Regulation of Human DAP10 Gene
Expression in NK and T Cells by Ap-1
Transcription Factors
This information is current as
of June 15, 2017.
Alina I. Marusina, Steven J. Burgess, Ishani Pathmanathan,
Francisco Borrego and John E. Coligan
J Immunol 2008; 180:409-417; ;
doi: 10.4049/jimmunol.180.1.409
http://www.jimmunol.org/content/180/1/409
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References
The Journal of Immunology
Regulation of Human DAP10 Gene Expression in NK and T
Cells by Ap-1 Transcription Factors1
Alina I. Marusina,2 Steven J. Burgess,2 Ishani Pathmanathan, Francisco Borrego,
and John E. Coligan3
T
he human NKG2D gene is located between the CD94 and
NKG2F genes within the NK gene complex on chromosome 12 and encodes a type II protein expressed by NK
cells, ␥␦ T cells, and CD8⫹ ␣␤ T cells (1, 2). NKG2D itself does
not possess signaling capacity. In humans, NKG2D exists on the
cell surface in complex with the DAP10 adaptor protein that contains a YxxM motif that, upon tyrosine phosphorylation after
NKG2D/DAP10 ligation, couples the receptor complex to the
PI3K/Grb2-Vav pathway (3, 4). Murine NKG2D is encoded by
two splice variants (5). The long isoform (mNKG2D-L) associates
only with DAP10, whereas the short isoform (mNKG2D-S) associates with DAP10 or DAP12 (5, 6). The pairing of NKG2D with either
the DAP10 or DAP12 adaptor proteins is a unique feature for murine
NKG2D, as there is no evidence for the short form of human NKG2D
(7). Moreover, the artificial creation of a short form of a human
NKG2D showed that it failed to associate with DAP12 (8).
NKG2D/DAP10 is an activating receptor that can trigger NK
cells and costimulate CD8⫹ T cells (9, 10). The ligands for human
NKG2D/DAP10 are structurally diverse and include the MHC
class I chain-related (MIC)4 proteins A and B and the UL16-binding proteins 1 through 4 (9, 11, 12). These ligands are usually
Receptor Cell Biology Section, Laboratory of Immunogenetics, National Institute of
Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852
Received for publication October 23, 2007. Accepted for publication October
23, 2007.
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 work was supported by funds from the Intramural program of the National
Institute of Allergy and Infectious Diseases.
2
absent on normal cells, but are often up-regulated on infected or
cancer cells, or by cells otherwise undergoing stress (13). The
expression of NKG2D/DAP10 ligands by such stressed cells
makes them susceptible to killing by NK and CD8⫹ T cells (9, 14,
15). This killing can be inhibited by blocking the NKG2D receptor
with mAb against NKG2D or its ligands (16). Recent studies have
revealed that tumors can evade immune recognition by NKG2D
bearing NK and CD8⫹ T cells by shedding soluble NKG2D ligands that mediate NKG2D/DAP10 internalization and degradation (17–19). In addition, elevated levels of TGF-␤ in cancer patients have been shown to inhibit the expression of NKG2D/
DAP10, thereby impairing NK cell cytotoxicity (20). In contrast to
the positive aspects of NKG2D/DAP10 ligand recognition for the
immune response to diseased cells, NKG2D/DAP10 ligand recognition can also have adverse effects for the host, particularly in the case
of certain autoimmune diseases. Inappropriate activation of NK/
CD8⫹ T cells by NKG2D/DAP10 ligation has been implicated in the
pathology of celiac disease (21), rheumatoid arthritis (22), and autoimmune diabetes in NOD mice (23). Thus, the modulation of
NKG2D/DAP10 receptor expression or blockade of NKG2D/DAP10
signaling has therapeutic implications for a variety of disease states.
Because of these observations, the determination of the factors
that regulate expression of NKG2D/DAP10 receptors is an area of
significant interest. In this study, we focused on the transcriptional
regulation of the human DAP10 gene. We identified multiple transcriptional start sites of the human DAP10 gene and localized its
promoter to a segment upstream of these sites. We demonstrated
that TCR stimulation up-regulates DAP10 promoter activity and
show that the Ap-1 proteins c-Fos and c-Jun play a key role in this
up-regulation. We also show that these transcription factors are
involved in regulating DAP10 expression by NK cells.
A.I.M. and S.J.B. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. John E. Coligan, Receptor Cell
Biology Section, Laboratory of Immunogenetics, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Twinbrook II, Room 205, 12441
Parklawn Drive, Rockville, MD 20852-1742. E-mail address: [email protected]
4
Abbreviations used in this paper: MIC, MHC class I chain-related; ChIP, chromatin
immunoprecipitation.
www.jimmunol.org
Materials and Methods
Abs and flow cytometry
To measure NKG2D cell surface expression, we used PE-conjugated antiNKG2D (R&D Systems). PE-conjugated isotype-matched control mAb
(eBioscience) was used to monitor background staining levels. Flow
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Human NKG2D/DAP10 is an activation receptor expressed by NK and subsets of T cells, whose ligands include MHC class I
chain-related (MIC) protein A and protein B and UL16-binding proteins that are often up-regulated by stress or pathological
conditions. DAP10 is required for NKG2D/DAP10 cell surface expression and signaling capacity. Little is known about the
mechanisms that regulate DAP10 gene expression. We describe the existence of multiple transcriptional start sites upstream of
DAP10 exon 1 and identify the location of the basic promoter upstream of these starting sites. The promoter is active in NK and
CD8ⴙ T cells, but not in CD4ⴙ T cells. We demonstrate TCR-mediated up-regulation of DAP10 transcription and found that a
40 bp region within the DAP10 promoter, containing an Ap-1 binding site, is largely responsible for this increased transcription.
Using pull-down and chromatin immunoprecipitation assays, we show that the DAP10 promoter interacts with Ap-1 transcription
factors in primary CD8ⴙ T and NK cells in vitro and in vivo. Overexpression of c-Jun or c-Fos in NK and T cells led to enhanced
DAP10 promoter activity and DAP10 protein expression. Taken together, our data indicate that Ap-1 is an important transcription
factor for regulating DAP10 gene expression in human NK and T cells, and that Ap-1 plays a key role in the transactivation of
DAP10 promoter following TCR stimulation. The Journal of Immunology, 2008, 180: 409 – 417.
410
cytometric analyses were performed on a FACSort cytofluorometer (BD Immunocytochemistry Systems). Purified anti-CD3 (clone UCHT1; eBioscience)
was used for cell stimulations. Anti-DAP10 (clone N-17; Santa Cruz Biotechnology), anti-c-Fos (Active Motif) and anti-␤-actin (Sigma-Aldrich) Ab were
used for Western blot analyses.
Cell isolation and culture conditions
RNA isolation, 5⬘-RACE, and RT-PCR
Before RNA isolation, cells were stored in RNAlater (Ambion) to prevent
RNA degradation. Total RNA was isolated using the RNAqueous-4PCR
kit (Ambion), including DNase treatment, according to the manufacturer’s
instructions. The 5⬘-RACE was conducted with the First Choice RLMRACE kit (Ambion), according to the manufacturer’s instructions. Primers
were 5⬘-RACE inner DAP10 5⬘-gat ggatcatggtggactgg-3⬘ and 5⬘-RACE
outer DAP10 5⬘-agagagagggacccacatcc-3⬘.
Synthesis of cDNA was performed with the iScript cDNA synthesis kit
(Bio-Rad). Samples were analyzed by real-time PCR (DNA Engine Opticon 2; MJ Research) using the iQ SYBR Green PCR kit (Bio-Rad). A
melting curve was performed at the end of each run to verify that there was
a single amplification product and a lack of primer dimers. Standard
curves, obtained using a serial 10-fold dilution of human primary CD8⫹ T
cell cDNA, were generated to determine the level of each amplified transcript,
and all samples were normalized to the amount of 18 S rRNA transcript
present in each sample. Primers for 18 S rRNA were from QuantiTect Primer
Assay for 18 S rRNA (Qiagen). Primers for DAP10 were (forward) 5⬘-tccat
ctgggtcacatcctcttcc-3⬘ and (reverse) 5⬘-gagtgatgatctctctcctggagtcgtctgagctg-3⬘.
Real-time PCRs were performed in triplicate.
lysates was measured with a Veritas Microplate Luminometer (Promega).
All luciferase assays were performed at least three times, each in triplicate.
The dual luciferase double reporter assay system and substrates were purchased from Promega.
For overexpression studies, 5 ⫻ 106 NKL or Jurkat T cells were transiently transfected with 5 ␮g of the pcDNA3, pcDNA3/c-Jun, or pcDNA3/
c-Fos plasmids together with 3 ␮g of the reporter plasmid and 0.15 ␮g of
the pRL-nul Renilla control vector using the Amaxa nucleofection system.
Cells were harvested at 24 h posttransfection, lysed, and analyzed using
luciferase assays.
Western blot analysis of DAP10 protein levels
Whole cell extracts were subjected to SDS-PAGE, followed by immunoblotting as previously described (24). Cells transfected with c-Jun or c-Fos
expressing plasmids were harvested at 36 h posttransfection, lysed, and
analyzed for protein expression by Western blot. Band intensity was quantified using UN-SCAN-IT gel software (Silk Scientific).
Pull-down assay
Primary CD8⫹ T cells stimulated with plate-bound anti-CD3 mAb (0.5
␮g/ml) for 16 h were harvested, washed, and lysed using a Nuclear Extract
kit (Active Motif). Nuclear extracts were reconstituted in binding buffer
containing 10 mM Tris-HCl, 50 mM NaCl, 1 mM MgCl, 5% glycerol, 0.5
mM EDTA, 5 mM DTT, 0.5% Nonidet P-40, 1% phosphatase inhibitor
mixture 1 (Sigma-Aldrich), 1% phosphatase inhibitor mixture 2 (SigmaAldrich), and 1% protease inhibitor mixture (Sigma-Aldrich). Cell lysates
were precleared by incubation with streptavidin-agarose beads (Dynabeads
M-280; Invitrogen Life Technologies) for 1 h at 4°C. Cell lysates were
incubated with streptavidin-agarose beads coupled to biotinylated doublestranded oligonucleotides (Sigma-Genosys) containing a wild-type (5⬘-tgg
tctctctgaccctcccccTGAGTTCgttcaccaaagg-3⬘) or mutated (5⬘-tggtctctct
gaccctcccccAAAAAAAAttcaccaaagg-3⬘) Ap-1 binding site (shown in
capital letters) from the DAP10 promoter region. The binding reactions
were performed for 1 h at 4°C. Unbound proteins were removed by six
washes with lysis buffer. After washing, the oligonucleotide-bound proteins were released in Laemmli sample buffer (Sigma-Aldrich), boiled for
5 min, resolved on 10% SDS-PAGE, and detected by immunoblotting as
described earlier.
Chromatin immunoprecipitation (ChIP) assay
All DNA fragments were derived by PCR using platinum Taq polymerase
(Invitrogen Life Technologies) with human genomic DNA (Promega) as
the template. A DAP10 luciferase construct was subcloned into the KpnI
and NheI restriction sites of the pGL3-basic reporter plasmid (Promega).
The primers used to generate the DAP10 luciferase constructs were: P
(reverse) NheI 5⬘-ctagctagcgaagaggatgtgacccagatg-3⬘; P1 (forward) KpnI
5⬘-ggggtaccccctccctctttctccatttc-3⬘; P2 (forward) KpnI 5⬘-ggggtaccttcttggc
cctacctcc-3⬘; P3 (forward) KpnI 5⬘-ggggtacctgagttcgttcaccaaaggc-3⬘; and
P4 (forward) KpnI 5⬘-ggggtacctcaacacacacaggaagc-3⬘. Mutant constructs
were generated with a QuikChange XL Site-directed Mutagenesis kit
(Stratagene), using a pair of overlapping internal primers that contained a
mutant sequence. The primers used for the promoter mutation constructs
were: Ap-1mut1 (forward) 5⬘-tctgaccctcccGGTACCttcgttcaccaa-3⬘ and Ap1mut2 (forward) 5⬘-cctccccctgagGGTACCcaccaaaggcag-3⬘. The pcDNA3/cJun and pcDNA3/c-Fos plasmids were gifts from Dr. N. Colburn (National
Cancer Institute, Frederick, MD).
All plasmid DNA used for transfections were purified with Qiagen Plasmid Maxi kits, according to the manufacturer’s protocol. Restriction enzymes were purchased from New England Biolabs. Custom synthesized
oligonucleotides were supplied by Sigma-Genosys. The correctness of the
plasmid inserts was verified by sequence analysis.
Chromatin preparation and immunoprecipitation were performed with a
EZ-ChIP kit (Upstate Biotechnology) with minor modifications. ProteinDNA cross-linking was achieved by incubating primary CD8⫹ T or NK
cells (2 ⫻ 106 cells per condition) with 1% formaldehyde for 10 min at
room temperature with gentle agitation. Cross-linking was blocked by adding glycine at a final concentration of 0.125 M and incubating at room
temperature for 5 min. Cells were collected by centrifugation and washed
in cold PBS containing 1% protease inhibitor mixture (Sigma-Aldrich).
Pelleted cells were resuspended in 200 ␮l of lysis buffer (Upstate Biotechnology) and incubated on ice for 10 min. Chromatin was then sonicated to
an average length of 0.2–1 kb using a Branson Sonifier 450 (Branson
Ultrasonics). Sonicated chromatin was then immunoprecipitated with antic-Fos, anti-c-Jun Ab (Santa Cruz Biotechnology) or isotype matching control Ab attached to microbeads using a protocol provided by Upstate Biotechnology. The bound protein-DNA complexes were eluted with freshly
prepared elution buffer (1% SDS and 0.1 M NaHCO3). After reversing the
protein-DNA cross-linking and subsequent DNA recovery, to detect
DAP10 promoter region sequences, samples were analyzed by quantitative
PCR (DNA Engine Opticon 2; MJ Research) with iQ SYBR Green
Supermix (Bio-Rad). The amount of DAP10 promoter related sequence
present in each reaction was calculated relative to a standard curve obtained using a serial 10-fold dilution of human genomic DNA (Promega).
Results were expressed as fold enrichment over control Ab. The following
primers were used in the ChIP assays: human DAP10 promoter, sense
5⬘-cagcaaattttcttggccctacctc-3⬘ and antisense 5⬘-gttactgcctttggtgaacga-3⬘;
and negative control primers, sense 5⬘-atggttgccactggggatct-3⬘ and antisense 5⬘-tgccaaagcctaggggaaga-3⬘. Negative control primers were from a
ChIP-IT kit (Active Motif).
Transfection and luciferase assay
Results
All cell types (3–5 ⫻ 106) were transiently transfected with 5 ␮g of the
reporter plasmid and 0.25 ␮g of the pRL-nul Renilla control vector (Promega) that acts as an internal control for the normalization of transfection
efficiency. Electroporation was performed using the Amaxa nucleofection
system, according to the manufacturer’s protocols. Cells were harvested at
16 –24 h posttransfection and lysed. Specific luciferase activity in the cell
Human DAP10 gene has multiple transcriptional start sites in
freshly isolated primary NK and CD8⫹ T cells
Plasmid construction
Sequence analysis of the 5⬘-flanking region of the human DAP10
gene with a transcriptional factor database search (TFSearch)
showed that it lacked a TATA box (data not shown), a feature
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Polyclonal human NK cells and CD8⫹ and CD4⫹ T cells were isolated by
negative selection from peripheral blood using NK cell or CD8⫹ or CD4⫹
T cell isolation kits (Miltenyi Biotec). All isolated cell populations were
routinely found to be ⬎97% in purity. Freshly purified cells were cultured
in IMDM (BioWhittaker) supplemented with 10% human AB serum (BioWhittaker), and 2 mM GlutaMax (BioSource International). The human
NK leukemia cell line, NKL, was cultured in RPMI 1640 (BioSource International) supplemented with 2 mM GlutaMax, 1 mM sodium pyruvate
(Invitrogen Life Technologies), 100 U/ml recombinant IL-2 (Biological
Resources Branch, National Cancer Institute, Frederick, MD), 5 ␮g/ml
plasmocin (InvivoGen), and 10% FBS (BioWhittaker). Jurkat T cells were
cultured in RPMI 1640 supplemented with 2 mM GlutaMax, 10 mM nonessential amino acids, 10% FBS, and 5 ␮g/ml plasmocin. All cells were
cultured at 37°C under an atmosphere of 5% CO2. Where appropriate, cells
were stimulated with plate-bound anti-CD3 or isotype-matched control
mAb (eBioscience) at different concentrations (0.05–1 ␮g/ml) for 2–5 days
before analysis.
DAP10 PROMOTER REGULATION BY Ap-1
The Journal of Immunology
411
FIGURE 1. Mapping the location of the human
DAP10 promoter. A, Genomic structure and location of
the multiple transcriptional start sites of the DAP10
gene. The location of the exons is shown (grayed box).
The sequence upstream of the translation initiation site
(ATG, ⫹1) is shown, as well as the location of the
5⬘-RACE identified transcriptional start sites (indicated
at arrows). B, Localization of the DAP10 promoter by
deletion mapping. The promoter constructs are shown
(left) with approximate positions of the 5⬘ and 3⬘ ends
for each fragment indicated relative to the ATG translation initiation codon at ⫹1. The luciferase activities
were measured 24 h after transfection and normalized
to the pGL3-basic control vector. Luciferase activities
are representative of three independent experiments
with each showing the same relative trend. Results
shown represent a mean value and SD of triplicate
experiments.
DAP10 promoter reflected the activity of the endogenous gene, we
concluded that the major regulatory elements necessary for cell typespecific DAP10 expression were present in the P2 promoter construct.
TCR ligation enhances DAP10 expression in T cells
The TCR plays a major role in defining the specificity of an immune response and together with coreceptors, such as CD28 and
Relative luciferase activity
A
Mapping of the human DAP10 promoter
To determine whether the sequence upstream of the first exon contained a promoter, we cloned a genomic fragment that began 0.7 kb
upstream and extended downstream through the ATG translational
start site (⫺698 to ⫹27, construct P1). A series of 5⬘ deletion mutant
constructs of this genomic fragment were created to locate the region
critical for DAP10 promoter activity. These constructs were inserted
into the pGL3-basic luciferase reporter vector, and transfected into
DAP10-expressing NKL and Jurkat T cells. Deletion of the segment
from ⫺698 to ⫺292 bp (construct P2) had a positive effect on DAP10
promoter activity in both cell types, but deletion from ⫺292 to ⫺180
bp (construct P3) resulted in a 3- to 5-fold reduction in luciferase
activity (Fig. 1B). The smallest deletion construct (⫺140 to ⫹27 construct P4) began only 5 bp upstream of the first transcription starting
site and possessed little promoter activity (1- to 3-fold over pGL3basic). These results indicate that the sequence between ⫺292 and
⫺140 bp contains the major promoter regulatory region important for
DAP10 gene transcription.
NK
CD8+ T
CD4+ T
PGL3-basic
B
NK
P2 promoter construct
CD8+T
CD4+T
DAP10
-actin
Cell type-specific activity of the DAP10 promoter in NK and
T cells reflects the activity of the endogenous gene
To test whether the genomic segment containing the DAP10 promoter
also contained elements responsible for controlling DAP10 cell typespecific expression in primary cells, we transfected the P2 DAP10
promoter construct, which possessed maximum promoter activity in
Jurkat T cells and NKL cells (see Fig. 1B) into primary NK cells and
CD8⫹ or CD4⫹ T cells isolated from healthy donors. The promoter
was active in NK and CD8⫹ T cells, but had little or no activity in
CD4⫹ T cells (Fig. 2A), which is consistent with the fact that endogenous DAP10 protein is expressed in NK and CD8⫹ T cells, but not
usually in CD4⫹ T cells (Fig. 2B). As the activity of the identified
FIGURE 2. Cell type-specific activity of the DAP10 promoter. A,
DAP10 promoter activity in primary NK cells and CD8⫹ or CD4⫹ T cells.
Freshly isolated human NK cells and CD8⫹ or CD4⫹ T cells were cultured
in 500 U/ml IL-2 for 4 days before transfection. The luciferase activities
were measured 16 h after transfection and normalized to the activity of the
pGL3-basic control vector. Luciferase activities are representative of three
independent experiments with each showing the same relative trend. Results represent a mean value and SD of triplicate experiments. B, Detection
of DAP10 protein in primary cells by Western blot analysis. Freshly isolated human NK cells and CD8⫹ or CD4⫹ T cells were cultured in 500
U/ml IL-2 for 4 days before lysis for Western blot analysis of DAP10
expression. Analysis of ␤-actin levels served as a loading control.
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often associated with the existence of multiple transcriptional start
sites (25). To investigate whether this was the case for the DAP10
gene and to identify the 5⬘ ends of the transcripts, RLM-RACE
using total RNA from freshly isolated human primary NK and
CD8⫹ T cells was performed. Sequence analyses of multiple 5⬘RACE clones from three different donors for each cell type demonstrated the existence of multiple transcriptional start sites (Fig.
1A). All transcriptional start sites in NK and CD8⫹ T cells occurred within a 136-bp region upstream of the DAP10 gene start
site of translation (ATG, ⫹1). Similar results were obtained using
cDNA from spleen and thymus (data not shown).
412
DAP10 PROMOTER REGULATION BY Ap-1
NKG2D/DAP10, controls T cell activation (26 –28). We were interested in determining the effect of TCR ligation on DAP10 expression. To make this determination, CD8⫹ T cells isolated from
healthy donors were activated with immobilized anti-CD3 mAb
for 2–5 days. NKG2D/DAP10 cell surface expression increased
significantly by exposure to anti-CD3 mAb compared with cells
cultured with immobilized control Ig mAb (Fig. 3A). This TCRmediated up-regulation of NKG2D/DAP10 was observed in T
cells isolated from multiple human donors (data not shown). To
check whether this increase in NKG2D/DAP10 cell surface expression correlated with an increase in DAP10 protein level, Western blot analyses were performed with cell lysates from freshly
isolated and anti-CD3-stimulated CD8⫹ T cells. An increase in the
DAP10 protein level was detected in CD3 activated CD8⫹ T cells
(Fig. 3B). This increase in the DAP10 protein level was observed
for a range of anti-CD3 mAb concentrations from 0.05 to 1 ␮g/ml
(data not shown). We also observed a very small but consistent
appearance of DAP10 protein level in CD4⫹ T cells after TCR
stimulation (data not shown). A significant increase in DAP10
transcripts after both 2 and 5 days of culture with immobilized
anti-CD3 mAb was also evident (Fig. 3C). After 5 days, we observed a ⬃5-fold induction in DAP10 transcripts over that observed of freshly isolated CD8⫹ T cells. Taken together, these
results demonstrate that TCR ligation positively regulates the expression of the DAP10 gene, which leads to enhanced DAP10
protein expression that, in turn, up-regulates NKG2D/DAP10 cell
surface expression.
The DAP10 promoter is a target for activation by TCR ligation
We examined in more detail how TCR signals regulate DAP10
transcription using the DAP10-positive Jurkat T cell line. Stimulation of Jurkat T cells with anti-CD3 mAb markedly increased
DAP10 protein levels, as well as mRNA levels (Fig. 4, A and B).
To study the effect of TCR stimulation on DAP10 promoter activity, we used the P2 construct (see Fig. 1B). As shown in Fig. 4C,
Jurkat T cells transfected with the P2 DAP10 promoter construct
had a consistent 2-fold increase in the promoter driven luciferase activity in the presence of immobilized anti-CD3 mAb compared with
transfected Jurkat cells treated with control mAb. Taken together,
these data indicate that TCR ligation enhances DAP10 mRNA and
protein levels through transactivation of the DAP10 promoter.
Role of an Ap-1-binding site in the DAP10 promoter activity
To localize the region of the DAP10 gene that mediates its induction in response to TCR ligation, each of the DAP10 deletion promoter constructs (Fig. 5A) was transiently transfected into Jurkat T
cells and the luciferase activity was measured with or without antiCD3 mAb stimulation. DAP10 promoter constructs P1 (⫺698 to
⫹27) (data not shown), P2 (⫺292 to ⫹27), and P3 (⫺180 to ⫹27)
showed similar response to TCR ligation, but the shortest promoter
construct P4 (⫺140 to ⫹27) showed only marginal up-regulation
after TCR ligation (Fig. 5A). Because the P3 construct was the
shortest construct showing TCR responsiveness, we concluded that
the 40 bp sequence located between ⫺180 and ⫺140 bp contains
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FIGURE 3. TCR stimulation of primary human T cells up-regulates NKG2D/DAP10 expression. A, Cell surface expression of human NKG2D/
DAP10 after CD3 stimulation of primary cells. CD8⫹ T cells were cultured with IL-2 (100 U/ml) and with immobilized anti-CD3 mAb or control
Ig mAb (500 ng/ml) for 4 days. Cell were harvested, stained with anti-NKG2D or control Ig mAb, and analyzed by flow cytometry. Cell populations
stimulated with anti-CD3 and stained with anti-NKG2D (thick line histogram), stimulated with anti-CD3 and stained with control Ig mAb (gray filled
histogram), and stimulated with control Ig mAb and stained with anti-NKG2D (dotted line histogram). B, DAP10 protein expression after CD3
stimulation of primary CD8⫹ T cells. Freshly isolated CD8⫹ T cells were cultured with immobilized anti-CD3 mAb (500 ng/ml) for 4 days, and
cell lysates were analyzed for DAP10 expression by Western blot. Analysis of ␤-actin levels served as a loading control. Data are representative
of three independent experiments. C, DAP10 mRNA expression levels with and without CD3 stimulation by primary CD8⫹ T cells. Freshly isolated
CD8⫹ T cells were cultured with immobilized anti-CD3 mAb (500 ng/ml). On days 2 and 5, cells were harvested, and total RNA was isolated.
Samples were analyzed for DAP10 transcript expression by quantitative PCR. Data shown are the mean fold induction and SD in DAP10 expression
taken from four independent experiments.
The Journal of Immunology
A
413
B
DAP10 mRNA in Jurkat T
Jurkat T
anti-CD3
DAP10
-actin
Jurkat T
P2
normalized to 18S rRNA
control
A
P3
anti-CD3
fold over control antibody
B
control
anti-CD3
C
Ap-1
C
control
anti-CD3
P4(-140)
P3(-180)
ctctctgaccctcccCctgagttcgttcaccaaaggcagtaacggagacacccCctcaacacaca
Jurkat T
Relative luciferase activity
control
P4
mutant
probe
wt
probe
beads nuclear
extract
c-Fos
% activity
D
PGL3-basic
P2 promoter
construct
a key regulatory segment important for the regulation of DAP10
promoter activity following TCR ligation.
The nucleotide sequence upstream of exon 1 plus a portion of
the exon 1 sequence is shown in Fig. 5B. A transcriptional factor
database search (TFSearch) of this sequence revealed the presence
of a putative binding site for Ap-1 transcription factors located
between ⫺180 and ⫺140 bp (Fig. 5B). Ap-1 transcription factors
are well-characterized mediators of T cell activation that can regulate the transcription of multiple cytokine, chemokine, and cell
surface receptor genes (29 –31).
Because the identified Ap-1 motif is a putative Ap-1 binding
site, we tested for its functionality in primary CD8⫹ T cells.
Ap-1 is a homodimer or heterodimer containing Fos (c-Fos,
FosB) and Jun (c-Jun, JunB, JunD) proteins that can be activated by TCR stimulation (29 –31). Using pull-down assays, we
investigated whether TCR activated primary CD8⫹ T cells contain c-Fos protein capable of binding to the putative Ap-1 binding site present in the DAP10 promoter region. To investigate,
CD8⫹ T cells isolated from healthy donors were activated with
immobilized anti-CD3 mAb for 16 h. Nuclear extracts were
incubated with streptavidin-agarose beads coupled to biotinylated double-stranded oligonucleotides containing either a wildtype or mutated Ap-1 binding site (see Materials and Methods)
designed from the DAP10 promoter. Fig. 5C shows that the
oligonucleotide containing the wild-type Ap-1 binding site was
significantly more efficient at binding c-Fos protein than the
Ap-1mut1
NKL
Jurkat T
Ap-1mut2
E
Jurkat T
P2
Ap-1mut1
Ap-1mut2
control
PGL3-basic
anti-CD3
Relative luciferase activity
FIGURE 5. Role of an Ap-1 binding site in DAP10 promoter activity.
A, Localization of a region critical for up-regulation of DAP10 promoter
activity after CD3 stimulation. The series of 5⬘ deletion promoter constructs are shown on the left. After transfection, Jurkat T cells were
cultured with immobilized anti-CD3 or control Ig mAb (1 ␮g/ml). The
luciferase activity was measured 24 h after transfection and normalized
to the pGL3-basic control vector. Results shown are the average and SD
of three independent experiments. B, An Ap-1 binding site is located
within the region associated with the TCR-mediated up-regulation of
the DAP10 promoter. The sequence upstream of exon 1, as well as a
portion of the exon 1 sequence, is shown. The putative Ap-1 binding
sequence is boxed. The 5⬘ ends of deletion constructs P3 and P4 are
indicated by an arrow. C, c-Fos protein from TCR stimulated CD8⫹ T
cells specifically binds to the Ap-1 binding site in vitro. CD8⫹ T cells
were cultured with immobilized anti-CD3 mAb (500 ng/ml) for 16 h.
Cells were harvested and nuclear cell extracts were incubated with biotinylated oligonucleotides probes containing the wild-type (wt) or mutant Ap-1 binding site and the complexes were pulled down with
streptavidin-agarose beads. c-Fos binding to beads in the absence of
probe is also shown along with analysis of c-Fos in the nuclear extracts.
c-Fos protein was detected by Western blot. Number at the top of each
lane represents relative quantification of band intensity. Data are representative of three independent experiments. D, Role of mutations in
the Ap-1 binding site in DAP10 promoter activity. The promoter constructs are shown (left). Jurkat T cells and NKL cells were transfected
with the indicated luciferase reporter plasmid constructs. Luciferase
activities were measured 24 h after transfection and normalized to the
pGL3-basic control vector. Results represent the average and SD of
three independent experiments. E, Role of mutations in the Ap-1 binding site on the DAP10 promoter activity induced by CD3 activation of
Jurkat T cells. The promoter constructs are shown (left). Jurkat T cells
were transfected with the indicated luciferase reporter plasmid constructs. After transfection, cells were cultured with immobilized antiCD3 or control Ig mAb. Luciferase activities were measured 24 h after
transfection and normalized to the pGL3-basic control vector. Luciferase activity shown is representative of three independent experiments
with each showing the same relative trend. Results represent a mean
value and SD of triplicate experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 4. Regulation of DAP10 expression following TCR stimulation in Jurkat T cells. A, DAP10 protein expression levels by Jurkat T cells
with and without CD3 stimulation. Jurkat T cells were cultured with immobilized anti-CD3 or control Ig mAb (1 ␮g/ml) for 16 h and analyzed for
DAP10 expression by Western blot. Analysis of ␤-actin levels served as a
loading control. B, DAP10 mRNA expression levels by Jurkat T cells with
and without CD3 stimulation. Jurkat T cells were cultured with immobilized anti-CD3 or control Ig mAb (1 ␮g/ml) for 16 h, followed by analysis
for DAP10 transcript expression by quantitative PCR. Data shown are the
mean fold induction in DAP10 expression taken from three independent
experiments. C, DAP10 promoter activity in Jurkat T cells. Jurkat T cells
were transfected with the indicated luciferase reporter constructs. After
transfection, cells were cultured with immobilized anti-CD3 or control Ig
mAb (1 ␮g/ml). Luciferase activity was measured 24 h after transfection
and normalized to the pGL3-basic control vector. Luciferase activity
shown is representative of three independent experiments performed with
each showing the same relative trend. Results represent mean luciferase
activity and SD of triplicate experiments.
P2
414
Ap-1 binding site is involved in the TCR-mediated regulation of
DAP10 promoter activity
Ap-1 transcription factors are able to transactivate the human
DAP10 promoter and up-regulate DAP10 protein expression
To determine whether c-Fos and c-Jun transcription factors can
enhance DAP10 promoter activity, the P2 DAP10 promoter construct was cotransfected with expression vectors for c-Jun or c-Fos
proteins into NKL or Jurkat T cells that were subsequently stimulated with immobilized anti-CD3 mAb. Overexpression of c-Jun
or c-Fos up-regulated DAP10 promoter activity ⬃1.5– to 2-fold in
both Jurkat T cells (Fig. 6A) and NKL cells (Fig. 6B). These results
provide further evidence that TCR ligation activates the DAP10
promoter in T cells via Ap-1 transcription factors and support the
notion that Ap-1 transcription factors are also involved in the transcriptional regulation of the DAP10 gene promoter activity in NKL
cells.
To assess whether overexpression of Ap-1 caused an increase in
endogenous DAP10 protein levels, Jurkat T cells and NKL cells
were transfected with c-Jun or c-Fos expression vectors, and after
48 h, levels of DAP10 protein were measured by Western blot
analyses (Fig. 6, C and D). Endogenous DAP10 protein levels
were increased 2.5-fold upon overexpression of c-Jun or c-Fos
compared with the control pcDNA3.1 vector in Jurkat T cells that
had been cultured with anti-CD3 mAb (Fig. 6C). In NKL cells, we
observed 1.3- or 2-fold up-regulation of DAP10 protein levels
upon overexpression of c-Jun or c-Fos transcription factors, respectively. Taken together these results provide evidence that Ap-1
transcription factors are biologically relevant regulators of DAP10
expression.
Ap-1 transcription factors interact with the DAP10 promoter
in vivo
To demonstrate that the up-regulation of DAP10 expression by
activated CD8⫹ T cells correlated with binding of Ap-1 transcription factors to the DAP10 promoter in vivo, we performed
ChIP assays. For CD8⫹ T cells, freshly isolated cells from multiple donors were cultured for 24 h with TCR (anti-CD3) stimulation. Fig. 7A shows that for all four donors significant amplification of DNA containing the relevant DAP10 Ap-1 site
could be detected in Ab-specific immunoprecipitates derived
B
NKL
Relative luciferase activity
Jurkat T
P2
PGL3-basic
Control
C
c-Fos
c-Jun
Control
D
Jurkat T
Control
DAP10
c-Jun
P2
PGL3-basic
c-Fos
NKL
Control
c-Fos
c-Jun
c-Jun
c-Fos
DAP10
-actin
-actin
FIGURE 6. Regulation of DAP10 gene expression by Ap-1 transcription factors. A, Regulation of the DAP10 promoter by c-Jun and c-Fos
transcription factors in Jurkat T cells. The indicated promoter constructs
were cotransfected into Jurkat T cells with expression vectors for c-Jun
or c-Fos, or the pcDNA3.1 control vector. After transfection, cells were
cultured with immobilized anti-CD3 mAb. Luciferase activity was measured 24 h after transfection and normalized to the pGL3-basic control
vector. Luciferase activity shown is representative of three independent
experiments performed with each showing the same relative trend. Results represent a mean value of triplicates and the SD is shown. B,
Regulation of DAP10 promoter by c-Jun and c-Fos transcription factors
in NKL cells. The indicated constructs were cotransfected into NKL
cells with expression vectors for c-Jun or c-Fos or the pcDNA3.1 control vector. Luciferase activity was measured 24 h after transfection and
normalized to the pGL3-basic control vector. Luciferase activity shown
is representative of three independent experiments performed with each
showing the same relative trend. Data shown represent a mean value
and SD of triplicate experiments. C, Up-regulation of DAP10 protein
levels by c-Jun and c-Fos transcription factors in Jurkat T cells. After
transfection with control, c-Jun, or c-Fos vectors, cells were cultured
with immobilized anti-CD3 mAb for 48 h. Expression of human DAP10
protein was analyzed by Western blot. Analysis of ␤-actin levels served
as a loading control. Number at the top of each lane represents quantification of band intensity relative to ␤-actin. D, Up-regulation of
DAP10 protein levels by c-Jun and c-Fos transcription factors in NKL
cells. After transfection with control, c-Jun, or c-Fos vectors, cells were
cultured for 48 h. Expression of human DAP10 protein was analyzed by
Western blot. The two different size bands observed for DAP10 in NKL
cells are likely due to the reported variability in O-linked glycosylation
(3). In most cell types, we have observed that the lower molecular size
band is less obvious. Analysis of ␤-actin levels served as a loading
control. Number at the top of each lane represents quantification of band
intensity relative to ␤-actin.
from these cells. No significant binding was observed in immunoprecipitates derived from freshly isolated T cells (data not
shown).
To investigate the in vivo interaction between Ap-1 transcription factors and the DAP-10 promoter in NK cells, primary NK
cells from multiple donors were cultured for 24 h in IL-2 (required for viability) before ChIP analyses. All four donors
showed significant amplification of DNA containing the relevant Ap-1 binding site in the c-Fos immunoprecipitates (Fig.
7B). No significant binding of relevant DNA was observed in
immunoprecipitates derived from freshly isolated NK cells
(data not shown). Taken together, these data demonstrate that
Ap-1 proteins interact with the DAP10 promoter in primary NK
and CD8 T cells in vivo.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
To investigate whether the Ap-1 binding site plays a role in the
induction of DAP10 gene expression following TCR ligation, the
P2 DAP10 promoter constructs (wild-type, Ap-1mut1, or Ap1mut2) were transfected into Jurkat T cells, followed by stimulation with immobilized anti-CD3 or control mAb (Fig. 5E). AntiCD3 stimulation resulted in a 2-fold increase in luciferase activity
for the wild-type P2 construct. In contrast, this up-regulation was
significantly diminished (over 50%) with the DAP10 promoter
constructs that had mutated Ap-1 binding sites (Fig. 5E). These
data indicate that the Ap-1 binding site is required for optimal
activation of the DAP10 promoter following TCR ligation.
A
Relative luciferase activity
oligonucleotide containing the mutated Ap-1 binding site.
These results indicate that the c-Fos protein from TCR activated
primary CD8⫹ T cells can specifically bind to the Ap-1 binding
site present in the DAP10 promoter region.
To investigate the involvement of this Ap-1 binding site in the
regulation of DAP10 promoter activity, we created two mutated
versions (Ap-1mut1 and Ap-1mut2) of the Ap-1 binding site in the
P2 DAP10 promoter construct. Each mutation significantly reduced the DAP10 promoter activity in both NKL and Jurkat T cells
(Fig. 5D), indicating that this site is involved in the transcriptional
regulation of the basal activity of the DAP10 promoter.
DAP10 PROMOTER REGULATION BY Ap-1
The Journal of Immunology
A
415
Fold over control antibody
CD8+T cells + anti-CD3
B
Fold over control antibody
NK cells
Discussion
Human NKG2D/DAP10 is a well-described activation receptor expressed by NK cells that is also capable of providing costimulatory
signals for CD8⫹ T cells (10, 28, 32). NKG2D/DAP10 has been
shown to play a role in controlling the progression of certain tumors and infectious diseases, as well as exacerbating certain autoimmune diseases (21, 22, 33, 34). Several mechanisms are
known to regulate the cell surface expression of the human
NKG2D receptor, including the differential action of cytokines
(IL-2/IL-15/IL-21, TNF-␣, and TGF-␤1) (22, 28, 35–39), interaction of the receptor with soluble and membrane bound ligands
(40 – 44) and availability of the DAP10 adaptor protein (3, 8). We
are interested in how the expression of this receptor is regulated.
Given that DAP10 is critical for NKG2D/DAP10 signaling capacity (3) and cell surface expression (3, 8), we focused our initial
study on the transcriptional regulation of the human DAP10 gene.
The human DAP10 gene is located on chromosome 19q13.1
only 130 bp downstream of the DAP12 gene in opposite transcriptional orientation (3). The human DAP10 gene is relatively small
(spans ⬍2 kb) and has four exons. DAP10 cDNA is ⬃500 bp and
encodes a 93 aa type I transmembrane protein (3). We show that
the DAP10 gene has multiple transcriptional start sites (⬃16), a
common feature of genes with TATA-less promoters (25). The
partner of DAP10, NKG2D, also has a TATA-less promoter and
multiple transcriptional start sites (45, 46), which is a feature common for other members of the NK complex, such as NKG2A (47–
49) and CD94 (50, 51). We did not see a predominance in use of
particular transcriptional start sites with NK cells compared with
CD8⫹ T cells, either in freshly isolated or IL-2 cultured cells (data
not shown). We then localized the DAP10 promoter to a region
between ⫺292 and ⫹27 bp (Fig. 1B). This location agrees with the
location of the transcriptional start sites, as most promoters occur
within 150 bp upstream of the transcriptional start sites. Using
deletion constructs, we localized two regions that affected DAP10
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 7. Interaction of the DAP10 promoter with Ap-1 transcription
factors in vivo. ChIP assays were performed to analyze Ap-1 binding to the
DAP10 promoter in primary CD8⫹ T cells stimulated in culture for 24 h
with immobilized anti-CD3 (1 ␮g/ml) (A), and in primary NK cells cultured in 100 U/ml IL-2 for 24 h (B). Samples were analyzed by quantitative
PCR. The amount of DAP10 sequence present in each reaction was calculated relative to a standard curve obtained using a serial 10-fold dilution
of human genomic DNA. Data shown are the fold enrichment over isotype
control Ab.
basal promoter activity. The region from ⫺698 to ⫺292 bp contains an element with a negative effect on DAP10 promoter activity, whereas the region from ⫺292 to ⫺140 bp contains an element
essential for DAP10 maximum promoter activity (Fig. 1B). Using
luciferase reporter assays, we showed that the defined DAP10 promoter was functional in NK and CD8⫹ T cells, but not in CD4⫹
T cells derived from healthy donors (Fig. 2A). This observation is
directly correlated with the DAP10 protein expression by those
cell types (Fig. 2B) and with the cell surface expression of NKG2D
on PBL from healthy donors (data not shown).
Under certain conditions, NKG2D/DAP10 can be expressed on
the surface of subsets of CD4⫹ T cells. For example, in some
cancer patients, a rare NKG2D⫹CD4⫹ T cell population has been
reported (52). This CD4⫹ subset was found in patients with MICpositive tumors and stimulation with MICA ligand was shown to
contribute to the expansion of this CD4⫹ T cell population. In
addition, NKG2D (as well as perforin) can also be induced on
CD4⫹ T cells in vitro following infection of PBMC from healthy
seropositive individuals with human CMV (53). Coligation of
NKG2D with TCR was shown to potently induce proliferation and
cytokine production by these cells. Of related interest, we have
observed that TCR-stimulated CD4⫹ T cells in culture are able to
express small amounts of DAP10 protein (data not shown). As
Ap-1 appears to be integral to CD4⫹ T cell function, the failure of
most CD4⫹ T to express DAP10 most likely relates to epigenetic
modulating events, such as chromatin modification or DNA methylation status.
We show that TCR ligation up-regulates NKG2D/DAP10 cell
surface expression by CD8⫹ T cells (Fig. 3A). This up-regulation
of NKG2D/DAP10 expression correlates with enhanced DAP10
mRNA and protein expression (Fig. 3, B and C). Taken together,
these results suggest that TCR ligation positively regulates expression of the DAP10 gene, which in turn promotes enhanced cell
surface expression of NKG2D/DAP10 in human T cells. Previous
studies have shown that TCR-induced stimulation of CD8⫹ T cells
increases cell surface expression of murine NKG2D within 4 days
(16, 54), which agrees with a reported increase in human DAP10
protein and mRNA levels by day 10 (37). However, our data show
that the up-regulation of DAP10 expression occurs after only 2–5
days of TCR stimulation.
The responsiveness of DAP10 promoter activity to TCR ligation
was mapped to the 40-bp segment of the promoter that contained
a Ap-1 binding site (Fig. 5B). In line with this observation, it is
known that the induction of promoter activity by coreceptors, such
as CD28, 4-1BB, and 2B4, is often dependent on activation by
transcription factors of the NFAT, NF-␬B, and Ap-1 families (55–
57). Mutations of the identified Ap-1 binding site lead to reduced
reporter transcriptional activity and a reduced response by the
DAP10 promoter construct to TCR stimulation in Jurkat T cells
(Fig. 5, D and E). The Ap-1 transcription factor is comprised of
Fos and Jun homodimers or heterodimers. Upon activation through
TCR ligation, they become phosphorylated, bind to their target
DNA binding sites, and act as potent transactivators of transcription (58 – 60). Using pull-down (Fig. 5C) and ChIP (Fig. 7A) assays, we show for primary CD8⫹ T cells that the DAP10 promoter
interacts with Ap-1 transcription factors in vitro and in vivo. Moreover, overexpression of c-Jun or c-Fos in Jurkat T cells led to
enhanced promoter activity, as well as increased DAP10 protein
expression (Fig. 6). Although the detailed mechanism of DAP10
induction by Ap-1 transcription factors remains to be defined, these
results provide evidence, for the first time, that Ap-1 transcription
factors are biologically relevant regulators of DAP10 expression in
human T cells. These results do not rule out the possibility that TCR
416
Acknowledgments
We thank Robert Valas for technical assistance and cell isolations, and Drs.
Madhan Masilamani, Xiaobin Tang, Gul’nar Fattakhova, and Sriram
Narayanan for thoughtful discussion.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Disclosures
The authors have no financial conflict of interest.
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ligation may regulate DAP10 gene transcription via additional
mechanisms.
Ap-1 also plays an important role in NK cell functions. NK cell
treatment with inhibitors of the Ap-1 pathway prevents NK cell natural cytotoxicity (61). Ap-1 regulates transcription of many genes,
such as ifn␥ (62, 63), il3 (64), granzyme B (65), il2 (66, 67), and il5
(68). Ap-1 binding sites are present in the NKG2A, 2B4, and proximal CD94 promoters (69). In NK cells, Ap-1 can be activated by
stimulation with cytokines such as IL-2 (70). Ap-1 activity is also
regulated at the posttranscriptional level by the activation of JNK (71).
A decrease in human NKG2D/DAP10 cell surface expression after
treatment of NK92 cells with JNK inhibitor has been recently reported
(72). Consistent with these observations and the fact that NKG2D/
DAP10 expression can be positively regulated by IL-2 stimulation in
NK cells, we found that the basal level of DAP10 promoter activity in
NKL cells was significantly decreased by mutations of the Ap-1 binding site. Moreover, using ChIP assays, we showed that in primary NK
cells c-Jun protein binds to the DAP10 promoter region in vivo. Ap-1
family members were also capable of up-regulating DAP10 promoter
activity, as well as the levels of endogenous DAP10 protein, in NKL
cells. However, unlike in CD8⫹ T cells, for reasons yet to be determined, we did not observe any significant binding of c-Fos protein to
DAP10 promoter in primary NK cells. Among the possible explanations for no significant binding observed is that different multiprotein
complexes are formed in the DAP10 promoter region in primary NK
and CD8⫹ T cells and NK cells may lack transcription factors necessary to form the complex containing c-Fos protein. This interpretation agrees with the fact that the Fos-Jun family members can form
over 15 different homodimers and heterodimers; moreover, interactions between various Fos-Jun family members and over 50 different
proteins have been reported (73). Another possibility is that other
epigenetic events, such as chromatin modifications or DNA methylation status, affect the regulatory specificities of Fos-Jun family proteins in NK and CD8⫹ T cells.
In summary, our studies clearly demonstrate that Ap-1 transcription factors play a significant role in regulating DAP10 expression
in human NK and T cells, and that in CD8⫹ T cells their role can
be enhanced by TCR ligation.
DAP10 PROMOTER REGULATION BY Ap-1
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