification of TCF-1␣, a T-cellspecific transcription factor that activates the T-cell receptor C␣ gene enhancer in
a context-dependent manner. New Biol. 2:621.
42. Akira, S., H. Isshiki, T. Sugita, O. Tanabe, S. Kinoshita, Y. Nishio, T. Nakajima,
T. Hirano, and T. Kishimoto. 1990. A nuclear factor for IL-6 expression (NF-IL6)
is a member of a C/EBP family. EMBO J. 9:1897.
43. Poli, V., F. P. Mancini, and R. Cortese. 1990. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper
proteins related to C/EBP. Cell 63:643.
44. Winning, T., E. Gemmell, B. Polak, N. W. Savage, G. J. Seymour, and
L. J. Walsh. 1996. Expression of CD1a monocytes cultured with supernatants
from periodontally diseased gingival epithelial cells. Oral Dis. 2:247.
45. Walsh, L. J., and G. J. Seymour. 1988. Interleukin 1 induces CD1 antigen expression on human gingival epithelial cells. J. Invest. Dermatol. 90:13.
46. Mizoguchi, A., E. Mizoguchi, H. Takedatsu, R. S. Blumberg, and A. K. Bhan.
2002. Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d up-regulation. Immunity 16:219.
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
26.
epitopic mapping of three CD1 molecules on normal human thymus cells. J. Immunol. 136:1752.
Kahn-Perles, B., J. Wietzerbin, D. H. Caillol, and F. Lemonnier. 1985. Delineation of three subsets of class I human T antigens (HTA) on Molt-4 cells: serologic
and regulatory relationship to HLA class I antigens. J. Immunol. 134:1759.
Colgan, S. P., V. M. Morales, J. L. Madara, J. E. Polischuk, S. P. Balk, and
R. S. Blumberg. 1996. IFN-␥ modulates CD1d surface expression on intestinal
epithelia. Am. J. Physiol. 271:C276.
Bonish, B., D. Jullien, Y. Dutronc, B. B. Huang, R. Modlin, F. M. Spada,
S. A. Porcelli, and B. J. Nickoloff. 2000. Overexpression of CD1d by keratinocytes in psoriasis and CD1d-dependent IFN-␥ production by NK-T cells. J. Immunol. 165:4076.
Salamone, M. C., G. A. Rabinovich, A. K. Mendiguren, G. V. Salamone, and
L. Fainboim. 2001. Activation-induced expression of CD1d antigen on mature T
cells. J. Leukocyte Biol. 69:207.
Ulanova, M., M. Torebring, S. A. Porcelli, U. Bengtsson, J. Magnusson,
O. Magnusson, X. P. Lin, L. A. Hanson, and E. Telemo. 2000. Expression of
CD1d in the duodenum of patients with cow’s milk hypersensitivity. Scand.
J. Immunol. 52:609.
Tsuneyama, K., M. Yasoshima, K. Harada, K. Hiramatsu, M. E. Gershwin, and
Y. Nakanuma. 1998. Increased CD1d expression on small bile duct epithelium
and epithelioid granuloma in livers in primary biliary cirrhosis. Hepatology
28:620.
Spada, F. M., F. Borriello, M. Sugita, G. F. Watts, Y. Koezuka, and S. A. Porcelli.
2000. Low expression level but potent antigen presenting function of CD1d on
monocyte lineage cells. Eur. J. Immunol. 30:3468.
Woolfson, A., and C. Milstein. 1994. Alternative splicing generates secretory
isoforms of human CD1. Proc. Natl. Acad. Sci. USA 91:6683.
Jenkinson, H. J., S. D. Wainwright, K. L. Simpson, A. C. Perry, P. Fotiadou, and
C. H. Holmes. 1999. Expression of CD1D mRNA transcripts in human choriocarcinoma cell lines and placentally derived trophoblast cells. Immunology
96:649.
Cramer, P., C. G. Pesce, F. E. Baralle, and A. R. Kornblihtt. 1997. Functional
association between promoter structure and transcript alternative splicing. Proc.
Natl. Acad. Sci. USA 94:11456.
Chen, Q. Y., N. Jackson, A. Vargas, S. Chalew, J. Rao, M. Batzer, M. S. Lan,
Y. H. Chang, M. Mokhashi, and D. Liu. 2003. Identification of three genomic
haplotypes 5⬘ to the human CD1D gene and their distribution in four ethnic
groups. Tissue Antigens 62:442.
Den Dunnen, J. T., and S. E. Antonarakis. 2001. Nomenclature for the description
of human sequence variations. Hum. Genet. 109:121.
Suzuki, Y., K. Yoshitomo-Nakagawa, K. Maruyama, A. Suyama, and S. Sugano.
1997. Construction and characterization of a full length-enriched and a 5⬘-endenriched cDNA library. Gene 200:149.
5521