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Division of Molecular Biology, Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Much of our understanding on the transcriptional control of MHC class II and class I gene expression has been derived from studies with cells obtained from patients with a MHC class II deficiency, also referred to as bare lymphocyte syndrome (2, 3, 4, 5). In bare lymphocyte syndrome patients, the genetic defect resides in the genes encoding transacting regulatory proteins required for transcriptional activation of MHC class II and class I promoters, i.e., in the genes encoding the MHC class II transactivator (CIITA)3 and the individual components of the regulatory factor X (RFX) complex (6, 7, 8, 9, 10). RFX manifests its role in transactivation through binding to the X1 box of the SXY regulatory module within the promoters of MHC class II genes (11). As such, RFX is crucial for the formation of a multimeric transcription factor protein/DNA complex with the CREB/activating transcription factor (ATF) proteins and the heterotrimeric complex NF-Y, which bind to the conserved X2- and Y-box promoter elements, respectively (12, 13, 14). CIITA functions as a coactivator by interacting with almost all of the components of this multimeric transcription factor complex bound to the SXY regulatory module, and together these regulatory proteins form a stable enhanceosome that drives transcription of MHC class II genes (15, 16, 17, 18, 19). This is achieved through interaction of the NH2-terminal activation domain of CIITA with components of the basal transcription initiation apparatus and by facilitation of chromatin remodeling through interactions with general coactivators that possess histone acetyltransferase activities (20, 21, 22, 23).
Whereas the DNA-binding components of the enhanceosome (including RFX)
are ubiquitously expressed, CIITA is only constitutively expressed in
professional APCs and is induced in nonprofessional APCs. The
expression of CIITA is controlled by four separate promoters, each
coding for distinct first exons (24). CIITA
promoter (CIITA-P)I is the promoter used in dendritic cells, and
CIITA-PII is expressed at insignificant levels and is as yet
functionally poorly understood. CIITA-PIII is constitutively expressed
in B lymphocytes and can drive CIITA expression after IFN-
stimulation in a number of different cell types, including endothelial
cells and fibroblasts (25). While the B cell-specific
expression of CIITA requires a small region directly upstream of the
initiation codon, the IFN- induction requires an additional region
located 
5 kb upstream of the transcriptional start site
(25). CIITA-PIV is the principal IFN--inducible
promoter (24, 25, 26, 27, 28, 29). The cellular and temporal diversity in
MHC class II expression is thus regulated via the differential usage of
the CIITA promoters.
Recently, several functional domains in CIITA-PIV and CIITA-PIII have
been characterized. For the IFN--mediated induction of CIITA-PIV
cooperative binding of Stat1 and upstream stimulatory factor-1 to a
IFN- activation site-like element, and binding of IFN regulatory
factor 1 to an IFN-stimulated response element-like sequence in
CIITA-PIV, are critical (25, 27, 28). Functional analysis
of CIITA-PIII showed that the region immediately upstream of the
translational start site is sufficient to confer B lymphocyte-specific
expression of a reporter gene construct (25, 30, 31).
Subsequently, the in vivo and in vitro footprinting analysis revealed
the existence of five sites that were found occupied in CIITA-PIII in
the B cell line Raji (31). Two of these protein-binding
DNA sites, activation response element (ARE)-1 and ARE-2, appeared to
be absolutely required for transactivation of CIITA-PIII in B
lymphocytes (31), whereas mutations in the protein-binding
sites A, B, and C did not have a major impact on CIITA-PIII promoter
activity in these type of cells.
Interestingly, activation of human T cells leads to the expression of MHC class II molecules on the cell surface. This MHC class II expression on activated T lymphocytes implies potential Ag presentation by T cells, which is supported by the observation that activated T cells are capable of Ag uptake and subsequent presentation of processed Ag to other T cells (32, 33, 34, 35, 36, 37, 38). It is believed that these T-T interactions play an important regulatory role in the immune response (37, 38).
To gain insight into the regulation of MHC class II expression in activated human T cells, CIITA promoter usage and the transcription factors involved in CIITA expression were determined. In this study, we show that CIITA expression in human activated CD4+ T cells is driven by CIITA-PIII only. Notably, despite the shared usage of CIITA-PIII, differences in CIITA-PIII occupation were observed between activated T cells and Raji B cells. Furthermore, EMSA studies showed differences in banding pattern in activated T cells and Raji B cells. This probably reflects the difference in CIITA activation pathways employed by T cells (induced expression) and Raji B cells (constitutive expression).
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Materials and Methods |
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The Jurkat cell line was purchased from the American Type Culture Collection (Manassas, VA; Jurkat clone E6-1, TIB-152) and cultured in RPMI 1640 (Life Technologies, Breda, The Netherlands) supplemented with 10% heat-inactivated FBS (Greiner, Alphen a/d Rijn, The Netherlands), 100 U/ml streptomycin, 100 U/ml penicillin, and 2 mM L-glutamine.
Naive CD4+ T cell isolation and stimulation
Peripheral blood was obtained from normal healthy donors by venapuncture procedure, and PBMCs were isolated using a Ficoll gradient (Pharmacy Leiden University Medical Center, Leiden, The Netherlands). To obtain naive and in vivo activated CD4+ T cells, PBMCs were stained with FITC-coupled HLA-DR (1:8) and PE-coupled CD4 (1:32) Abs (both from BD Biosciences, Mountain View, CA) in PBS containing 5% heat-inactivated FBS (Greiner) for 30 min at 4°C. CD4+DR- (naive CD4+ T cells) and CD4+DR+ (in vivo activated CD4+ T cells) cells were separated and collected using the FACSVantage SE (BD Biosciences). To increase cell numbers, the in vivo activated T cells were, after isolation, further propagated with 10 µg/ml PHA and irradiated allogenic PBMCs (3000 rad) in RPMI 1640 (Life Technologies) supplemented with 10% human serum, 10 U/ml IL-2, 100 U/ml streptomycin, 100 U/ml penicillin, and 2 mM L-glutamine. To obtain in vitro activated CD4+ T cells, naive CD4+ T cells were, after isolation, stimulated once with 1 µg/ml anti-CD3, 1 µg/ml anti-CD28 (both from Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands), and irradiated allogenic PBMCs (3000 rad), and further propagated in RPMI 1640 (Life Technologies) supplemented with 10% human serum, 10 U/ml IL-2, 100 U/ml streptomycin, 100 U/ml penicillin, and 2 mM L-glutamine.
Isolation of human primary B cells
Primary B cells were isolated from human spleen using Dynabeads CD19 pan B (Dynal Biotech, Oslo, Norway), and after positive selection the beads were removed from the cells by DETACHaBEAD CD19 (Dynal Biotech) according to the manufacturer’s instructions.
RNA isolation and RT-PCR analysis
Total RNA was isolated from the different T cell populations using the RNAzol extraction method (Cinna/Biotecx Laboratories, Houston, TX). RNA samples (2 µg) were transcribed into cDNA using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). The cDNA was then subjected to PCR using oligonucleotide primers specific for CIITA-PI, CIITA-PIII, CIITA-PIV, and GAPDH: CIITA-PI sense, 5'-GGTGAGCGGAGATTCCAGGCACTGG-3'; CIITA-PIII sense, 5'-GATTCCTACACAATGCGTTGCCTGG-3'; CIITA-PIV sense, 5'-AGCTGGCGGGAGGGAGAGGCCACC-3'; CIITA-PI/PIII/PIV antisense, 5'-CATACTGGTCCAGTTCCGCGATATTGG-3'; GAPDH sense, 5'-GGTCGGAGTCAACGGATTTG-3'; and GAPDH antisense, 5'-ATGAGCCCCAGCCTTCTCCAT-3'.
One microliter of cDNA was amplified in 1x PCR buffer II (Roche Diagnostics, Mannheim, Germany), 0.3 mM NucleixPlus PCR nucleotide mix (Amersham Pharmacia Biotech, Piscataway, NJ), 0.4 µM of both sense and antisense primers, 2.5 U AmpliTaq DNA polymerase (Roche), and MgCl2 (Roche) in a total volume of 50 µl. The final concentration of MgCl2 was for 1.25 mM CIITA-PI, 1.5 mM CIITA-PIII, 1.5 mM CIITA-PIV, and 4 mM GAPDH. The PCR conditions used for CIITA-PI, CIITA-PIII, and CIITA-PIV were as follows: denaturation for 1 min at 94°C, annealing for 1 min at 61°C, and extension for 1 min at 72°C for a total of 35 cycles; the PCR conditions used for GAPDH were as follows: denaturation for 1 min at 94°C, annealing for 1 min at 60°C, and elongation for 1 min at 72°C for a total of 25 cycles. The PCR products were visualized on an ethidium bromide-stained agarose gel, and colors were reversed for reasons of clarity.
IVGF analysis
Dimethyl sulfate treatment of cells, preparation of genomic DNA, and ligation-mediated PCR were performed as described by Mueller and Wold (39), with minor modifications (40). Three CIITA locus-specific primers and 2.5 µg of methylated genomic DNA were used to amplify cleaved fragments from the noncoding strand of CIITA-PIII: P3.31, 5'-GCAACGCATTGTGTAGGAATCCC-3'; P3.32, 5'-GCCCAGCTCAGAAGCACACAGCCTCATCAC-3'; P3.33, 5'-GCACACAGCCTCATCACTAGCCTCA-3'.
Electrophoretic mobility gel shift assay
Nuclear extracts. Purified nuclear extracts from naive CD4+ T cells, in vivo and in vitro activated CD4+ T cells, Raji B cells, and primary B cells were obtained as described by Kaminuma et al. (41). In vivo and in vitro activated T cells and primary B cells were derived as described above; naive CD4+ T cells were isolated from Ficoll-purified PBMCs using the human CD4+ T cell enrichment columns (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.
EMSA. Two microliters of nuclear extracts, containing approximately 4 µg/µl nuclear proteins, were incubated in the following binding buffer: 10 mM HEPES (pH 7.9), 60 mM KCl, 10% v/v glycerol, 1 mM EDTA, 10 mM Na3PO4, 1 mM DTT, 3 mM MgCl2, 1 µg of poly(dI-dC), and 1 µg of sonicated salmon sperm ssDNA, with 2 ng of 32P-labeled dsDNA probe for 30 min at 4°C. The samples were run on a 6% polyacrylamide gel in 0.25x Tris-borate-EDTA buffer at 200 V for 150 min. The following oligonucleotides were used as probe: CRE consensus, sense 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' and antisense 5'-CTAGCTCTCTGACGTCAGGCAATCTCT-3'; wt-PIII-ARE-2, sense 5'-GAGTTTTTTTGATGATCCCTCACTTGT-3' and antisense 5'-ACAAGTGAGGGATCATCAAAAAAACTCTC-3'; Wt-PIII-ARE-1, sense 5'-GAGGAGGGCTTAAGGGAGTGTGGTAAAATT-3' and antisense 5'-AATTTTACCACACTCCCTTAAGCCCTCCTC-3'; site A, sense 5'-TTAGCTTGGCGGGCTCCCAACTGG-3' and antisense 5'-CCAGTTGGGAGCCCGCCAAGCTAA-3'; NF-1 consensus, sense 5'-TTTTGGATTGAAGCCAATATGATAA-3' and antisense 5'-TTATCATATTGGCTTCAATCCAAAA-3'; site B, sense 5'-ACTTGTTTCTTTGCATGTTG-3' and antisense 5'-CAACATGCAAAGAAACAAGT-3'; site C, sense 5'-CAGTCCACAGTAAGGAAGTGAAATTA-3' and antisense 5'-TAATTTCACTTCCTTACTGTGGACTG-3'; mutant site C, sense 5'-CAGTCCACAGTATCCTAGTGAAATTA-3' and antisense 5'-TAATTTCACTAGGATACTGTGGACTG-3'; the changed nucleotides are underlined. Due to the limited amount of in vivo activated T cell nuclear extract, we performed the EMSA with site A and site B oligonucleotides only with nuclear extract from in vitro activated T cells, as these elements were found not to be critical for CIITA-PIII activation in Jurkat T cells.
Supershift assays were performed by incubating nuclear extract and oligonucleotide for 30 min on ice, after which 1 µg of Ab was added and incubation was continued for an additional 60 min on ice. The following Abs were used for supershift assays: ATF/CREB (sc-270) reactive with ATF-1 p35, CREB-1 p43, and cAMP responsive element modulator-1; CREB-1 (sc-271) reactive with CREB-1 p43; ATF-1 (sc-243) reactive with ATF-1 p-35; PU.1 (sc-352); Pip (sc-6059; all obtained from Santa Cruz Biotechnology, Santa Cruz, CA), acute myeloid leukemia (AML)1 (PC284); AML2 (PC286); AML3 (PC287) (all obtained from Oncogene Research Products, San Diego, CA).
Reporter and expression constructs
The pGL3-CIITA-PIII reporter construct was generated by cloning a 668-bp region of pGL2-CIITA-PIII, which contains the sequence -545 to +123 (obtained by J. P.-Y. Ting, University of North Carolina, Chapel Hill, NC) into the pGL3-basic luciferase reporter plasmid (Promega). The pGL3-CIITA-PIV construct was previously described by van den Elsen et al. (42). Several 5' truncations of CIITA-PIII were generated. The -145- to +123-bp or the -113- to +123-bp fragment of CIITA-PIII was created by PCR using the GL2 primer (Promega) and either the pIII-145 sense primer (5'-TAAGGGAGTGTGGTAAAATT-3') or the pIII-113 sense primer (5'-AGATCTGAAACAGAAATCTGACCGCTTGG-3') and pGL3-CIITA-PIII as template. The PCR products were subsequently cloned into the pGL3-basic luciferase reporter plasmid (Promega). Mutations in ARE-1, ARE-2, and site C were introduced by site-directed mutagenesis using overlapping extension PCR (43). Mutation primers for the PU.1 site in site C are described above. Mutation primers for the AML site at ARE-1 were as follows: AML.M sense, 5'-CTTAAGGGAGAAGCTTAAAATTAG-3'; AML.M antisense, 5'-CTAATTTTAAGCTTCTCCCTTAAG-3'. Mutation primers for ARE-1 were as follows: ARE-1.M sense, 5'-GGGCTTAAGTCTAGATGGTAAAATT-3'; ARE-1.M antisense, 5'-AATTTTACCATCTAGACTTAAGCCC-3'. Mutation primers for the CRE site in the ARE-2 binding site were as follows: ARE-2.M sense, 5'-GAGTTTTTTTGACCGGGCTCACTTGTTTC-3'; ARE-2.M antisense, 5'-GAAACAAGTGAGGCCCGGTCAAAAAAACTC-3', the changed nucleotides underlined. All constructs were verified by DNA sequencing at the Leiden Genome Technology Center (Leiden, The Netherlands).
Transient transfections
The human T lymphocyte cell line Jurkat was transfected by electroporation (Genepulser; Bio-Rad, Richmond, CA) at 250 V and 960 µF, and harvested after 72 h. Electroporations were performed with 8 x 106 cells and 10 µg of luciferase pGL3-reporter construct together with 2.5 µg of SV40-Renilla pGL3-reporter construct (Promega) as an internal control. Luciferase and Renilla luciferase activity were measured using the Dual Luciferase Assay kit according to the manufacturer (Promega) and normalized for transfection efficiency with the Renilla luciferase activity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First we have investigated which of the CIITA promoters is used to
drive CIITA expression in
CD4+HLA-DR+ T cells. Two
populations of CD4+HLA-DR+
T cells were obtained, which we designated in vivo activated
CD4+ T cells and in vitro activated
CD4+ T cells. In vivo activated
CD4+ cells were obtained by gating
CD4+HLA-DR+ from PBMC, and
to increase cell numbers these cells were propagated in vitro using
PHA, IL-2, and irradiated allogenic PBMCs. In vitro activated
CD4+ T cells were derived from
CD4+HLA-DR--gated PBMC and
stimulated in vitro using CD3 and CD28 Abs, IL-2, and irradiated
allogenic PBMCs. To detect which CIITA promoter was used in activated T
cells, a RT-PCR with primer sets specific for CIITA-PI, CIITA-PIII, and
CIITA-PIV was applied. The results of these analyses showed that both
the in vivo and the in vitro activated CD4+ T
cell populations employ CIITA-PIII only (Fig. 1
). This implies that CIITA-PIII is thus
not exclusively used in B cells but is also the principal promoter
employed in activated T cells.
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To compare promoter occupation between naive and activated T
cells, and between activated T cells and Raji B cells, in vivo genomic
footprinting (IVGF) analysis was employed to visualize the protein/DNA
interactions on CIITA-PIII. The lower strand from nucleotide -250 to
+10 of the CIITA-PIII promoter was analyzed using dimethyl sulfate as a
modifying agent. Analysis of equal amounts of methylated genomic DNA by
IVGF analysis showed clear differences in promoter occupation between
naive and activated T cells (Fig. 2A, summarized in Fig. 2B). In the screened region, naive T cells have no
protection, as its footprint pattern is comparable with that of Jurkat
DNA, in which the proteins were removed before methylation (see Fig. 2A, in vitro methylated DNA).
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A second strong hypermethylation in activated T cells is seen at the ARE-2. Again the protection pattern at the ARE-2 site differs between activated T cells and Raji B cells; nucleotide -64 is enhanced in activated T cells, while this guanine residue is partially protected in Raji B cells. Furthermore, the guanine at position -61 is enhanced in Raji B cells, while in activated T cells this guanine is neither enhanced nor protected.
A third region with strong protection in activated T cells is seen between site A (nucleotide -13 to -25) and site B (-38 to -45). At site A, a cluster of four protected guanine residues is found in activated T cells, while in Raji B cells only two guanine residues are partially protected. Moreover, directly downstream of site B, protected guanine residues were detected in activated T cells. Also, we found a few protections or enhancements of promoter DNA upstream of the ARE-1 binding site and at site C. Overall, the CIITA-PIII in activated T cells shows more protected guanine residues outside the described Raji B cell binding regions, and within these areas the protection pattern differs between activated T and Raji B cells.
Transcription factor binding to CIITA-PIII in T cells
The IVGF analysis showed that the protection pattern at several CIITA-PIII binding sites differs between activated T cells and Raji B cells. This difference in protection pattern might be due to the binding of different transcription factors to these regulatory elements in activated T and Raji B cells. To determine which transcription factors bind to the different regulatory elements, EMSA with DNA sequences of these elements was utilized. Naive T nuclei possess low numbers of nuclear proteins (approximately seven times fewer proteins than activated T cells), which makes it difficult to observe qualitative differences. Therefore, equal amounts of nuclear proteins were used in the assays described below instead of equal numbers of cells.
ARE-1
Incubation of an oligonucleotide containing the ARE-1 binding site
and nuclear extract from naive T cells, in vivo activated T cells,
primary B cells, or Raji B cells revealed a complex, complex 1,
specifically present in activated T cells (Fig. 3, lane 6, arrow 1), and a
complex, complex 2, specifically present in naive T cells and Raji B
cells (Fig. 3, lanes 1 and 11, arrow 2). This
complex was difficult to detect in primary B cells. No differences were
found in banding pattern between in vivo and in vitro activated T cells
(not shown). The ARE-1 site displayed a high homology with the binding
site for proteins of the AML/core-binding factor (CBF)
/Runx family.
In a supershift assay, addition of the AML1 Ab did not reveal the
presence of AML1 in the complex (Fig. 3, lanes 3,
8, 13, and 17), whereas AML2 and AML3
were readily detected. Addition of AML2 Ab shifted complex 1 in
activated T cells (Fig. 3, lane 9, from arrow 1 to arrow
2*). Moreover, in naive T cells and Raji B cells, complex 2 is also
shifted with this Ab (Fig. 3, lanes 4 and 14,
from arrow 2 to arrow 2*). These two different AML2 complexes might
reflect the presence of an isoform of AML2 or an additional factor in
the complex. Inclusion of the AML2 Ab also resulted in the formation of
an additional complex in primary B cells. Interestingly, addition of
the AML3 Ab to the binding reaction resulted in the formation of a
large complex in naive and activated T cells, but not with primary B
cells or Raji B cell nuclear extract (Fig. 3, lanes 5,
10, 15, and 19, arrow 3*). However, it
is not clear which DNA-protein complex is shifted by the AML3 Ab. Thus,
AML3 binds the ARE-1 site in naive and activated T cells, but not in B
cells, and a specific AML2/ARE-1 complex is formed in activated T
cells.
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Next, an oligonucleotide spanning the ARE-2 binding site was
used to test transcription factor binding to this site. Three specific
complexes were seen using the nuclear extracts of naive T cells, in
vivo activated T cells, Raji B cells (Fig. 4, lanes 1, 5, and
9, indicated by arrows), and in vitro activated T cells and
primary B cells (not shown). The ARE-2 sequence shows homology with
binding sites for members of the CREB/ATF family of transcription
factors, and in Raji B cells it has been shown that CREB/ATF can bind
to ARE-2 (N. Van der Stoep, E. Quinten, and P. J. van den Elsen,
unpublished observations). Also in T cells, Abs recognizing CREB/ATF
proteins were able to supershift the complexes (Fig. 4, bracket). The
upper complex can be supershifted with an Ab specific for CREB-1 (Fig. 4, lanes 2, 6, and 10), while the
lower complex supershifts with a specific ATF-1 Ab (Fig. 4, lanes
4, 8, and 12). All three complexes can be
supershifted with an Ab broadly reactive with CREB and ATF members
(Fig. 4, lanes 3, 7, and 11).
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The site A sequence formed a complex (Fig. 5A, arrow 1) with a factor
predominantly present in in vitro activated T cells. A more slowly
migrating complex (Fig. 5A, arrow 2) can be seen using Raji
B cell nuclear extracts, but not in an activated T cell extract. This
later complex was previously suggested to be a NF-1 (related)
transcription factor (31). Indeed, an oligonucleotide
containing the consensus NF-1-binding sequence was able to eliminate
the upper complex in Raji B cells (Fig. 5A, arrow 2), but
not the lower complex in T cells (Fig. 5A, arrow 1; data not
shown).
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B, bracket). The sequence of site B displays weak homology
with an octamer factor-binding site. However, it was shown previously
that only very weak binding of octamer-binding transcription factor-1
could be detected at site B (31). Because site B has no
further apparent homology with any transcription factor-binding site
known to date, it remains unclear which proteins bind to site B in
activated T cells and Raji B cells. Site C
Incubation of a nuclear extract from in vivo and in vitro
activated T cells with an oligonucleotide containing the sequence of
site C resulted in the formation of a specific activated T cell complex
(Fig. 6, lane 2, arrow 1; and
data not shown), which is not detectable in nuclear extracts from naive
T cells or Raji B cells (Fig. 6, lanes 1 and 3).
Furthermore, in Raji B cells, two specific complexes are formed (Fig. 6, lane 3, arrows 2 and 3). Site C has a high homology, with
the binding site of transcription factors belonging to the Ets family.
An oligonucleotide containing site C with a mutated Ets binding site
was not able to form both complex 1 in activated T cells and complex 3
in Raji B cells (Fig. 6, lanes 7 and 10),
indicating that these complexes are formed by Ets motif-binding
factors. Because the Ets protein containing complex 1 is formed only in
activated T cells, mutation of this Ets binding site results in the
same banding pattern with naive and activated T cell nuclear extract.
Furthermore, in Raji B cells, we now demonstrate that the Ets family
member PU.1 and its activating partner Pip/IFN regulatory factor 4/IFN
consensus sequence binding protein activated T cells are able to bind
to site C. Incubation of wild-type site C in the presence of PU.1 or
Pip-specific Abs showed that complex 2 is formed by Pip (Fig. 6, lane 11) and complex 3 by PU.1 (Fig. 6, lane 12).
In accordance with the fact that PU.1 is not expressed in T cells, the
PU.1 Ab was not able to change the binding pattern in T cells (Fig. 6, lane g). Thus, in Raji B cells PU.1 and Pip are capable of
binding to site C, while in activated T cells site C probably binds an
Ets-like protein.
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To correlate protein/DNA interactions with CIITA-PIII
transcriptional activity in T cells, several 5' truncated promoter
constructs and mutant promoter constructs were generated and
transiently transfected in the Jurkat T cell line (Fig. 7). The activity of these truncated and
mutant CIITA-PIII promoter constructs was compared with the activity of
a wild-type CIITA-PIII promoter fragment encompassing nucleotides -545
to +123. The activity of CIITA-PIII-145, lacking site C, was about 75%
of the wild-type CIITA-PIII promoter activity. Mutation of the PU.1
site in wild-type CIITA-PIII site C results in a decreased CIITA-PIII
activity to 60%, which is comparable with the reduction in activity
seen with CIITA-PIII-145. Deletion of the promoter region containing
both site C and ARE-1 in CIITA-PIII-113 caused a dramatic drop in
promoter activity to 18%. When in wild-type CIITA-PIII the AML-binding
sequence at ARE-1 was mutated, promoter activity decreased to 38% (not
shown). Moreover, mutation of the entire ARE-1 site in wild-type
CIITA-PIII completely abolished CIITA-PIII activity. Also, mutation of
the CRE site at the ARE-2 annihilated CIITA-PIII activity, revealing
that both ARE-1 and the ARE-2/CRE site are of crucial importance for
CIITA-PIII promoter activity in Jurkat T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Moreover,
naive T cells displayed an almost bare CIITA promoter occupancy, as
analyzed by IVGF (Fig. 2A). This lack of transcription
factor binding might relate to the low amount of nuclear protein per
naive T cell. However, when in the EMSA analysis equal amounts of
nuclear protein were used, different banding patterns were observed in
naive T cells and activated T cells with ARE-1, and site A, B, and C
oligonucleotides (Figs. 3, 5, and 6). Thus, naive and activated T cells
not only differ from each other with respect to the amount of nuclear
protein per cell, but also differ qualitatively with respect to the
proteins able to interact with the various regulatory elements. It was found that of the four CIITA promoters, only CIITA-PIII is used to drive CIITA expression in both in vivo and in vitro activated T cells. CIITA-PIII is thus, besides by B cells, also employed by activated T cells. However, B cells have constitutive CIITA expression, while in activated T cells CIITA expression is induced, suggesting the presence of alternative activation pathways of CIITA-PIII. In accordance, IVGF analysis revealed differences in promoter occupation between activated T cells and Raji B cells. A prominent difference was seen in promoter occupation at the ARE-2 site. For Raji B cells, it has already been shown that the ARE-2 element is a binding site for members of the CREB/ATF family of transcription factors (N. Van der Stoep, E. Quinten, and P. J. van den Elsen, unpublished observations). We now show that in activated T cells CREB/ATF family members also can bind the ARE-2 element of CIITA-PIII. Although the promoter occupancy differs at the ARE-2 site between activated T cells and Raji B cells, EMSA analysis did not reveal any differences in the specific banding pattern between these cell types. On the one hand, it is possible that the conditions used in our EMSA analysis did not support the formation of protein/DNA complexes that occurs in activated T cells in vivo. On the other hand, the IVGF analysis showed that CIITA-PIII upstream of ARE-2, in contrast to Raji B cells, is highly occupied in activated T cells. Factors binding to this region might interact with CREB/ATF at the ARE-2 site, causing differences in protection pattern in the genomic footprint compared with Raji B cells, or, alternatively, occupation of this region leads to a conformational change of the DNA and to a change in methylation pattern. Besides in activated T cells and Raji B cells, in primary B cells and naive T cells CREB/ATF binding also was demonstrated with the ARE-2 sequence. Yet, because the IVGF analysis revealed that the ARE-2 is not occupied in naive T cells, these data suggest that in naive T cells CREB/ATF is already present in very low amounts in the cell but is recruited to ARE-2 following T cell activation. This might be achieved through the activities of coactivators and/or, as discussed above, by the increased amounts of CREB/ATF in the nuclei of activated T cells.
Also, differences in promoter occupation between B and T cells were found at the ARE-1 site. The ARE-1 site in CIITA-PIII was proposed to be the binding site of a transcriptional enhancer factor-2-like transcription factor, as suggested by competition with a transcriptional enhancer factor-2 consensus oligonucleotide (31). Using specific Abs, we now show that the ARE-1 sequence can be bound by AML2, and that the banding pattern differs among activated T cells and naive T cells, primary B cells, or Raji B cells. It suggests that in activated T cells ARE-1 is bound by an AML2 isoform or, alternatively, that AML2 is complexed at ARE-1 with additional factors in naive T cells and Raji B cells. Notably, we also show that binding of AML3 to the ARE-1 site was specific for T lymphocytes because no AML3 Ab supershift is observed with nuclear extracts of Raji B cells or primary B cells.
All members of the AML family are capable of forming a heterodimer with
the partner subunit CBF
and form the CBF complex. Cooperative
binding of CBF and Ets-1 to their separate binding sites has been
demonstrated (45, 46), and this cooperative binding is
relatively independent of the orientation and distance between the two
binding sites (45). In this respect, it is of interest to
note that CIITA-PIII contains both a CBF and an Ets binding site. An
AML binding site has been located next to ARE-1, and site C has been
shown to be capable of binding Ets family members (PU.1 in Raji B
cells, and an Ets-like protein in activated T cells). The CIITA-PIII
activity in activated T lymphocytes and Raji B cells might be the
result of cooperative binding of these Ets and AML transcription
factors. However, this cooperative binding of these factors may not be
of crucial importance for CIITA-PIII activation because mutation of the
Ets motif at site C and the AML site at ARE-1 has only moderate effects
on CIITA-PIII activity. However, mutation of the entire ARE-1 sequence
totally annihilates promoter activity, showing that ARE-1 is conclusive
for CIITA-PIII activity in T cells. Also, CREB/ATF
transcription factor binding is of crucial importance for CIITA-PIII
activation, because deletion of the CRE site in ARE-2 totally
diminishes CIITA-PIII activity in T cells.
Previously, it has been shown that also in Raji B cells, mutation of
the ARE-1 sequence results in a strong reduction of CIITA-PIII activity
(31). In Raji B cells the CIITA-PIII activity is reduced
to 25%, while in Jurkat T cells the same mutation completely abolishes
CIITA-PIII activity, suggesting a more potent role of the ARE-1 binding
site in T cells than in B cells. Mutation of the ARE-2 binding site in
CIITA-PIII results in complete loss of promoter activity in both Raji B
cells (31) and Jurkat T cells, indicating that this site
is equally important for both T and B cell CIITA-PIII activity.
Considerable difference is seen between B and T cells when site C is
mutated. In Raji B cells there is an increase of promoter activity to
130% (31), while in Jurkat T cells there is a
reduction to 60% of the wild-type promoter activity. It should be
noted that the mutation in site C, as described in this study,
specifically affects the PU.1 binding site and therefore differs from
that designed by Ghosh et al. (31), which affects the
complete nucleotide sequence of site C.
Although it is a recognized fact that activated human T lymphocytes express MHC class II molecules on their membrane, the precise function of these molecules on T cells is not yet fully established. It has been demonstrated that MHC class II molecules expressed on activated T cells are capable of Ag presentation. However, there are some controversies concerning MHC class II expression and Ag presentation by T cells. On the one hand, it is reported that activated T cells act as professional APCs, in that they deliver costimulatory signals to other T cells (38). Also, the expression of CD80/B7-1 at the surface of activated T cells implies that T cells can stimulate bystander T lymphocytes. On the other hand, it has been suggested that activated T cells provide down-regulatory signals for the immune response by inducing anergy in activated T cells and cytotoxicity in resting T cells (47). Besides potential APC activity, it is also reported that MHC class II molecules can transduce signals into T lymphocytes, which results in protein kinase C membrane translocation and inositol phosphate accumulation (48). Moreover, simultaneous triggering of CD3 and MHC class II molecules also leads to an increase in CD3-mediated T blast proliferation (48). Thus, MHC class II expression on T cells indirectly affects the activity of bystander T lymphocytes but can also provide signals that further enhance T cell proliferation and activity.
Recent findings have shown that CIITA, besides its central role in the transcriptional regulation of Ag presentation, either directly or indirectly, also affects the transcriptional expression patterns of other genes. For instance, it has been demonstrated that CIITA modulates IL-4 expression through interference with the general coactivator CREB binding protein (49). In addition, it has been reported that CIITA is capable of suppressing the Fas:Fas ligand pathway by inhibiting transcription of the Fas ligand gene through its promoter (50), and that expression of CIITA in Jurkat T cells enhances HIV-1 replication and transcriptional activity of the HIV long terminal repeat (51). All these data suggest additional functions of CIITA in the regulation of T cell-mediated control of the immune response.
In conclusion, CIITA-PIII is the principal CIITA promoter employed by human activated T cells to drive expression of CIITA. While the IVGF analysis of naive T cells showed no transcription factor binding on CIITA-PIII, several DNA/protein interactions were found to be specific for activated T cells compared with Raji B cells. In accordance, using band shift assays, differences in factor binding were found among activated T cells, naive T cells, primary B cells, and Raji B cells at established regulatory sites. Some of these DNA binding factors were identified as CREB/ATF, AML/Runx, and Ets family members.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter J. van den Elsen, Division of Molecular Biology, Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail address: pjvdelsen{at}lumc.nl
3 Abbreviations used in this paper: CIITA, class II transactivator; CIITA-P, CIITA promoter; AML, acute myeloid leukemia; ARE, activation response element; ATF, activating transcription factor; CBF, core-binding factor; IVGF, in vivo genomic footprinting; RFX, regulatory factor X.
Received for publication September 18, 2001. Accepted for publication November 15, 2001.
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