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* Curriculum in Genetics and Molecular Biology and
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599;
H. Lee Moffitt Comprehensive Cancer Center, Department of Biochemistry and Molecular Biology, University of Southern Florida, Tampa, FL 33612;
Department of Pediatrics and Center for Environmental Medicine and Lung Biology, University of North Carolina, Chapel Hill, NC 27599; and
¶ Department of Basic Sciences, Mercer University School of Medicine, Macon, GA 31207
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induces MHC class II Ags on
Th1 cells (3). These Ags are readily found on activated
human T cells, such that they are generally accepted as activation
markers of T cells (4, 5). Human T cells can
be induced to expressMHC class II by mitogens, Ags, PMA, calcium
ionophore, and various Abs directed at surface receptors expressed by T
cells (6). Additionally, MHC class II-expressing T cells
are found in a wide repertoire of clinical scenarios, including
autoimmune diseases, inflammation, infection, and oncogenic
transformation (7, 8, 9). Although the exact function of MHC
class II on T cells is not completely resolved, activated human T cells
are known to exhibit characteristics of APCs, as they can capture,
process, and present Ag on newly synthesized MHC class II molecules.
In addition, they are able to deliver costimulatory signals to
other T cells. Although initiation of the T cell response is dependent
on professional APCs, it is likely that with time and under conditions
of crowding, T cells can costimulate each other and become autonomous
from DCs (10, 11). Recently, T cell APC activity has also
been suggested to be a mechanism for self tolerance
(12).
The class II trans-activator (CIITA) is a master
transcriptional regulator encoded by the MHC2TA gene, and it
controls the expression of MHC class II as well as Ii and DM molecules
(13, 14, 15). It is defective in group A, type II bare
lymphocyte syndrome patients, who express little or no MHC class II
(15). Cell-specific expression of human CIITA results from
the differential usage of four CIITA promoters. These promoters are
numbered PI–PIV by the location of their transcriptional start site
from 5' to 3'. Promoter I (PI) is expressed in DCs (15).
Promoter II (PII) is expressed at very low levels, is functionally not
well characterized, and is absent from the mouse sequence
(16). Promoter III (PIII) is constitutively active in B
cells and DCs and is induced by IFN- in other cell lines or types
(16, 17, 18). The promoter region that is required for
activity in B cells is contained within the first 319 bp
(19), while the region required for the IFN- response
requires an additional 5 kb of distal sequences (18).
Finally, promoter IV (PIV), is expressed in response to IFN- in
various cell types, and controls MHC class II in murine nonhemopoietic
cells (16, 20, 21). Most studies have focused on PI, PIII,
and PIV, because of the low level of transcripts derived from PII. PI,
PIII, and PIV each have their own unique transcriptional start site,
resulting in the production of CIITA isoforms that are distinguished by
their most N-terminal sequences (16).
Previous studies have shown that MHC class II gene expression in a human transformed T cell line is correlated with the presence of CIITA. Transfection of the MHC2TA gene into HLA-DR- T cell lines can cause MHC class II expression (22). Analysis of MHC class II gene control in primary T cells has been limited to the delineation of DR promoter usage (23). Beyond these observations, the molecular pathway by which MHC class II expression is achieved in T cell lines, much less primary T cells, is unclear This study focuses on CIITA gene control in primary human blood T cells and a human T cell line. We show here that the PIII CIITA isoform, but not PI nor PIV, is expressed in activated human T lymphocytes purified by immunoselection. Transfection of a promoter-reporter construct for PIII shows the utilization of this promoter in both activated T cells and a DR+ T cell line. PIII usage is enhanced by molecular activators of T cells, such as the cAMP response element (CRE) binding protein (CREB) (24). Additionally, two elements within PIII, activation response element-1 (ARE-1) and ARE-2, are required for PIII activation. While the HLA-DR+ T cell line constitutively exhibits ARE-2 DNA-binding activity, primary T cells exhibit a major departure, in that ARE-2 binding is undetectable in resting T cells, but is induced upon mitogenic stimulation. This indicates that primary T cells require activation signals to cause ARE-2 binding activity, leading to CIITA expression and the subsequent induction of MHC class II. In contrast, the rare DR+ transformed T cell line, H9, is frozen in its activation state, where ARE-2 is persistently expressed, as is MHC class II. This strict correlation between ARE-2 binding activity and MHC class II gene expression in normal and transformed T cells indicates that the recognition of ARE-2 by transcription factor(s) is crucial for PIII activation in these cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Constructs containing sequential deletions of PIII were cloned as previously described (18). The site-specific PIII mutants were generated as previously described (19). Rous sarcoma virus (RSV)-CREB was provided by S. Kenney (University of North Carolina Lineberger Cancer Center, Chapel Hill, NC). RSV-ATF-1 contains the activating transcription factor (ATF)-1 cDNA in the RSV-pECE vector (gift from M. Green) (25). All plasmids were purified using a Qiagen column (Qiagen, Chatsworth, CA) before transfection.
Cell lines and growth conditions
The H9 HLA-DR+ human T cell line, the Raji
human B cell line, and THP-1 human monocyte cell lines were maintained
in RPMI (Life Technologies, Gaithersburg, MD) supplemented with 2 mM
L-glutamine, 10% FBS (Sigma-Aldrich, St. Louis, MO), and
100 U/ml penicillin and streptomycin (Life Technologies). DCs were
prepared from PBMCs as previously described (26). THP-1
cells were treated with 500 U/ml IFN- (PeproTech, Rocky Hill, NJ)
24 h before RNA isolation.
Generation of primary human T cells
PBMCs were isolated by Ficoll-Hypaque density-gradient centrifugation (ICN Biomedicals, Aurora, OH) from anti-coagulated venous blood. T cells were isolated by stringent immunomagnetic negative selection using the Pan T cell Isolation kit (Miltenyi Biotec, Auburn, CA). This kit includes Abs against CD11b, CD16, CD19, CD36, and CD56 to remove monocytes, DCs, NK cells, B cells, early erythroid cells, platelets, and basophils. After purification, cells were cultured for 3 days in RPMI supplemented with 2 mM L-glutamine, 10% FBS, 100 U/ml penicillin, and 2 µg/ml PHA (Sigma) for stimulation. The lack of B cell and macrophage contamination was verified by RT-PCR for CD19 and CD14, respectively (see below).
Flow cytometry
Cells were pelleted, washed twice with 15 ml PBS, resuspended in 1 ml PBS, and then incubated with 70 µl of a 3 mg/ml solution of mouse Ig (IgG fraction) on ice for 10 min. Cell suspension (50 µl) was added to tubes (USA Scientific, Ocala, FL) containing panels of fluoresceinated or phycoerythrinated mAbs to CD19, CD14, CD3, or HLA-DR (BD Biosciences, San Jose, CA; Caltag Laboratories, South San Francisco, CA) as well as to tubes containing either isotype controls or no Abs. Cells were incubated on ice for 15 min. All samples were washed with 0.5 ml PBS with 2% BSA (Sigma-Aldrich) and fixed in 0.5 ml 1% Ultrapure formaldehyde (Polysciences, Warrington, PA). Forward and side scatter and two colors of fluorescence were measured for each sample on a FACScan flow cytometer (BD Biosciences). Ten thousand events were analyzed in each sample. Data were analyzed using FlowJo data analysis software (TreeStar, Stanford, CA).
Transfection and luciferase assays
H9 cells were electroporated with 10 µg of a luciferase reporter construct in a 300-µl final volume of serum-free RPMI containing 3 x 106 cells. Primary human T cells were electroporated with 20 µg of a luciferase reporter construct in a 300-µl final volume of serum-free RPMI containing 3 x 106 cells (23). Cells were harvested 24 h post-transfection. Luciferase assays were performed using an LB 953 AutoLumat (EG&G, Berthold, Germany), as previously described (27).
RT-PCR
RNA was prepared using the SV Total RNA Isolation System (Promega, Madison, WI). RT-PCR was performed using the Access RT-PCR System (Promega) with the following oligonucleotides: PI sense, 5'-TACCACTGCACTCTGCTCCATGAG-3'; PIII sense, 5'-CCTGGCTCCACGCCCTG-3'; PIV sense, 5'-GAGCTGGCGGCAGGGAG-3'; PI, PIII, and PIV antisense, 5'-GAACTGGTCGCAGTTGATG-3'; CD14 sense, 5'-CTGACACTGGACGGGAATC-3'; CD14 antisense, 5'-GACTCCCCTGAAGCCAAG-3'; CD19 sense, 5'-CAAGTCCCCAAGATTCACAC-3'; CD19 antisense, 5'-AGGAATACAAAGGGGACTGG-3'; GAPDH sense, 5'-CCATGGAGGAAGGCTGGGG-3', and GAPDH antisense, 5'-CAAAGTTGTCATGGATGACC-3'. Reactions were run for 35 cycles with an annealing temperature of 60°C. Amplification products were analyzed by gel electrophoresis through 2% agarose gels.
Real-time PCR
cDNA was synthesized from total RNA using random hexamers, Moloney murine leukemia virus reverse transcriptase (Life Technologies), and RNasin RNase inhibitor (Promega). Real-time PCR was performed using the ABI PRISM 7900 sequence detection system (PerkinElmer, Foster City, CA). Primers and probes (Oligo Facility, University of North Carolina) were designed to span exon-intron junctions. CIITA probes were labeled at the 5' end with the reporter dye FAM and at the 3' end with the quencher dye TAMRA. The 18S rRNA probe was labeled at the 5' end with the reporter dye TET and at the 3' end with the quencher dye TAMRA. Primer and probe sequences are as follows: PI probe, 5'-CAGGCCCTCTTGGACAACCTGCT-3'; sense primer, 5'-GCCATCCTGACTTCAGGTGAGAA-3'; antisense primer, 5'-CATGGAGCAGAGTGCAGTGGTAT-3'; PIII probe, 5'-CCACGCCCTGCTGGGTCCTA-3'; sense primer, 5'-GGCTGGGATTCCTACACAATG-3'; antisense primer, 5'-CAACTCCATGGTGGCACACT-3'; PIV probe, 5'-CTGTGAGCTGCCGCTGTTCCC-3'; sense primer, 5'-GGGAGAGGCCACCAGCAG-3'; antisense primer, 5'-GCTCCAGGTAGCCACCTTCT-3'; and 18S rRNA probe, 5'-CAAATTACCCACTCCCGACCCG-3'; sense primer; 5'-GCTGCTGGCACCAGACTT-3'; and antisense primer, 5'-CGGCTACCACATCCAAGG-3'. PCR of cDNA specimens and standards were conducted in a total volume of 15 µl with 2x Platinum Quantitative Supermix-UDG (Invitrogen, San Diego, CA). Thermal cycler parameters included 2 min at 50°C, 2 min at 95°C, and 40 cycles involving denaturation at 95°C for 15 s and annealing/extension at 56°C for 1 min. Values were calculated based on standard curves generated for each gene. Normalization of samples was determined by dividing copies of CIITA by copies of 18S rRNA.
Nuclear extract preparation and EMSA
Nuclear extracts were prepared as described previously (28). The blood T cells were prepared by negative selection using immunomagnetic negative selection with the Pan T cell beads (Miltenyi Biotec) described above. Gel-shift analysis was performed as described previously (29) using synthetic oligonucleotides and 700 ng of the nonspecific competitor poly(dI:dC). The ARE-2 oligonucleotide spans from -66 to -51 bp of the promoter relative to the transcription start site and is 5'-GATCCTTGATGATCCCTCACTAGATC-3'. Sequences of the competitor probes are as follows: mouse ARE-2 oligonucleotide, 5'-GATCCCAGGTGGTCCCTTGCTA-3'; consensus PU.1 oligonucleotide, 5'-GGGCTGCTTGAGGAAAGTATAAGAAT-3'; and consensus B cell-specific activator protein (BSAP.CD19), 5'-CCGCAGACACCCATGGTTGAGTGCCCTCCAGGCCC-3'. For the supershift assay, activated T cell nuclear extract was incubated with p-CREB-1 goat polyclonal IgG (sc-7978) specific for phosphorylated CREB-1; ATF-1 mouse monoclonal IgG (sc-270) reactive with ATF-1p35, CREB-1p43, and CREM-1; CREB-2 rabbit polyclonal IgG (sc-200), specific for CREB-2 and ATF-4; and SP-1 goat polyclonal IgG (all from Santa Cruz Biotechnology, Santa Cruz, CA) or with no Ab for 2 h on ice. Following this, 32P-labeled ARE-2 probe was added to the reaction mix and incubated at room temperature for 30 min. DNA/protein complexes were then resolved on a gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary human T cells were isolated from PBMCs by negative
selection with a magnetic cell separator in which the non-T cells were
labeled with a mixture of Abs coupled to magnetic bead (see
Materials and Methods). The use of magnetic beads was
necessary to insure the purity of the cells, and extensive analyses
verified this purity (see below). The use of negative selection was
necessary to insure that the resting T cells are not activated. After
isolation, the T cells were activated with PHA for 3 days. PHA
activation is necessary to induce MHC class II expression, because
human T cells do not express MHC class II constitutively
(11). Flow cytometric analysis of isolated T cells
confirms that the population is primarily T cells, as assessed by
surface staining for CD3 (Fig. 1
A). As expected, these T
cells do not express abundant class II MHC, since they have not been
activated (Fig. 1B). Staining for CD14 and CD19 indicates
that the T cells are free from macrophage/monocyte and B cell
contamination, two primary sources of contaminating MHC class II Ags
(Fig. 1, C and D). To further assess the purity
of the T cell population with higher sensitivity, we used RT-PCR
analysis. RT-PCR analysis performed on RNA isolated from a purified
population of primary human T cells using primers for CD14 and CD19
indicates that the population of T cells is free of macrophage and B
cell contamination, respectively (Fig. 2B, top and
middle panels, lanes 4 and 5). RT-PCR
analysis was also used to determine the CIITA isoform that is expressed
in T cells. The four promoters and primer sets are depicted in Fig. 2A. It was important to confirm that the T cell population
was pure, because B cells use predominantly PIII, while
macrophage/monocytes use PI, PIII, and also PIV (16, 30).
If contaminating B cells and monocytes were present in the preparation,
they would greatly alter the integrity of the data. RT-PCR results of
the purified T cell preparation show that the transcript driven by
PIII, but not by PI or PIV, is the primary one expressed in these
purified, activated primary human T cells (Fig. 2B,
middle panel, lanes 1–3). Resting T
cells do not express any detectable CIITA isoforms (Fig. 2B,
top panel, lanes 1–3). RT-PCR analysis of RNA
harvested from positive controls for each primer set includes primary
human DCs for PI, Raji B cell line for PIII, and THP-1 (monocyte cell
line) induced with IFN- for PIV. All the positive controls produced
the expected products, indicating that these primer pairs can amplify
the proper product (Fig. 2B, bottom panel).
RT-PCR analysis of CIITA expression in the H9 class II-positive T cell
line indicated usage of PIII as well as minor PIV (data not shown). As
H9s are a transformed cell line, it is possible that expression differs
slightly from primary T cells. For more quantitative results and higher
sensitivity, we used real-time PCR analysis to confirm the RT-PCR
findings (Fig. 2C). As detected by RT-PCR, PIII CIITA is the
predominant form expressed in activated T cells, although a hint of PI
is also seen. DCs express both PI and PIII and a small amount of PIV,
Raji express primarily PIII and a slight amount of PIV, while
IFN--treated THP-1 monocytes express predominantly PIV, but also a
significant amount of PIII. The same RNAs used for RT-PCR were
used here.
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To delineate the promoter region of PIII that is required for
expression in T cells, luciferase reporter constructs containing
sequential deletions of PIII were transiently transfected in H9 or
primary T cells (Fig. 3A).
Cell lysates were assayed by luciferase assay to assess expression
levels of these constructs. The results of the transfection and
luciferase assay analysis in H9, an MHC class II-expressing T cell
line, indicate PIII expression and show that PIII promoter activity
increases slightly from the 113-bp to the 195-bp construct (Fig. 3B). Transfection of activated primary human T cells also
shows that PIII activity is modestly increased when the promoter is
lengthened from 113 to 195 bp. Activity does not increase significantly
with the transfection of constructs containing more upstream PIII
sequences (Fig. 3C).
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A).
Luciferase reporter constructs containing mutations at each of these
sites were generated based on the PIII.545 construct, as previously
described (19). Transfection of the site-specific mutant
constructs into H9 and activated primary human T cells followed by
luciferase assay analysis shows that mutations in site A, B, or C had
little effect on PIII activity, but mutations of ARE-1 or ARE-2
significantly reduced activity (Fig. 4, B and C).
An ARE-1 or ARE-2 mutation obliterated reporter expression in H9 cells
in the composite of three experiments shown in this figure, although in
other experiments some residual expression remained. Residual
expression is also seen in blood T cells with the ARE-1 and ARE-2
single mutation. In blood T cells, mutation of site A, B, or C actually
resulted in a slight enhancement of reporter activity, which may be due
to the possibility that the sites are repressor elements. Nonetheless,
these results taken together indicate that, like B cells, the
expression of PIII CIITA in a T cell line and blood T cells depends on
intact ARE-1 and ARE-2 sites.
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Recent studies have found an important role for the CREB in T cell
activation. Low levels of CREB have been observed in resting, naive
CD4+ T cells and were elevated following TCR
stimulation (31). CREB is also shown to be a critical
activator of the IL-2 promoter (32), is induced by the
CD28 costimulator (33), and has a role in T cell
differentiation by down-regulating IL-4 (3, 34). To
determine whether CREB may enhance PIII activity in primary T cells,
the PIII.545 luciferase reporter construct and an expression vector
containing CREB were cotransfected into these cells (Fig. 5). The inclusion of CREB caused a 3- to
4-fold enhancement of PIII activity (wild-type PIII.545 activation
levels reported as relative light units (RLU) per micrograms differ
from those in Fig. 4
due to the use of a different luminometer). Since
we showed that ARE-2 is important for PIII activity and the ARE-2 site
is the only one of the five known sites with any homology to a CREB
binding site (CRE), we decided to test whether CREB activated PIII by
binding ARE-2. The enhancement of PIII by CREB is not dependent on an
intact ARE-2 site, as mutation of the ARE-2 site did not affect this
enhancement, although this mutation did decrease the overall level of
expression as expected (Fig. 5). We further explored whether a CRE-like
site in the -254 to -248 bp region (relative to the transcription
start site) of PIII could be mediating the effects of CREB. However,
mutating both this site and ARE-2 did not affect the activation of PIII
by CREB (data not shown).
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EMSA was performed on nuclear extracts from H9 and primary human T
cells to determine whether the ARE-2 element interacts with DNA-binding
factors from cells of the T cell lineage. Nuclear extracts from blood T
cells were prepared by immunomagnetic negative selection using Pan T
cell Miltenyi magnetic beads, and each preparation from one blood bag
gives at most 4 x 107 cells and usually
much less than that. For this reason, each nuclear extract preparation
is only sufficient for one experiment, and each experiment required a
separate donor and cell preparation, attributing to some variations in
the appearance of the EMSA. Nonetheless, EMSA was used to determine 1)
differences or similarities between transformed and primary blood T
cells, and 2) differences between resting and activated blood T cells.
EMSAs using nuclear extract from Raji cells and competitive probes were
included as controls (Fig. 6, lanes
1–4). The mouse ARE-2 oligo included as a negative control did
not eliminate the ARE-2 band, as it is substantially different from the
human ARE-2 site in three of 11 bases (Fig. 6, lane 3). The top band is
a specific band that was competed with the ARE-2 competitor, while the
bottom band is nonspecific. Incubation of H9 nuclear extracts with an
oligonucleotide containing the ARE-2 element resulted in a similar
complex formation (Fig. 6, lane 5). Addition of ARE-2
competitor oligonucleotide eliminated this complex (Fig. 6, lane
7), but the addition of an unrelated oligonucleotide did not (Fig. 6, lane 6). EMSA analysis of extracts prepared from primary
blood T cells showed two complex formations only in extracts from
activated T cells, but not resting T cells (Fig. 6, lanes 8
and 9). This is the first circumstance where ARE-2 binding
activity is inducible. A probe for NF-Y was used on both resting and
activated T cell extracts to indicate that the absence of an ARE-2
complex in resting T cells was not due to decreased levels of resting T
cell extract (Fig. 6, lanes 10 and 11). EMSA
analysis of T cell extracts for ARE-1 is not shown because the
gel-shift band is not clearly visible despite a variety of optimization
protocols. This is similar to previous findings that the in vitro ARE-1
complex in B cells is very weak (19). Due to limitation in
the amount of nuclear extract, another T cell preparation was prepared
to show competition of gel-shift complex formation with an ARE-2
oligonucleotide, but not an identical oligonucleotide with mutations in
the ARE-2 site, a CREB sequence or a B cell-specific activator
protein sequence.
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(lanes 8 and
9), but only two bands are specific for ARE-2, because
addition of the ARE-2 competitor oligonucleotide removes both upper
bands but not the lower band (Fig. 7, compare lanes 1 and 2). A CREB-1 oligonucleotide
eliminated the formation of the top band, but not the second one. A
nonspecific oligonucleotide encoding the PU.1 sequence is shown in lane
4, and the intensity of the second band is comparable in lanes
3 and 4. A supershift analysis using an array of Abs
was then performed. The top band is specific for the ATF-1 Ab, which
also recognizes CREB-1, CREM-1, and ATF-1 family members. Incubation
with this Ab significantly reduced the intensity of the top band and
caused the formation of a shifted band. The lower band was not altered
by this Ab (compare lane 6 to the nonspecific serum control,
lane 9). This suggests that an ATF-1/CREB-1/CREM-1 family
member constitutes the upper band formed with ARE-2. The lower band is
specific for an unidentified protein that binds ARE-2. An
anti-CREB-2 Ab did not significantly alter the two bands.
Phosphorylation of the serine 133 residue of CREB is required for its
ability to activate transcription (24). Therefore, we
analyzed another CREB-1 Ab (lane 8), which is
specific for the form that contains the phosphorylated serine 133. This
Ab also did not supershift either band, suggesting that phosphorylated
CREB-1 is not associated with the ARE-2 binding complex (Fig. 7, lane 4). These results suggest that an ATF-1/CREB-1/CREM
family member may be associated with the ARE-2 binding complex, while
another unknown protein is also associated with this sequence. However,
the luciferase assay shown in Fig. 5 indicates that CREB does not
require ARE-2 for trans-activation of the PIII reporter, and
phosphorylated, transcriptionally active CREB-1 was not detected by
supershift assay (Fig. 7).
ATF-1 was originally identified as a factor that bound to CRE-like
elements present in adenovirus promoters and is 65% identical with
CREB in primary structure. Like CREB family members, it is a
transcription factor that binds to CREs (24). Since the
supershift assay (Fig. 7) identified an ATF-1/CREB-1/CREM-1 family
member binding to ARE-2, it is important to functionally assess whether
ATF-1 activates PIII through ARE-2 by a luciferase assay. Both H9 (Fig. 8A) and activated primary T
cells (Fig. 8B) were cotransfected with a vector containing
the ATF-1 gene or its empty vector control together with wild-type or
mutant ARE-2 PIII reporter. Our data show that ATF-1 activates PIII
CIITA 3- to 4-fold (Fig. 8), an activation level achieved with the
cotransfection of CREB and PIII reporter (Fig. 5). Similar to the
results from the CREB activation assay (Fig. 5), ATF-1 activation of
PIII is not mediated through the ARE-2 sequence because activation
remained unchanged when the ARE-2 site was mutated. Thus, both CREB and
ATF-1 appear to activate PIII CIITA, but not through binding of the
ARE-2 site. Additional experiments are necessary to identify the other
ARE-2 binding proteins.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Results from this report show congruent findings in transformed and primary human T cells, in that the PIII isoform of CIITA is expressed, and this expression is dependent on the ARE-1 and ARE-2 elements within this promoter. ARE-1 and ARE-2 were previously identified as two sites that are most important for PIII expression in B cell lines (16). ARE-1 is a transcription enhancer factor-2-like sequence that belongs to the Kruppel family of transcription factors, which includes proteins that bind to CTCCC motifs and are ubiquitously expressed. The identity of the ARE-2 binding protein has remained elusive, and at least one component may represent a novel protein. ARE-1 and ARE-2 binding sites are also occupied in both immature and mature DCs, implying that the proteins that recognize these two sequences are constitutively expressed at different stages of DC maturation (15). These results underscore the importance of ARE-1 and ARE-2, which have now been broadly observed in three prominent DR+ cell types; B cells, activated T cells, and DCs.
Although our findings of the T cell line, H9, agree with the analysis of PIII in B cell lines (19), primary T cells show a major departure, in that PIII is only expressed in activated human T cells. Correlatively, ARE-2 binding activity is only found in activated human T cells. This indicates that ARE-2 binding activity is induced upon T cell activation and strongly implies that ARE-2 binding is a critical step for the control of PIII CIITA in primary T cells. The identification of ARE-2 binding protein is crucial in further understanding how PIII is regulated in a variety of cell types.
One candidate protein that we pursued as a possible factor that
recognizes ARE-2 is CREB. This is particularly relevant, as a recent
report published during the review of this manuscript concludes that
ARE-2 is a CREB/ATF binding site (38). CREB is a protein
known to be involved in T cell activation, and it was initially
included in this study based on its role in T cell activation
(32, 33). Data from our gel shifts indicate that CREB-1 or
other family members, ATF-1 and CREM (Fig. 7, lane 6), can
bind ARE-2 or associate with the ARE-2 binding complex. However, it is
unlikely that CREB activates through association with the ARE-2 site
because 1) the phosphorylated form of CREB, which is the form required
for transcriptional activation, was not detected by gel supershift; and
2) CREB does not activate a PIII reporter through the ARE-2 site. Based
on data from the gel shift (Fig. 7), we also used a luciferase reporter
assay to assess whether ATF-1 activates PIII by its binding to ARE-2.
We show that similar to CREB, ATF-1 activated PIII, but this is
independent of an intact ARE-2 site (Fig. 8). Therefore, it is unlikely
that ATF-1 is the protein that binds ARE-2. It is crucial to
exhaustively test other CREB, ATF, and CREM family members in a
functional assay to assess their role in the activation of PIII in
primary T cells.
We also attempted to further identify the CREB recognition site in this
promoter. A potential CREB binding site is present in the -252 to
-248 region of PIII. Mutating this sequence had no effect on the
activation of PIII by CREB. One possible explanation for these findings
is that the activation of PIII by CREB occurs via an indirect pathway.
For example, the binding of CREB to promoter elements of the IFN-
gene was observed in distinct T cell populations, suggesting that CREB
may play a role in regulating IFN- gene transcription
(31). CREB may enhance PIII CIITA by inducing IFN-
expression, which then causes the up-regulation of PIII CIITA
expression.
A comparison of the recently published report shows data that are in
agreement with our findings (38). They found a
predominance of PIII usage and genomic footprints over the PIII
promoters in blood T cells. They additionally found that AML-2 can bind
to the ARE-1 site. Both papers show the functional importance of ARE-1
and ARE-2 in PIII using T cell lines, and this paper extended the
analysis to activated blood T cells. Our work shows RT-PCR and
real-time PCR analyses of different promoter usage and indicates a
prominent PIII usage, although a slight amount of PI usage is also
observed. In DCs we detected usage of all three (PI, PIII, and PIV);
however, the last is a minor population. The usage of PI, PIII, and PIV
in DCs has been recently reported by Landmann et al. (15).
Finally, the prominent use of PIII in Raji has been reported (17, 18), although we saw slight PIV usage as well. In THP-1 cells
both PIII- and PIV-induced transcripts were found, confirming previous
findings that both promoters are IFN- responsive (16, 18, 39).
In summary, this report examines the molecular control of CIITA in primary and transformed T cells to further our understanding of MHC class II gene activation/expression in these cells. Our results show a crucial role for both ARE-1 and ARE-2 in this activation. It further shows the induction of an ARE-2 binding complex in activated primary T cells, which is correlated with the importance of ARE-2 in PIII expression in these cells. This provides the first evidence of a system where ARE-2 binding is inducible and provides strong impetus to identify the ARE-2 binding protein. It also points out a notable difference between transformed and nontransformed T cells and underscores the significance of studying the latter to obtain the most physiologically relevant data.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jenny P.-Y. Ting, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599. E-mail address: panyun{at}med.unc.edu
3 Abbreviations used in this paper: DC, dendritic cell; ARE, activation response element; CIITA, class II trans-activator; CRE, cAMP response element; CREB, CRE binding protein; PI, promoter I; RLU, relative light unit; RSV, Rous sarcoma virus; ATF, activating transcription factor.
Received for publication January 7, 2002. Accepted for publication July 15, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-regulated expression of the class II transactivator gene. J. Immunol. 160:233. requires cooperative interaction between Stat1 and USF-1. Immunity 8:157.[Medline]
regulation of the type IV class II transactivator promoter in astrocytes. J. Immunol. 162:4731.
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) of the PIII isoform. B,
RT-PCR analysis of CIITA promoter expression. Primary human T cells
were immunoselected using a magnetic cell separator. RNA was harvested
from the T cells after 3 days of stimulation with PHA. RT-PCR was
performed on RNA harvested from resting (top panel) and
activated (middle panel) T cells using primers for PI,
PIII, and PIV CIITA. CD14 and CD19 primers were used to detect any
macrophage or B cell contamination. Positive control RNA from the
primary human dendritic cells (PI, PIII), THP-1 human monocyte cell
line (PIV, CD14), and Raji human B cell line (CD19, GAPDH) were
included in the RT-PCR analysis (bottom panel). Bands
shown are from different gel lanes that were cut and aligned
artificially for presentation, although they are of different sizes.
PCR products sizes are as follows: PI, 346 bp; PIII, 225 bp; PIV, 240
bp; CD14, 212 bp; CD19, 174 bp; and GAPDH, 228 bp. C,
Real-time PCR analysis of CIITA promoter expression. cDNA made from the
same RNA samples used for the RT PCR were quantified by real-time PCR.
Values are normalized with respect to 18S rRNA levels and expressed as
fold activation over resting T cells. All reactions were performed in
triplicate.
, Wild-type
PIII.545 control. Data represent three independent experiments in which
each data point was determined in triplicate. RLU values were
normalized against amount of protein in extract. C,
ARE-1 and ARE-2 are important for PIII activity in primary human T
cells. Primary human T cells were electroporated with luciferase
reporter constructs containing PIII site-specific mutations. Cells were
harvested for luciferase reporter assays 24 h post-transfection.
