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Prepublished online as a Blood First Edition Paper on December 12, 2002; DOI 10.1182/blood-2002-09-2924.
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Blood, 15 April 2003, Vol. 101, No. 8, pp. 3058-3064
IMMUNOBIOLOGY
Regulation of human  2-microglobulin
transactivation in hematopoietic cells
Sam J. P. Gobin,
Paula Biesta, and
Peter J. Van den
Elsen
From the Department of Immunohematology and Blood
Transfusion, Leiden University Medical Center, Leiden, the
Netherlands.
 |
Abstract |
 2-Microglobulin (2m) is a
chaperone of major histocompatibility complex (MHC) class I (-like)
molecules that play a central role in antigen presentation,
immunoglobulin transport, and iron metabolism. It is therefore of
importance that 2m is adequately expressed in cells that
perform these functions, such as hematopoietic cells. In this study, we
investigated the transcriptional regulation of 2m in
lymphoid and myeloid cell lines through a promoter containing a
putative E box, Ets/interferon-stimulated response element
(ISRE), and  B site. Here we show that upstream stimulatory factor 1 (USF1) and USF2 bind to the E box and regulate 2m
transactivation. The nuclear factor B (NF-B) subunits
p50 and p65 bind to the B box and p65 transactivates
2m. Interferon regulatory factor 1 (IRF1), IRF2, IRF4,
and IRF8, but not PU.1, bind to the Ets/ISRE, and IRF1 and IRF3 are
strong transactivators of 2m. Together, all 3 boxes are
important for the constitutive and cytokine-induced levels of
2m expression in lymphoid and myeloid cell types. As such, 2m transactivation is under the control of
important transcriptional pathways, which are activated during injury,
infection, and inflammation.
(Blood. 2003;101:3058-3064)
© 2003 by The American Society of Hematology.
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Introduction |
2-Microglobulin (2m)
is a ubiquitously expressed 12-kDa glycoprotein that associates
with major histocompatibility complex (MHC) class I (-like) molecules
that are of great importance in antigen presentation, IgG transport,
and iron metabolism.1 As such, 2m
is linked to a variety of human diseases because of its association
with immunologically and hematologically relevant molecules.
2m is best known for its association with the MHC class
I heavy chain, which is essential for the stable expression of these
antigen-presenting molecules. Classical MHC class I molecules (HLA-A,
-B, and -C) are ubiquitously expressed in most somatic cells. They are
essential in the immune response because they present antigen-derived
peptides to cytotoxic T lymphocytes and are important in protection
against natural killer (NK) cell-mediated cytotoxicity.2
2m also associates with MHC class Ib or class I-like
molecules, such as HLA-E, -F, -G, and CD1, which have a more restricted
tissue distribution and have more specialized functions in antigen
presentation.3-5 2m is also a partner of HFE (formerly called HLA-H), an MHC class I-like molecule that is
important for transferrin-mediated iron uptake.6-9
Patients suffering from hereditary hemochromatosis have been found to
bear a mutation in the HFE gene that specifically
disrupts its association with 2m, resulting in a
strongly compromised function.6,7 This is
characterized by iron accumulation in parenchymal cells in various
organs but a paucity of iron in Kupffer cells and
macrophages.6-9 Clinical consequences include liver
cirrhosis, diabetes, arthritis, and heart failure. Furthermore,
2m is also able to form a dimer with the neonatal Fc
receptor, which is important for fetomaternal transport of
IgG.10,11 Finally, a role for 2m has also
been suggested in amyloidosis. Patients undergoing long-term dialysis often develop amyloidosis, which in turn affects bone cell metabolism by inducing bone mineral dissolving and enhancing osteoblast
proliferation.12,13 This is thought to be related to the
binding of 2m with  2-macroglobulin and
heparin sulfate.14,15
Because 2m is essential for the functioning of molecules
central in antigen presentation, IgG transport, and iron metabolism, a
tight control of 2m expression is essential to secure
the expression in a variety of cell types, such as hematopoietic,
parenchymal, and syncytiotrophoblast cells. The basal level of
2m expression can be enhanced by cytokines to meet local
requirements for an adequate immune response and possibly also in
fulfilling any of its other functions. The transcriptional
regulation of 2m is thought to be similar to that of MHC
class I genes. Both 2m and MHC class I genes possess the
SXY module, a set of regulatory elements shared with MHC class II
genes, and are regulated through an MHC-specific
enhanceosome.16,17 This multiprotein complex, containing
RFX, CREB/ATF, and NF-Y transcription factors, is the basis for
transactivation driven by the class II transactivator (CIITA).17 Other potential regulatory elements that could
mediate the cytokine-induced transactivation have been identified
further upstream in the human 2m promoter, but these
elements have never been fully characterized. Therefore, we
investigated the transcriptional regulation through these upstream
promoter elements that could control the 2m expression
in lymphoid and myeloid cell lines, central in immunologic and
hematologic functions.
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Materials and methods |
Cell culture
The cell lines used in this study were the acute T-cell leukemia
Jurkat, the Burkitt lymphoma B-cell line Raji, the acute lymphoblastic
leukemia B-cell line SB, the monocytic cell line THP-1, the cervical
carcinoma cell line HeLa, the teratocarcinoma cell line Tera-2 (all
from American Type Culture Collection, Manassas, VA), and Epstein-Barr
virus (EBV)-transformed B cells MSH. These cell lines were grown in
Iscove modified Dulbecco medium (IMDM; Life Technologies, Paisley,
Scotland) supplemented with 10% (vol/vol) heat-inactivated fetal calf
serum (Life Technologies), penicillin (100 IU/mL), and streptomycin
(100 µg/mL). Where indicated, cells were treated with tumor necrosis
factor (TNF-; 500 U/mL; Bender Medsystem, Vienna,
Austria), interferon 2c (IFN-2c; 500 U/mL; Bender
Medsystem), IFN-1a (500 U/mL; Avonex/Biogen, Cambridge, MA),
or IFN- (500 U/mL; Boehringer Ingelheim, Alkmaar, the Netherlands).
Preparation of nuclear extracts and electrophoretic mobility
shift assay
Nuclear extracts were prepared as previously
described.16 Nuclear extracts (about 5 µg protein) were
incubated in DNA/protein-binding buffer (20 mM HEPES
[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.9, 50 mM
KCl, 10% vol/vol glycerol, 0.5 mM dithiothreitol [DTT], 0.1 mM EDTA
[ethylenediaminetetraacetic acid]), with 200 ng
poly(dI·dC), 200 ng sonicated single-stranded herring sperm DNA, and
1 ng [32P]-radiolabeled probe for 30 minutes at 4°C.
The samples were run on a 6% nondenaturing polyacrylamide gel in
0.25 × TBE (Tris-borate EDTA) buffer at 200 V for 2 hours.
The gels were fixed with a 10% methanol and 10% acetic acid solution,
dried onto Whatmann 3M paper and exposed to an x-ray film.
The following ds-oligonucleotides were used as probes for the putative
E box, Ets/interferon-stimulated response element (ISRE), and B site
of 2m: E: 5'-AAACATCACGAGACTCT-3'; Ets/ISRE:
5'-TAAGAAAAGGAAACTGAAAACG-3'; B:
5'-ACGGGAAAGTCCCTC-3'. The following probe was used as
control Ets/ISRE site: 5'-CAGTCCACAGTAGGAAGTGAAATTA-3'.
For supershift assays, 1 µg of each antibody (Ab) specifically
directed against the different transcription factors was added 20 minutes after the nuclear extract had been incubated with the probe and
this mixture was incubated for an extra 30 minutes at 4°C. The
antibodies used were directed against upstream stimulatory factor 1 (USF1; sc-229), USF2 (sc-861), E47 (sc-763), interferon regulatory
factor 1 (IRF1; sc-497), IRF2 (sc-498), IRF3 (sc-9082), IRF4 (sc-6059),
IRF7 (sc-9083), IRF8 (sc-6058), PU.1 (sc-352), Ets1/2 (sc-112), Spi-B
(sc-5944), signal transducer and activator of transcription 1 (STAT1;
sc-345), p50 (sc-114), p65 (sc-109), c-Rel (sc-71), RelB (sc-226); all
were from Santa Cruz Biotechnology (Santa Cruz, CA).
Plasmids
Luciferase reporter plasmids used were generated by cloning
genomic promoter fragments into pGL3-Basic (Promega, Madison, WI).
These constructs contain a polymerase chain reaction (PCR)-generated promoter fragment of 2m of, respectively, 302 bp
(pGL3-2m), 193 bp (pGL3-2m193), 180 bp
2m (pGL3-2m180), 157 bp 2m
PCR (pGL3-2m157), 145 bp (pGL3-2m145),
and a 269-bp AspI-AhaII HLA-B7 promoter fragment (pGL3-HLA-B). The
mutant promoter constructs of 2m containing a mutation
either in the E box, ISRE, or B site were generated by overlap
extension PCR.16 These mutant promoter constructs are
identical to the wild-type constructs (pGL3-2m) except
for a 2- to 3-bp mutation in the core sequence of the individual boxes
(Figure 1). The Renilla
luciferase constructs pRL-actin was used as internal control for
transfection efficiency. pRL-actin was generated by cloning a
PCR-generated 1-kb human -actin promoter fragment into pRL-null
(Promega).17
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| Figure 1.
Promoter structure of human 2m.
Schematic representation of the promoter of 2m,
depicting the position and order of the E box, ISRE, and B site.
Underneath, the nucleotide sequence of the upstream region of
2m containing the 3 boxes is shown, indicating the
mutations in the putative E box, Ets/ISRE, and B site that are
introduced for the mutant promoter constructs. Human
2m promoter region accession number
AF092744.
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The expression vectors pSG5-USF1 and pSG5-USF2 were a kind gift
of Dr M. Sawadogo.18 The pRc/RSV expression vectors of
IRF1, IRF2, IRF4, and IRF8 were generated by cloning the PCR-amplified cDNAs of IRF1, IRF2, IRF4, and IRF8 into pRc/RSV. Also PU.1 was cloned
into pRc/RSV (kindly provided as pCDNA3-PU.1 by Dr M. Fenton). The
expression vectors pCMVBL/IRF3, pCMVBL/IRF3 N,
pCMVBL/IRF3(5D), pCMVBL/IRF7 and pCMVBL/IRF7(2D) were a kind gift of Dr
J. Hiscott,19,20 and these inserts were also cloned into
the expression vector pRc/RSV.
Transient transfection
Adherent cells were transfected by the calcium phosphate
coprecipitation method as described previously.16 In each
of 4 wells of a 6-well plate, 0.2 × 106 cells were
transfected with a DNA mix containing 1 µg firefly luciferase pGL3
reporter plasmid and 0.2 µg Renilla luciferase pRL-SV40
control plasmid (Tera-2). For cotransfection 1 µg pRc/RSV expression
vector was used. For cytokine induction experiments, cells were treated
with 500 U/mL TNF-, IFN-, IFN-, or IFN- for 48 hours after
transfection. Nonadherent cells (Jurkat, Raji, THP-1) were transfected
by electroporation,17 with 10 µg firefly luciferase pGL3
reporter plasmid and 1 µg Renilla luciferase pRL-actin control plasmid. To measure promoter activity, cells were harvested 3 days after calcium phosphate transfection or 2 days after
electroporation. Luciferase activity was determined using the
dual-luciferase reporter assay system (Promega) and a luminometer
(Tropix, Bedford, MA).
| |
Results |
Promoter structure of the human 2m gene
The proximal promoter region of 2m contains a set
of regulatory elements that form the SXY regulatory module, a module
that is shared with MHC class I and class II genes.16 The
SXY module is the basis for an MHC-specific enhanceosome that
is important in the CIITA route of transactivation.17
Computer-aided inspection of the human 2m promoter
region upstream of the SXY module revealed the presence of a putative E
box, ISRE, and B site (Figure 1). This is similar to the promoter structure of the 2m promoter in the mouse except that
the E box, located upstream of the ISRE, is not found in the mouse
2m promoter.21,22 The ISRE and B boxes
are also found in MHC class I promoters, but the order of these boxes
is reversed, that is, the ISRE in the 2m promoter is
positioned 5' of the B site. Furthermore, unlike MHC class I
promoters, the ISRE region of 2m consists of 2 overlapping ISREs including a putative Ets-binding site (GGAA), which therefore classifies as a potential combined Ets/IRF-response element (Figure 1).
The E box, ISRE, and B site are important for the constitutive
and cytokine-induced 2m promoter activity
To test for the importance of the potential E box, ISRE, and B
site for a constitutive level of promoter activity, we generated promoter constructs, which were mutated in each of the 3 regulatory sites (Figure 1). Transient transfection of these 2m
promoter constructs in Raji B cells, Jurkat T cells, and THP-1
monocytes revealed that mutation of the E box, ISRE, or B site
significantly reduced the basal promoter activity (Figure
2A). Mutation of the ISRE resulted in the
most dramatic reduction in basal promoter activity (reduced to about
10%-15%), whereas mutation of the E box or B site resulted in
reductions to 20% to 50% of wild-type. Therefore, all 3 potential
regulatory sites play a role in the basal promoter activity in lymphoid
and monocytic cells.
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| Figure 2.
The importance of the E box, ISRE, and B site in the
constitutive and cytokine-induced 2m promoter activity.
(A) Transient transfection of wild-type 2m and E box-,
ISRE-, or B-mutated reporter constructs in Jurkat, Raji, and THP-1
cells revealing the importance of each regulatory site to the
constitutive promoter activity in lymphoid and monocytic cells. (B)
Transient transfection of the 2m reporter construct in
Tera-2 cells induced with TNF-, IFN-, IFN-, or IFN- (each
500 U/mL) for 48 hours. 2m is induced by all cytokines
of which IFN- is the most potent. The induction ratios are indicated
above the histogram. (C) Transient transfection of wild-type
2m and E box-, ISRE-, or B-mutated reporter
constructs in Tera-2 cells induced with TNF- or IFN- (each 500 U/mL) for 48 hours. All boxes are important in the TNF-- and
IFN--induced 2m promoter activity. The induction
ratios are indicated above the histogram. The luciferase activity
values were normalized with the Renilla luciferase activity
values and are expressed as mean ± SD of 4 experiments.
RLU indicates relative light units.
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Because the ISRE and B site are mediators of cytokine-induced
transactivation, we tested whether 2m promoter activity
could be up-regulated by IFN and TNF-. In transient transfection
experiments in the cytokine-responsive teratocarcinoma cell line
Tera-2, promoter activity of 2m was induced in response
to TNF- and to IFN-, IFN-, and IFN- (Figure 2B). Of these
cytokines IFN- was the most potent inducer of 2m
transactivation. Using the mutant constructs, we demonstrated that
mutation not only of the ISRE or B site, but also of the E box, in
general compromised the IFN- and TNF- induction of
2m promoter activity (Figure 2C). Together, these results strongly suggest an important regulatory role for the E box,
ISRE, and B site in constitutive and cytokine-induced 2m transactivation.
Transactivation of 2m is controlled by upstream
stimulatory factors binding to the E box
The binding capacity of the putative regulatory sites was
investigated by electrophoretic mobility shift assay (EMSA) analysis. Transcription factor binding to putative E box was tested using nuclear
extracts of the B-cell line Raji, T-cell line Jurkat, and monocytic
cell line THP-1. The probe encompassing the E box bound a complex,
which supershifted with Abs directed against USF1 and USF2. We also
tested for the presence of the lymphoid/myeloid-specific factor E47,
but did not detect E47 with the Ab used (Figure
3A). Furthermore, using the Ab for either
USF1 or USF2, the complex was almost completely supershifted, which
implies that the complex consists almost entirely of the USF1/USF2
heterodimer. It should also be noted that in the 3 cell lines the
intensity of the bands representing the complex was similar, indicative
of a comparable protein content in the different nuclear extracts.
Similar results were found using nuclear extract of nonlymphoid cells
such as HeLa or Tera-2 cells, underscoring the ubiquitous expression
and binding of these USF transcription factors to the E box.
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| Figure 3.
Transcription factor binding and transactivation
capacity of the E box of 2m.
(A) EMSA showing binding of complexes to the E box of
2m. Using specific Abs, the complex binding to the E box
was shown to contain USF1 and USF2 in Jurkat, Raji, and THP-1 cells.
The presence of E47 was not detected. Arrowheads indicate the USF1/USF2
complex; *, supershifted complex(es). (B) Transient transfection of
wild-type 2m- and E box-mutated reporter constructs
with USF1 and USF2 expression vectors (1 µg) in Tera-2 cells. The
luciferase activity values were normalized with the Renilla
luciferase activity values and are expressed as mean ± SD of 4 samples. The induction ratios are indicated above the
histogram. RLU indicates relative light units.
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Because the E box was shown to be a binding site for USF1 and USF2, we
tested whether 2m promoter activity was controlled by
USFs with transient transfection experiments in Tera-2 cells. Cotransfection of the 2m promoter construct with
exogenous USF1 or USF2 enhanced promoter activity of 2m
(Figure 3B), demonstrating that USF1 and USF are positive regulators of
2m promoter activity. Mutation of the E box reduced the
elevated promoter activity of 2m, revealing that the E
box contributes to the USF-mediated transactivation of
2m (Figure 3B).
Transactivation of 2m is regulated by NF-B
through its B site
Next, the binding capacity of the B site was investigated. The
B site was shown to bind protein complexes containing p50 and p65
using nuclear extracts of Jurkat and THP-1 cells. This was similar to
nonlymphoid cells, where p50 and p65 were supershifted in EMSA using
nuclear extract from HeLa cells induced with TNF- (data not shown).
However, supershift analysis using Raji nuclear extracts revealed that
the Ab against p50 gave a strong supershifted complex, whereas the
supershift with the p65 Ab was weak. Using Abs against c-Rel and RelB
resulted in a slight reduction of the protein/DNA complex, which could
suggest that c-Rel and RelB are also present in the complex (Figure
4A). Similarly, in other B-cell lines a
predominance of p50 binding to the B site was found combined with a
weak supershift for p65, c-Rel, and RelB (Figure 4A; data not
shown).
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| Figure 4.
Transcription factor binding and transactivation
capacity of the B site of 2m.
(A) EMSA showing binding of complexes to the B site of
2m. Using specific Abs, the complex binding to the B
was shown to contain p50 and p65. The presence of c-Rel and RelB was
weakly detectable in Raji and MSH B cells. Note the difference
in the quantity of complex formation with an equal loading as in Figure
3. Arrowheads indicate NF-B complexes binding the B site; *,
supershifted complex(es). (B) Transient transfection of wild-type
2m- and B site-mutated reporter constructs with p50
and p65 expression vectors (1 µg) in Tera-2 cells. The luciferase
activity values were normalized with the Renilla luciferase
activity values and are expressed as mean ± SD of 4 samples. The induction ratios are indicated above the
histogram. RLU indicates relative light units.
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The transactivation of 2m by nuclear factor B
(NF-B) was tested in transient transfection experiment in Tera-2
cells. Cotransfection of the 2m promoter construct with
p50 and p65 resulted in enhanced promoter activity (Figure 4B).
Mutation of the B site strongly impaired the induced promoter
activity by NF-B (Figure 4B). Together this demonstrates that this
site is a functional B site.
IRFs regulate 2m transactivation through the
ISRE
Binding activity to the ISRE region was tested in EMSAs, using
nuclear extract of Raji, Jurkat, and THP-1 cells. Several protein/DNA complexes were observed in EMSAs with nuclear extracts from Jurkat, Raji, and THP-1 cells. The supershift with the Ab directed against IRF2
was strongest (Figure 5A), indicating
that this was the predominant factor in the complex. Only a weak
supershift was observed with the Ab directed against IRF1, indicating
that the presence was less abundant in this transcription factor
complex binding the ISRE region (Figure 5A). In Jurkat and Raji cells
we could not detect binding of lymphoid/myeloid-specific factors IRF4
and IRF8. In contrast to Raji B cells, in EMSAs with nuclear extract
from MSH and SB we observed a strong supershift with the IRF4-specific Ab and a weak supershift with IRF8-specific Ab (Figure 5A). In addition, IRF4 and IRF8 were also present in the complex binding to the
ISRE region in THP-1 cells. Moreover, induction of THP-1 cells with
IFN- resulted in a strong increase in IRF1 binding and a mild
increase in IRF8 and reduction of IRF2 binding (Figure 5A). In these
experiments, we could not detect the binding of IRF3 or IRF7 in any of
the cell lines (Figure 5A and data not shown). It is not clear whether
this is due to the absence of these factors or caused by a poor quality
of these Abs. Although the ISRE contains a potential Ets-binding site
(GGAA), we could not detect any binding of PU.1, Ets1/2, or Spi-B in
THP-1 nuclear extracts (Figure 5A and data not shown). This was an
unexpected finding because IRF4 and IRF8 are considered to form a
complex with PU.1/Ets factors for optimal binding. The presence of PU.1 in the nuclear extracts and the quality of the PU.1 Ab were verified with another ISRE/Ets probe that served as control (data not shown). Because the supershift with the IRF Abs reduced only marginally the
protein/DNA complex, it is possible that other transcription factors
are also present in the complex. It is of interest to note that there
was more complex formation with nuclear extracts from Raji cells and
IFN--induced THP-1 cells than Jurkat and noninduced THP-1 cells,
which is not due to loading difference (compare binding to the E probe
in Figure 3A). In nonlymphoid cells, the binding of IRFs to the
ISRE was more restricted. Using nuclear extract from HeLa cells, IRF2
and little IRF1 were present in the complex binding to the ISRE,
whereas upon IFN- treatment we observed a strong increase of IRF1 in
the complex (data not shown). No STAT1 binding to the ISRE of
2m was detected in any of the cell lines (data not
shown). Together, this indicates that IRF2 and in certain B cells also
IRF4 and IRF8 are important factors binding to the ISRE in lymphoid
cells. Furthermore, IRF2, IRF4, and IRF8 are important factors binding
to the ISRE in nonactivated monocytic cells, whereas IRF1 becomes
important after IFN- induction of monocytic cells.
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| Figure 5.
Transcription factor binding and transactivation
capacity of the ISRE of 2m.
(A) EMSA showing binding of complexes to the ISRE of 2m.
Using specific Abs, the complex binding to the ISRE was shown to
contain in IRF2, and little IRF1, in Jurkat T and Raji B cells. In the
B cells MSH and SB there was, in addition, a strong presence of IRF4
and a weak presence of IRF8. IRF4 and IRF8 were also found in THP-1
cells, whereas in THP-1 cells induced with IFN-, there was an
additional strong presence of IRF1. Arrowheads indicate the
complex containing IRF factors; *, supershifted complex(es). (B)
Transient transfection of the 2m reporter construct with
IRF1, IRF2, IRF3(5D), IRF4, IRF8, and PU.1 expression vectors (1 µg)
in Tera-2 cells, as indicated. IRF1 or IRF3(5D) could barely
transactivate the ISRE-mutated reporter construct in Tera-2 cells
(3-fold by IRF1 and 2-fold by IRF3(5D); data not shown). The luciferase
activity values were normalized with the Renilla luciferase
activity values and are expressed as mean ± SD of 4 samples. The induction ratios are indicated above the
histogram. RLU indicates relative light units.
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Next, we investigated the ability of IRF factors to transactivate
2m in Tera-2 cells, because this cell line does not
express IRFs constitutively. IRF1 was shown to be an important factor binding to the ISRE in IFN--induced THP-1 cells. In transient transfection assays, IRF1 strongly enhanced 2m promoter
activity (64-fold induction; Figure 5B). In addition, IRF2, which
constitutively binds to the ISRE in monocytic cells, moderately
enhanced 2m promoter activity (4-fold induction; Figure
5B). Cotransfection of IRF1 with IRF2 did not further enhance
2m promoter activity but rather tempered the IRF1
induction. Although in EMSA we could not detect any binding activity of
IRF3 or IRF7, we also tested their capacity to transactivate
2m. These IRFs posses an autoinhibitory domain and are
present in a nonactive form. Therefore, we used the constitutively
active forms IRF3(5D) and IRF7(2D), which bear a mutation in their
autoinhibitory domain.19,20 Transient transfection assays
with these mutant constructs showed that IRF3(5D), but not IRF7(2D),
was a potent inducer of 2m promoter activity (83-fold induction by IRF3(5D) versus 1-fold induction by IRF7(2D); Figure 5B
and data not shown). Cotransfection of IRF3(5D) with IRF7(2D) did not
change the strength of 2m transactivation by IRF3(5D) (data not shown). The wild-type forms of IRF3 and IRF7 did not transactivate 2m and served as controls (data not
shown). Furthermore, transactivation of ISRE-mutated 2m
promoter with IRF1 or IRF3(5D) was negligible (3-fold and 2-fold,
respectively; data not shown). Next, we tested the lymphoid and
myeloid-specific factors IRF4 and IRF8 because in some B and monocytic
cell lines the protein complex binding to the ISRE also contained IRF4
and IRF8. Neither IRF4 nor IRF8 alone was able to enhance
2m transactivation (Figure 5B). Interestingly, the
combination of IRF2 with IRF4 or IRF8 enhanced the weak induction
2m by IRF2 alone (Figure 5B). Because these IRFs often
require PU.1 or other Ets factors as binding partner for their
activity, we tested their transactivation potential in combination with
PU.1. However, in EMSA we had not detected PU.1 binding to the ISRE. In
line with this finding, IRF4 or IRF 8 was not able to transactivate
2m in combination with PU.1 (Figure 5B). For genes such
as ISG15, it has been described that IRF4 and IRF8 can also
bind in a complex with IRF1 and IRF2, which positively regulates gene
activation.23 To test this possibility we determined the
joint transactivation capacity of IRF1, IRF2, IRF4, and IRF8. However,
IRF2, IRF4, and IRF8 tempered the IRF1-induced 2m
transactivation, indicating that there was no obvious additive or
synergistic effect of the joint expression and binding of these IRFs
(Figure 5B).
| |
Discussion |
As chaperone of MHC class I (-like) molecules, 2m
is essential for the functioning of molecules central in antigen
presentation, IgG transport, and iron metabolism. This necessitates a
tight control of 2m transactivation to secure an
adequate expression in a variety of tissues and cell types. In
particular, hematopoietic cells fulfill an important role in these
functions.2,5,9 The clinical consequences of an aberrant
2m expression are exemplified in studies with
2m knock-out mice that have a greatly compromised immune
response due to the lack of antigen presentation and have a disturbed
iron metabolism similar to patients with hereditary hemochromatosis.6-8
In the present study, we investigated the regulatory elements upstream
of the SXY regulatory module that could provide for the constitutive
and cytokine-induced transactivation of 2m. In the human
2m promoter, this region includes a (putative) E box, an
Ets/ISRE, and a B box. It is noteworthy that the mouse 2m promoter differs from the human promoter in that it
lacks the E box and the Ets site within the ISRE.21,22,24
All 3 sites were important for the constitutive levels of
2m transactivation because mutation of either regulatory
site strongly reduced the basal level of 2m promoter
activity in lymphoid and monocytic cell lines. Based on the level of
reduction, the ISRE is the strongest contributor to the basal promoter
activity of 2m. In addition, 2m
transactivation is up-regulated by several cytokines, such as TNF-,
IFN-, IFN-, and IFN-. This is of biologic importance for the
coordinate cytokine-regulated expression of all genes involved in the
MHC class I antigen presentation pathway, including 2m
and MHC class I molecules, during inflammation or
infection.25-28 In this context, it would be of interest
to investigate whether the expression of other molecules that
2m associates with, such as HFE and the neonatal Fc
receptor, are also regulated by cytokines. As predicted, mutation of
the ISRE almost entirely abolished the transactivation induced by
IFN-, as did the mutation of the B site for the transactivation
induced by TNF-. Interestingly, mutations of the ISRE compromised
the TNF--induced promoter activity and, conversely, mutation of the B site compromised that by IFN- in absolute numbers. This can be
explained by the induction of NF-B by IFN- and the induction of
IRF1 by TNF-.29-32 In addition, mutation of the E box
compromised 2m transactivation by both IFN- and
TNF-. This suggests cooperation between the different boxes, which
could be brought about by interactions between transcription factors of
the different families or through joint recruitment of a general coactivator.
More detailed analysis revealed that the E box, positioned upstream of
the ISRE in the 2m promoter region, is almost
exclusively bound by USF1 and USF2. These 2 USFs, which most likely
form a heterodimer, are transactivators of 2m and
mutation of the E box diminished the USF-enhanced level of
2m transactivation. The fact that USF1 and USF2 could
still moderately enhance 2m promoter activity when the E
box was mutated may be due to an additional E box positioned in the X
box in the SXY module. This second E box was able to bind USF1 and USF2
(S.J.P.G. et al, unpublished results, January 2002) and may
also contribute to the 2m promoter activity controlled
by USFs. Because USF1 and USF2 are ubiquitously expressed factors, they
fulfill a more general rather than a lymphoid/myeloid-specific role in
the regulation of 2m transcription.
The B site, flanking the ISRE at the 3' site, is the binding site
for both p50 and p65 in T cells and monocytic cells. These NF-B
subunits most likely form a heterodimer,33 and together strongly induced 2m transactivation. B cells displayed a
slightly different binding profile in that they bind predominantly p50 and less p65. In addition, the binding of RelB and c-Rel was also detected but was very weak. Nevertheless, this opens the possibility that in B cells different heterodimers are formed and bind the B
site of 2m, although it remains to be determined whether
also other dimers are important for the transactivation of
2m in this cell type. NF-B is a crucial
transcriptional regulator of genes, which products are essential in the
immune functions fulfilled by lymphoid and myeloid
cells.33 In this context it is of relevance that also
2m is under the control of NF-B.
Perhaps the most important regulatory site of the upstream region of
the 2m promoter is the ISRE because mutation of this site dramatically reduced the basal and cytokine-induced promoter activity in lymphoid and monocytic cells. The ISRE of 2m
is in fact built up of 2 overlapping ISREs and contains a putative
Ets-binding site. The ISRE is bound by several general and
lymphoid/myeloid-specific IRFs. Based on supershift analysis we
observed a difference in presence and redundancy of IRFs in the complex
binding the ISRE. In B and monocytic cell lines, IRF2, IRF 4, and IRF8
were detected in the complex binding to the ISRE. However, in Jurkat T
and Raji B cells we could not detect the binding of IRF4 or IRF8. In
the case of Jurkat cells, this is most likely due to the fact that expression of these lymphoid/myeloid-specific IRF factors requires an
activation step.34,35 Furthermore, little IRF1 was present in the ISRE-binding complex using nuclear extracts of the different cell lines. However, IFN- induction of THP-1 cells greatly increased the presence of IRF1 in the complex. The binding of IRF3 and IRF7 was
not detected, although we could not exclude the possibility that this
was due to the quality of the Abs. The binding of several IRF factors
allows the formation of different protein complexes. In transient
transfection assays, IRF1 and IRF3 were found to be strong
transactivators of 2m. Combinations of IRF1 or IRF3 with
other IRFs tempered their transactivation potential. IRF2 alone weakly
induced 2m promoter activity, but in combination with
IRF1 it compromised the activation strength of IRF1. IRF1 is a general
transactivator of genes bearing a conventional ISRE.36,37 These include MHC class I genes, which are also regulated by
IRF1.26,38 Similar to the promoters of RANTES and ISG56,
2m possesses an extended ISRE with several GAAA
tandem-repeats and is also regulated by IRF3.39,40 In
contrast, IRF7 did not induce 2m promoter activity,
which may be due to the fact that the 3 GAAA repeats in the ISRE
(GAAAA, GAAAC, and GAAAA,
respectively) are preferentially or specifically bound by IRF3 and not
IRF7.41 The MHC class I gene HLA-B, which does
not posses an extended ISRE, is only marginally activated by IRF3
(5-fold) and not by IRF7 (S.J.P.G. et al, unpublished results,
July 2002). Genes that have a combined Ets/ISRE are
cooperatively bound and regulated by the lymphoid/myeloid-specific factors IRF4, IRF8, PU.1, and Spi-B.35,42-44 Because the
ISRE of 2m also contained a potential Ets site (GGAA),
we investigated binding of PU.1 and other Ets factors to this
ISRE.43,45 Despite a perfect Ets core sequence, we could
not detect the binding of PU.1, Ets1/2, or Spi-B. In the case of PU.1,
this was not related to the quality of the Ab, because the Ab could
supershift PU.1 bound to a control Ets/ISRE probe. It is possible that
the lack of binding of PU.1 to this Ets site is related to the flanking sequences.46 This excludes PU.1 as binding partner for
IRF4 or IRF8, but it is possible that other Ets factors could
bind the Ets site and form a complex with the IRF factors.
Despite the binding of IRF4 and IRF8 to the ISRE of 2m,
they did not activate the 2m promoter, although the
combinations of IRF2 with IRF4 or IRF8 weakly induced 2m
promoter activity. However, this was tested in a nonlymphoid/myeloid
cell line and it is therefore possible that IRF4 and IRF8 require
lymphoid/myeloid-specific partners to be able to control
2m transactivation. Alternatively, it is also possible
that the observed transactivation by IRF4 and IRF8 is mediated by IRF2,
which is expressed in all lymphoid/myeloid cells types. It is therefore
of importance to determine the exact role of IRF4 and IRF8 in the
transactivaiton of 2m in lymphoid and myeloid cells.
Interestingly, functional interactions of IRF4 and IRF8 with E47 are
reported for the Ig 3' enhancer, which contains a flanking ISRE and
E box.47,48 It is tempting to speculate that IRF4 and IRF8
could interact with E box-binding proteins, although in our experiments
and with the Ab used we could not detect E47 binding.
In conclusion, 3 juxtaposed regulatory sites, the E box, ISRE, and B
site in the upstream promoter region of 2m mediate the
constitutive and cytokine-induced regulation of 2m
transactivation (Figure 6). The E box is
a binding site for the ubiquitous factors USF1 and USF2 (Figure 6). The
B box is bound by the NF-B subunits p50 and p65, and specifically
in B cells weakly bound by c-Rel and RelB (Figure 6). The ISRE was
bound by the ubiquitous factors IRF1 and IRF2. The binding of IRF3 was
not detected but both IRF1 and IRF3 are strong transactivators of
2m (Figure 6). The lymphoid/myeloid-specific factors,
IRF4 and IRF8 (but not PU.1) bound to the ISRE, but their role in
2m transactivation is not clear. Thus, all 3 boxes are important for the constitutive and cytokine-induced levels of 2m expression in lymphoid and myeloid cell types. Thus,
similar to MHC class I molecules, 2m transactivation is
under the control of important transcriptional pathways that are
activated during injury, infection, and
inflammation.25,33,34,49,50 This is of general physiologic
importance for adequate antigen presentation, IgG transport, and iron
metabolism during these circumstances.
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| Figure 6.
Lymphoid/myeloid-specific binding of transcription
factors to the E box, ISRE, and B site of 2m.
Schematic representation of the 2m promoter and
transcription factors binding to the 3 adjacent regulatory sites in the
promoter region upstream of the SXY regulatory module. The E box is
bound by ubiquitous factors USF1 and USF2. The ISRE is a binding site
for IRF1 and IRF2 and the lymphoid/myeloid factors IRF4 and IRF8.
Because IRF3 is also a potent transactivator of 2m, also
IRF3 is likely to bind the ISRE. The B site is bound by the NF-B
subunits p50 and p65, and in B cells also marginally by c-Rel and
RelB.
|
|
| |
Acknowledgments |
The authors thank Dr M. Fenton, Dr J. Hiscott, and Dr M. Sawadogo
for providing expression plasmids.
| |
Footnotes |
Submitted September 25, 2002; accepted November 27, 2002.
Prepublished
online as Blood First Edition Paper, December 12, 2002; DOI
10.1182/blood-2002-09-2924.
Supported by the Netherlands Foundation for the Support of
Multiple Sclerosis Research no. 96-248 MS and the Dr Gisela Thier Foundation. S.J.P.G. is a research fellow of the Royal Netherlands Academy of Arts and Sciences.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Peter J. Van den Elsen, Department of
Immunohematology and Blood Transfusion, Leiden University Medical
Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands; e-mail:
pjvdelsen{at}lumc.nl.
| |
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Abstract
Introduction