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B1
Department of Immunohematology and Blood Bank, Leiden University Medical Center, Leiden, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B motifs of enhancer A
(B1 and B2) of the classical and nonclassical HLA class I genes
were investigated for their binding properties of transcription factors
and tested for their contribution to the NF-B-induced route of
transactivation. It was shown that NF-B-induced transactivation
through enhancer A is most important for the HLA-A locus,
which contains two NF-B binding sites. Although the enhancer A of
HLA-B contains only one NF-B binding site (B1), there
was still a moderate transactivation by NF-B. Since
HLA-F, which also possesses one NF-B binding site but
lacks protein binding to its B2 site, was not transactivated by
NF-B, the NF-B-mediated transactivation through the B1 motif
in HLA-B is most probably facilitated by binding of the
transcription factor Sp1 to the upstream B2 site. Thus,
transcriptional regulation of HLA class I genes by NF-B is
restricted to the HLA-A and HLA-B
loci. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The regulation of MHC class I gene expression is mediated by several
conserved cis-acting regulatory elements clustered in the
promoter region of MHC class I genes. These include the enhancer A
element, the IFN-stimulated response element (ISRE) and site 
(reviewed in Refs. 2, 7, and 8–10).
Enhancer A is the target binding site for transcription factors of the
NF-B/Rel family and is thought to be essential for constitutive and
cytokine-induced gene expression. The B motif GGGGATTCCCC in
enhancer A of the HLA class I gene promoters is highly conserved,
particularly in the HLA-A and HLA-B loci (2).
This is a symmetrical variant of the more divergent B site in the
promoter of the Ig -light chain gene (GGGACTTCC (11)). Although the
B motif is the principal target sequence for proteins of the
NF-B/Rel family, it is also bound by several other DNA-binding
proteins, such as the high mobility group protein I(Y)
(HMG I(Y))3 and proteins that
belong to the leucine zipper family of transcription factors (reviewed
in Refs. 12 and 13). The NF-B/Rel family of transcription factors
comprises at least five members, p50, p65 (also termed RelA), p52,
c-Rel, and RelB, that bind DNA either as homo- or heterodimers
(reviewed in Refs. 12 and 13). These dimers posess different binding
affinities for B sites and their half-sites, as well as different
transcriptional properties (12, 13, 14, 15, 16).
The p50-p65 heterodimer, termed NF-B, is present in virtually all
differentiated cells and is the most abundant of the NF-B/Rel dimers
(12, 13). NF-B is normally retained in the cytoplasm complexed to
the inhibitory protein IB (reviewed in Refs. 16–18). Following
activation by stimuli such as cytokines and phorbol esters, IB is
inactivated and degraded, after which NF-B is released and
subsequently translocated to the nucleus (reviewed in Refs. 12 and 13).
Interaction of NF-B with the B site results in transactivation of
MHC class I and a variety of other genes, including those encoding
cytokines and viral genes.
The level of gene transcription of the various MHC class I loci is
determined by tissue-specific levels of expression of the NF-B/Rel
family proteins, their differential binding affinity for a particular
B site, and the transactivation capacities of the different dimers
(reviewed in Refs. 12 and 13). The NF-B subunits p65 and c-Rel both
have a transactivation domain, although p65 is the more potent of the
two. The NF-B subunit p50, lacking such transactivation domain, is
thought to have no transactivating capacity as a homodimer but rather
to fulfill an ancillary function for the p65 and c-Rel subunits.
However, since in cell-free assays the p50 homodimer can transactivate
MHC class I (19), its exact role in transactivation is still unclear.
The human classical and nonclassical MHC class I genes differ in their
level of constitutive expression in different cell types and also in
their cytokine-induced expression patterns (reviewed in 2 .
Locus-specific variation in the two putative B sites of the enhancer
A elements (2, 20, 21) determines their binding capacity and
functioning. Locus-specific contribution of the enhancer A element to
transactivation may be an important mechanism in the differential
regulation of the constitutive and cytokine-induced levels of classical
and nonclassical HLA class I expression in various cell types.
In this study, we assessed the capacity of the two putative B sites
within the enhancer A region in the promoter of the classical and
nonclassical HLA class I genes to bind proteins of the NF-B/Rel
family of transcription factors and to mediate transactivation by
NF-B. The NF-B-mediated transactivation of HLA class I genes is
restricted to the HLA-A and HLA-B loci and seems
to require binding not only of NF-B to the B1 site, but also of
either NF-B or Sp1 to the B2 site.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cells used were the teratocarcinoma cell line Tera-2 and the B lymphoblastoid cell line CCRF-SB (American Type Culture Collection, Manassas, VA). The cells were grown in Iscove’s modified DMEM supplemented with 10% (v/v) heat-inactivated FCS (Life Technologies, Paisley, Scotland), penicillin (100 IU/ml), and streptomycin (100 µg/ml).
Plasmids
Reporter constructs pGL3-A230 and pGL3-A140 were generated by
cloning a 228-bp BglI-AhaII HLA-A*0201promoter fragment or a 143-bp PpuMI-AhaII
HLA-A*0201 promoter fragment, respectively, upstream of the
firefly luciferase gene in pGL3-Basic (Promega, Madison, WI). Enhancer
A-containing reporter constructs were generated by cloning
ds-oligonucleotides of the enhancer A sequence (containing both the
B1 and B2 motifs) from the various HLA class I genes upstream of
the 143-bp HLA-A*0201 promoter fragment in pGL3-A140.
The expression vector constructs of pRSV-p50 and pRSV-p65 (a kind gift
of Dr. L. Struyk, Academic Medical Centre, Amsterdam, The Netherlands)
contain the 1.3-kb cDNA and 2.6-kb cDNA fragments encoding NF-B-p50
and NF-B-p65, respectively, and were generated by inserting the
cDNAs into pRSVNeo (22) from which the NeoR gene
has been deleted.
Transient transfection
Cells were transfected by the calcium phosphate coprecipitation
method of Chen and Okayama (23). In each of four wells of a six-well
plate, 0.2 x 106 Tera-2 cells were transfected with a
DNA mix containing 2.5 µg of pGL3 reporter plasmid, 2.5 µg of
pRSV-lacZ plasmid, and 5 µg of pRSV (control) or 2.5 µg of pRSV-p50
and pRSV-p65 (NF-B) each and harvested 2 days after
transfection. Luciferase activity was determined using a luminometer
(Tropix, Badford, MA) and corrected for transfection efficiency by
measuring ß-galactosidase activity. Transfection of pGL3-Basic was
used as a reference to correct for the possible effects of
cotransfection with the expression vectors. The relative luciferase
values are given as mean relative light units ± SD
(n = 4).
Preparation of nuclear extracts
Nuclear extracts were prepared from 10 x 106 cells. The cells were harvested, washed with PBS, taken up in 300 µl (three cell volumes) of hypotonic solution (20 mM HEPES, pH 8.0, 10 mM KCl, 0.15 mM EGTA, 0.15 mM EDTA, 1 mM DTT, 0.5 mM 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF)), and then left on ice for 15 min. The cells were lysed with Nonidet P-40 (final concentration of 0.1% for CCRF-SB cells; 0.2% for Tera-2) for 3 to 5 min. Then, 80 µl (80% of the cell volume) of a sucrose solution (50 mM HEPES, pH 8.0, 10 mM KCl, 0.25 mM EDTA, 1 mM DTT, 0.5 mM AEBSF, 70% (w/v) sucrose) was added, and the nuclei were centrifuged at 5000 rpm for 5 min at 4°C. The supernatant was discarded, and the pellet was gently taken up in 300 µl (three cell volumes) of solution B (10 mM HEPES, pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM AEBSF, 25% (v/v) glycerol) and centrifuged at 5000 rpm for 5 min at 4°C. After the supernatant was discarded, the cell pellet was taken up in 200 µl (2 cell volumes) of extraction solution (10 mM HEPES, pH 8.0, 400 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM AEBSF, 25% glycerol) and left on ice for 30 min with intermittent vortexing. The extracted nuclei were centrifuged at 14,000 rpm for 5 min at 4°C, and the supernatant was aliquoted and stored at -80°C. The total amount of protein was determined using the BCA (bicinchoninic acid) Protein Assay Reagent kit (Pierce, Rockford, IL) according to the manufacturer’s instructions.
Electrophoretic mobility gel shift assay (EMSA)
Nuclear extracts (
5 µg protein) were incubated in
DNA/protein-binding buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 10% (v/v)
glycerol, 0.5 mM DTT, 0.1 mM EDTA), with 250 ng poly(dI · dC), 100
ng sonicated herring sperm ssDNA, and 1 ng 32P-radiolabeled
probe for 15 min at 4°C. The samples were run on a 6% nondenaturing
polyacrylamide gel in 0.25x TBE (0.89 M Tris-borate, 0.89 M boric
acid, and 0.02 M EDTA) at 200 V for 2 h. The gels were
fixed with a 10% methanol, 10% acetic acid solution, dried onto
Whatmann 3M paper, and exposed to an x-ray film. The
ds-oligonucleotides containing the individual B1 and B2 sites
from the various HLA class I genes were used as probes (see Table I
).
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B/Rel family of transcription factors was
added to the nuclear extract and probe mixture and incubated for 1
h at 4°C. The Abs used were anti-p50 Ab (sc-114), anti-p65 Ab
(sc-109), anti-c-Rel Ab (sc-71), anti-Sp1 Ab (sc-59),
anti-Fra1 Ab (sc-605), anti-Fra2 Ab (sc-171), anti-Fos Ab
(sc-413), anti-Jun Ab (sc-44), and anti-IRF-1 Ab (sc-640), all
from Santa Cruz Biotechnology (Santa Cruz, CA), and an anti-HMG
I(Y) antiserum (24) (kindly provided by Dr. Dimitris Thanos, Columbia
University, New York, NY). |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel protein binding to the B
sites in enhancer A of the classical and nonclassical HLA class I genes
The promoter regions of classical and nonclassical HLA class I
genes contain two putative B sites, B1 and B2, in their
enhancer A elements. The B1 site in enhancer A of HLA-A(GGGGATTCCCC; Table I
) is the most conserved throughout the
MHC class I loci and was used as reference to test for binding of
proteins of the NF-B/Rel family in EMSAs. Using nuclear extracts
from the B lymphoblastoid line CCRF-SB, which constitutively expresses
proteins of the NF-B/Rel family, this B motif was found to bind
several protein complexes: an upper complex consisting of two closely
migrating bands, designated complex 1 and complex 2; and a lower
complex, designated complex 3 (Fig. 1A). Employing antisera
specific for p50, p65, and c-Rel (25, 26), the proteins contained in
these complexes could be identified (Fig. 1, A and
B). The uppermost band in the duplex (complex 1) was shown
to contain p65. This protein/DNA complex probably represents the
p50-p65 heterodimer, although the presence of p65-p65 homodimers cannot
be excluded. The lower band of the two closely migrating protein/DNA
complexes (complex 2) was shown to contain c-Rel. Complex 2 could be
either the p50-c-Rel or the p65-c-Rel heterodimer. Finally, the lower
band (complex 3), shown to contain the p50 subunit, is most probably
the p50-p50 homodimer (Fig. 1, A and B). No
supershift was obtained with an irrelevant Ab (Fig. 1C).
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B motifs of all
HLA class I loci (Table I) was used to perform a complete analysis of
their binding properties for proteins of the NF-B/Rel family using
nuclear extracts from B cells. In Figure 2, A and B, it is
shown that, similar to the conserved B1 site of the HLA-A
locus, the B1 site of the HLA-B locus (GGGGATTCCCC), was
bound by the complexes containing the p50, p65, and c-Rel subunits. The
B2 site in HLA-A (GGGGAGTCCCA) was also bound by these
complexes, but with an apparently weaker binding affinity (Fig. 2, A and B). No binding of proteins of the
NF-B/Rel family was detected for the B2 sites of HLA-B(GGGGAGGCGCA) and the B1 and B2 sites of HLA-C(GAGGATTCTCC and GGGGAGGCGCC, respectively) under these
experimental conditions (Fig. 2A). EMSAs with B1 and
B2 probes of the nonclassical HLA class I genes revealed that the
B1 site in HLA-F (GAGAATTCCCC) was bound by the p50, p65,
and c-Rel subunits (Fig. 2A), while both the B1 and B2
sites in HLA-G (GGGGATTCTCT and GGGGAGGCCCC, respectively)
were bound only by the p50-containing complex (complex 3; Fig. 2, A and B); binding of the p65-containing complex
was hardly detectable and only after long exposure times (data not
shown). No NF-B complex could be detected binding to the B2 site
of HLA-F (TTGGAAGGCTC) nor to the B1 and B2 sites of
HLA-E (GGGAAACTCTG and AAGAACTGCTG, respectively) (Fig. 2A).
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B site probes were seen to bind proteins not belonging to
the NF-B family of transcription factors for which we were testing.
For example, a slowly migrating complex (referred to as complex A) was
found to bind the B2 sites of HLA-B, HLA-C,
and HLA-G, which all share the
GGGGAGGCG/CC sequence (Table I). The
specificity of the binding was tested by competition with cold B
probes. Cold B2 probe of HLA-C or of HLA-G
competed with the B2 site of HLA-C for binding of complex
A, whereas cold B1 probe of HLA-A did not compete (Fig. 3A). Conversely, binding of
complex A to the B2 site of HLA-G was inhibited when
using cold B2 probe of HLA-C or of HLA-G, but
not when using the B1 probe of HLA-A (data not shown).
Since the sequence of these B2 sites is homologous to an Sp1 binding
site (27, 28, 29), we tested for binding of Sp1 to the B2 site of
HLA-C. Using an anti-Sp1-specific Ab in supershift
assays, complex A was shown to contain Sp1 (Fig. 3B). The
complex could not be supershifted with an irrelevant Ab (Fig. 3B). Similarly, Sp1 binding could also be demonstrated to
the B2 sites of HLA-B and HLA-G (data not
shown).
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B1 sites of HLA-C and HLA-G (Fig. 2, A and B), which share the GGATTCTC sequence. The
specificity of the binding was also tested by competition with cold
B probes, and binding of complex B was competitive when using the
cold B2 probe of HLA-C or of HLA-G (containing
the homologous sequence), but only weakly competitive with the
divergent B1 probe of HLA-A (data not shown). These
characteristics suggest that complex B contains the high mobility group
I(Y) protein (HMG I(Y)). Using an HMG I(Y)-specific Ab (24), a
supershift was obtained (data not shown), strongly suggesting that this
complex contains HMG I(Y) protein.
Recently, the Fos/Jun family protein Fra-2 has been shown to bind the
B binding site of porcine MHC class I (30). Since this B binding
site is homologous to the B2 site of HLA-A, we tested for
binding of members of the Fos/Jun family of transcription factors to
the B1 and B2 sites of HLA-A. However, we were unable
to detect Fra-2 or other factors of the Fos/Jun family in the complex
binding to the B1 site or B2 site of HLA-A, using
nuclear extracts of CCRF-SB or EBV-transformed B cell nuclear extracts
(data not shown).
Locus-specific HLA class I transactivation mediated by enhancer A
Having determined the binding properties of NF-B/Rel proteins
to the B1 and B2 sites of enhancer A, we evaluated the
contribution of the enhancer A elements of the various HLA class I loci
to transactivation in transient cotransfection experiments. For these
experiments, reporter constructs were generated containing the enhancer
A sequence of the various HLA class I genes in front of a minimal
promoter fragment of HLA-A2.1 (Fig. 4A).
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B
subunits using pGL3-A230, containing the 228-bp HLA-A2.1
promoter fragment. As shown in Figure 4B, cotransfection
with the p50 subunit alone did not result in an increase of
HLA-A2.1-driven transcription in Tera-2 cells, whereas
cotransfection with p65 resulted in a 6-fold induction. Together, p50
and p65 were able to induce HLA-A2.1-driven transactivation
to an even higher level (14-fold), which supports the suggestion that
they act synergistically (Fig. 4B).
Subsequently, the reporter constructs containing the enhancer A
sequence of the various HLA class I genes in front of a 143-bp
HLA-A2.1 promoter fragment were transiently cotransfected
with expression vectors of p50 and p65 in Tera-2 cells. Figure 4C shows a typical transient cotransfection experiment.
Enhancer A of HLA-A, containing two B sites able to bind
NF-B, gave rise to a significant transcriptional activity when
cotransfected with p50 and p65 (15-fold induction), whereas
transactivation of enhancer A of HLA-B (containing only one
B binding sequence) was relatively low (3-fold induction; Fig. 4C). The enhancer A of HLA-C did not give rise to
any significant NF-B-induced transactivation activity, which is in
accordance with the inability of the B1 and B2 motifs of
HLA-C to bind NF-B subunits. None of the enhancer A
elements of the nonclassical HLA class I genes HLA-E,
HLA-F, or HLA-G gave rise to any significant
NF-B-induced transactivation activity (Fig. 4C). Despite
the binding activity of the p50 and p65 to the B1 of
HLA-F, no transactivation of HLA-F was observed;
this could be attributed to the lack of binding to the B2 site. As
expected, binding activity of p50 to both the B1 and B2 of
HLA-G did not result in transactivation. Similar results
were obtained in HeLa cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B binding sites
determines both the binding affinity of the various NF-B dimers and
conformational changes in the nucleoprotein complex (19, 31, 32, 33, 34). Since
the various NF-B complexes differ in their transactivation
capacities (12, 13), this could result in differences in activation of
gene transcription. Therefore, locus-specific variations in the
nucleotide sequences of the B binding sites in HLA class I genes
could be at the heart of their differential NF-B-mediated regulation
via enhancer A. In this study, the B sites in enhancer A of the
promoter region of classical and nonclassical HLA class I genes were
investigated for their capacity to bind proteins of the NF-B/Rel
family of transcription factors and their contribution to
transactivation.
Locus-specific binding properties of NF-B to the B1 and B2
sites in enhancer A of HLA class I
The enhancer A element in HLA class I contains a palindromic
B site, GGGGATTCCCC, referred to as B1. This site is conserved in
the HLA-A and HLA-B loci but is also found,
albeit with nucleotide alterations, in the promoters of the other HLA
class I loci. In addition, upstream of the B1 site is the B2 site
GGGGAGTCCC, which is found in the HLA-A alleles and
also, with minor to major nucleotide variations, in the
promoters of the other HLA class I loci. Both the B1 and B2 sites
of the enhancer A region of HLA-A have been shown to be
occupied, as determined by in vitro footprinting (35). In this study,
the B1 site of HLA-A and HLA-B (GGGGATTCCCC)
was found to bind three complexes containing members of the NF-B/Rel
family of transcription factors, which is in general agreement with
findings by others (21, 29, 36). Supershift assays indicated that the
three complexes that were found to bind the B1 site contained p50,
p65, and c-Rel subunits. These complexes could represent the p50-p65
heterodimer, the p50-c-Rel or p65-c-Rel heterodimer, and the p50-p50
homodimer. Although in HLA-A the B2 site
(GGGGAGTCCCA) differs by only two
nucleotides (underlined) from the B1 site, it displayed a reduced
binding affinity for all three complexes under these experimental
conditions. The central nucleotides (ATT) in a B site function as
the binding site for HMG I(Y) proteins, which play a role in the DNA
bending and the ultimate structure of the nucleoprotein complex
and also act as coactivators in NF-B-mediated transactivation (24, 37). These central nucleotides are rarely guanines, and it is therefore
likely that the central guanine in the B2 site of HLA-Ais responsible for this weaker binding affinity. No binding of any
NF-B complexes could be detected to the B2 site of
HLA-B or to either of the B sites of HLA-C.
Two of the nonclassical HLA class I genes were bound by NF-B
complexes. The B1 site of HLA-F
(GAGAATTCCCC) was bound by the three p50-,
p65-, and c-Rel-containing complexes, despite two nucleotide
differences from the consensus sequence. The B1 site of HLA-G(GGGATTCTCT) showed binding of only p50,
whereas binding of the other NF-B subunits (p65 and c-Rel) was
barely detectable. The specificity of the B2 site of HLA-Gfor p50 is in agreement with in vitro DNA binding studies in which
it was shown that the otherwise perfect B motif
GGGGAGGCCCC is bound only by p50 (33). No binding of
any B-specific complexes could be detected to both B sites of
HLA-E or to the B2 site of HLA-F. Together,
these data argue that only the B2 site of HLA-A is a true
NF-B binding site and that the putative B2 site in the promoter
of the other HLA class I loci cannot be termed a B site.
Two other complexes, designated complex A and B, were found to
bind to some of the B motifs of HLA class I loci; these complexes
did not contain any of the B/Rel family proteins for which we were
testing. The slower migrating complex A had an apparent binding
specificity for the GGGGAGGCG/CC sequence. This
sequence has been proposed as a binding site for Sp1 in HLA-B(28, 29). Furthermore, both half-sites of this sequence are
homologous to zinc finger binding sites: the 5' half-site is homologous
to the binding site for the myeloid zinc finger protein MZF1 (38), and
the 3' half site is homologous to the binding site for the zinc finger
protein ZFX, identified in the promoters for HLA-A genes
(39, 40). In this study, we demonstrated that complex A contained Sp1;
but since the supershift was not complete with the Ab used, the
possibility cannot be excluded that other (zinc finger) proteins are
contained in the complex. The faster migrating complex B had an
apparent binding specificity for the GGATTCTC sequence. These
characteristics, and the fact that a supershift could be obtained using
an anti-HMG I(Y) antiserum (24), strongly suggest that complex B
consists of HMG I(Y) proteins (24, 37). It is possible that in the
absence of NF-B binding, such as to the B1 sites of
HLA-C and HLA-G, formation of a complex with HMG
I(Y) proteins is more prominent.
NF-B-induced transactivation through enhancer A is limited to
the HLA-A and HLA-B loci
Transactivation by NF-B dimers is dependent on the subunits
they contain. Both the p65 and c-Rel subunits possess transactivation
domains, but p65 has been shown to be the more potent transactivator of
the two (12, 13). Experiments in mice have shown that a transgene
driven by a promoter containing multiple B sites was not transcribed
in organs in which only the p50 subunit was expressed; the transgene
was found to be transcribed only in those organs in which both p65 and
p50 were expressed (41). Moreover, p50 alone does not activate
transcription in transient transfection assays (this study and Refs.
29, 42, and 43). The p50 subunit has therefore been attributed an
ancillary function in DNA binding of the dimer and in transactivation
by the p65 or c-Rel subunits.
The enhancer A elements of classical HLA class I genes
HLA-A and HLA-B were, in accordance with their
ability to bind the p50-p65 heterodimer, mediators of NF-B-induced
transcription. Enhancer A of HLA-A, which contains two B
sites binding the different NF-B dimers, displayed the strongest
transactivation capacity of all of the HLA class I loci. Multiple B
binding sites that are occupied by NF-B, as is the case for
HLA-A, can lead to a synergistic transcriptional effect (44)
and are thought to be required for transcriptional induction (21, 45).
Less marked was the transactivation through the enhancer A region of
HLA-B, which contains only one NF-B binding site (B1).
In this locus, the B2 site is bound by Sp1 (this study and Refs. 28
and 29), a transcription factor that is shown able to interact with
NF-B (46, 47). Sp1 binding to the B2 site may assist in the
NF-B-mediated transactivation through the B1 site by allowing
protein/protein interactions, leading to transactivation through the
enhancer A of HLA-B (28, 48). This would explain why there
is still transactivation through the single NF-B-occupied B1 site
in this locus (this study and Refs. 28 and 29). In support of this
hypothesis is the lack of NF-B-mediated transcriptional activity
through the B1 site in the enhancer A region of HLA-F,
because unlike the HLA-B gene, HLA-F does not
bind transcription factors to the upstream B2 site. These
findings are corroborated by the observation that the single B site
in the bidirectional promoter of the TAP1 and
LMP2 genes has been found to depend on the flanking
Sp1 site for TNF-induced transcription by NF-B (49, 50). The
other HLA class I loci were not found to be regulated by NF-B, which
is in accordance with the lack of NF-B binding to the B1 site
(HLA-C, HLA-E) or to the B2 site
(HLA-F), as explained above. Finally, the lack of
NF-B-induced transactivation of HLA-G can simply be
explained by a lack of transactivating activity by the p50-p50
homodimer (this study and Refs. 29 and 43).
In this study, it is shown that two classical, but none of the
nonclassical, HLA class I genes are transactivated by NF-B.
Transactivation was most important in the HLA-A locus, which
contains two NF-B binding sites in the enhancer A region. Although
the enhancer A of HLA-B contains only one NF-B binding
site (B1), there was still a moderate transactivation by NF-B.
Binding of Sp1 to the upstream B2 site seems to contribute to this
transactivation through the B1 motif in HLA-B, because
HLA-F, which also has one NF-B binding site but lacks an
NF-B or Sp1 binding site flanking the B1 site, was not
transactivated by NF-B. Thus, NF-B-mediated transactivation of
the HLA class I genes seems to require the binding not only of the
B1 site by NF-B, but also of the B2 by either NF-B or Sp1.
Since transcriptional regulation of HLA class I genes by NF-B is
restricted to HLA-A and HLA-B genes, the
expression and activation of NF-B contribute only to the
developmental and tissue-specific expression patterns of these
classical HLA class I genes.
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Acknowledgments |
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B expression vectors, and
anti-HMG I(Y) antiserum, respectively. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. S. J. P. Gobin, Department of Immunohematology and Blood Bank, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
3 Abbreviations used in this paper: HMG I(Y), high mobility group I(Y); IRF-1, IFN regulatory factor-1; EMSA, electrophoretic mobility gel shift assay; Sp1, specificity protein 1; AEBSF, 4-(2-aminoethyl)-benzenesulfonylfluoride.
Received for publication October 24, 1997. Accepted for publication April 30, 1998.
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