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, STAT1, and TGF-ß-Regulated Expression of the Class II Transactivator Gene1
University of North Carolina Lineberger Comprehensive Cancer Center, Department of Microbiology-Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible expression of class II MHC genes.
Although the role of CIITA is greatly appreciated, the mechanisms
underlying constitutive and IFN--induced expression of CIITA are not
understood. The study of CIITA induction is extremely important, but
has been fraught with difficulty. This study describes for the first
time a large (7-kb) fragment of 5' flanking sequences that mediates the
B cell-specific, IFN--induced, and TGF-ß-suppressed expression of
CIITA. This pattern of expression matches the authentic expression of
the endogenous gene. Within the 7-kb fragment, sequences that lie
between nucleotides -545 and -113 relative to the transcriptional
start site are critical for constitutive promoter expression in B
cells. In contrast, inducible activation of CIITA by IFN- requires
sequences contained in an additional 4 kb of upstream DNA. This region
mediates an IFN- response when linked to either the endogenous CIITA
promoter or a heterologous promoter. A role for STAT1 in regulation of
the CIITA promoter is shown by the rescue of IFN- induction by
expression of STAT1 in STAT1-defective U3A cells. TGF-ß significantly
inhibits IFN--mediated induction of the CIITA promoter in 2fTGH
fibroblasts, which indicates that the promoter is a target for TGF-ß.
This inhibition is achieved by suppression of the basal promoter. This
study provides a focal point for understanding the mechanism of B
cell-specific, IFN--induced, and TGF-ß-suppressed expression of
CIITA. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induces their expression in a wide variety of cell
types (5, 6, 7, 8, 9, 10). As none of the known IFN--inducible DNA-binding
factors binds to class II MHC promoter sequences, the existence of an
alternative pathway for the induction of these genes by IFN- has
been postulated. Recently, a gene termed the class II transactivator (CIITA) was cloned using genetic complementation of the in vitro derived HLA-DR-negative mutant B cell line, RJ2.2.5 (11). All current evidence shows that CIITA is absolutely essential for class II MHC gene expression. CIITA expression is lacking in a subset of patients suffering from bare lymphocyte syndrome, a severe combined immunodeficiency disease characterized by the lack of class II MHC expression (4, 11). The pivotal role of CIITA in class II MHC gene expression also has been well established in vitro in DR-negative mutant cell lines (12, 13, 14), and most recently in vivo in CIITA-/- gene knockout mice (15; Y. Ito-Lindstrom, J. F. Piskurich, and J. P.-Y. Ting, unpublished observations).
Structure-function analysis of CIITA has revealed several critical domains. The N-terminal region of the CIITA protein is rich in acidic residues and contains a transcriptional activation domain (11, 16, 17). CIITA is an unusual transcription factor in that it contains a GTP-binding domain that is critical for its function (18). CIITA is also unusual in that it does not bind any of the regulatory elements that are involved in class II MHC expression. However, activation by CIITA has been shown to require regulatory elements contained in class II MHC genes (16, 17, 19). This observation has led to the hypothesis that CIITA acts as a transcriptional coactivator via protein-protein interactions with other DNA-binding proteins that bind to the class II MHC promoter or other coactivators that may regulate the promoter. Recent reports have indeed shown interactions between CIITA and Bob1 as well as RFX5 (19, 20).
The CIITA gene per se is expressed similarly to class II MHC
genes. CIITA is required for both constitutive expression of class II
MHC in B cells and IFN--inducible class II MHC expression in other
cell types (12, 13, 14, 15). The CIITA mRNA is itself induced by IFN-
(12, 13, 14). As expected, induction of CIITA expression by IFN- has
been shown to precede that of class II MHC. However, the upstream
events leading to CIITA induction have not been well defined. It
recently has been shown that induction of CIITA mRNA expression by
IFN- requires STAT1 protein (21), and is down-regulated by TGF-ß
(21, 22). These findings are supported by the observations that
STAT1-/- gene knockout mice are defective in CIITA gene
expression (23) and that TGF-ß1-/- mice
develop severe autoimmune disease accompanied by increased expression
of class II MHC (24, 25). Interestingly, mutant cell lines that are
selectively defective in the IFN- induction of CIITA have been
reported by us and others (12, 13), which suggests that the mechanism
of CIITA induction may include novel components.
This study shows the first isolation and analysis of a 7-kb region
within the 5' flanking sequence of the human CIITA gene that contains
sequences that are important in constitutive, IFN--inducible, and
TGF-ß-suppressible expression of CIITA. Using chimeric reporter
plasmids in which fragments of the CIITA gene were fused to the
luciferase reporter gene, we identified distinct CIITA gene fragments
that are necessary for the constitutive expression of CIITA in B cells
and the induction of the CIITA gene by IFN-. These sequences are
regulated similarly to the intact CIITA gene. Induction by IFN- is
defective in U3A, a cell line defective in STAT1, and activated by
overexpression of STAT1, which indicates a role for STAT1 in regulation
of the CIITA gene at the level of the promoter. Pretreatment with
TGF-ß, which is known to inhibit class II MHC expression,
significantly inhibits induction of the CIITA promoter in the absence
or presence of IFN-. These 5' flanking DNA sequences can be used to
further delineate the mechanism of B cell-specific, IFN--induced,
and TGF-ß-suppressed expression of CIITA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Raji is a human EBV-transformed Burkitt’s lymphoma cell line
that constitutively expresses a high level of class II MHC Ags. Raji
cells were grown in RPMI 1640 supplemented with 8% heat-inactivated
FCS, 2 mM L-glutamine, and 100 U/ml penicillin and
streptomycin. U373-MG is a human glioblastoma multiforme line that
expresses both class II MHC molecules and CIITA after treatment with
IFN- (7, 13). U373-MG is grown in McCoy’s 5A medium supplemented
with 10% FCS, 2 mM L-glutamine, and 100 U/ml penicillin
and streptomycin. The 2fTGH cell line is derived from HT 1080 human
fibrosarcoma cells that do not constitutively express class II MHC Ags,
but express a high level of these Ags after IFN- induction. U3A
(generously provided by Dr. George Stark, Cleveland Clinic Foundation
Research Institute, Cleveland, OH) is a STAT1-defective cell line
derived from 2fTGH (26). U3A and 2fTGH cells were maintained in DMEM
(Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 2 mM
L-glutamine, and 100 U/ml penicillin and streptomycin.
Isolation and characterization of human CIITA genomic clones
To isolate genomic clones containing the 5' flanking region of
human CIITA, approximately one million recombinants from a commercially
prepared 
FIX II human fibroblast genomic library (catalog 946204;
Stratagene, La Jolla, CA) were screened as previously described (27).
The probe was simultaneously amplified and radiolabeled using PCR
(Perkin-Elmer Cetus, Norwalk, CT) in the presence of
[32P]dCTP (DuPont NEN, Boston, MA). Oligonucleotide
primers used for the PCR correspond to bp 91 to 111
(5'-GCTGCCTGGCTGGGATTCCTA-3') and the reverse complement of bp 375 to
395 (5'-CCTCCCTGGTCTCTTCATCAC-3') of the human CIITA cDNA sequence,
as reported by Steimle et al. (11). A human CIITA cDNA was used as
template for the PCR (13). Twelve overlapping CIITA genomic clones were
isolated, plaque purified, and mapped by Southern blot analyses. DNA
prepared from each clone by precipitation with polyethylene glycol (28)
was digested with NotI to excise the genomic insert from the
vector DNA and subsequently digested with HindIII. All
restriction enzymes were from New England Biolabs (Beverly, MA). After
electrophoresis in 1% agarose, the DNA was depurinated in 2.5 M HCl,
denatured in 1.5 M NaCl, 0.5 M NaOH, and transferred to Nytran
(Schleicher & Schuell, Keene, NH) by capillary blotting. Filters were
baked at 90°C for 1 h, and probed as described above. Duplicate
filters were prehybridized for 1 h at 42°C in 6x SSC, 5x
Denhart’s solution, 1% SDS, and 100 µg/ml boiled salmon sperm DNA;
then hybridized at 42°C for 18 h in the same solution with
3 x 10-12 mol of 32P end-labeled
5'-untranslated region oligonucleotide added. The 5'-untranslated
region oligonucleotide, 5'-AGGATGCCTTCGGATGCCCAGCTCAGAAGC-3', which
corresponds to the reverse complement of bp 15 to 44 of the human CIITA
cDNA sequence described above (also see Fig. 1
B), was
5' end labeled using T4 polynucleotide kinase (New England Biolabs) and
[-32P]ATP (3000 Ci/mmol; Dupont NEN) (29). These
duplicate filters were washed in 6x SSC, 0.1% SDS at 42°C and
visualized by autoradiography using an intensifying screen and Kodak
XAR-5 film.
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A). This
fragment was subcloned into pBluescript II (SK-)
(Stratagene), and approximately 1 kb from the 3' end of the fragment
was sequenced by the dideoxy chain-termination method (30), using
Sequenase (United States Biochemical Corp., Cleveland, OH) (Fig. 1B). The 3' end of exon 1 of human CIITA was
determined by a comparison of the genomic DNA sequence with the
sequence of the human CIITA cDNA (11), and the 5' end of exon 1 was
determined by mapping the transcriptional start site in Raji cells, as
described below. Identification of CIITA transcriptional start site by primer extension
Primer extension analysis of the CIITA mRNA was performed using 20 µg of poly(A)+ RNA isolated from Raji cells, as previously described (31), using the 5'-untranslated region oligonucleotide described above. Absolute product lengths were obtained by comparison with a sequencing ladder. One predominant transcriptional start site for the human CIITA gene was identified. This start site is located very close (within 16 bp) to the end of the human CIITA cDNA reported by Steimle et al. (11).
Constructs
TfiI digestion was used to liberate a fragment
containing 123 bp of 5'-untranslated sequences and approximately 1200
bp of 5' flanking sequences of the human CIITA gene. This
TfiI fragment was blunted and cloned into the
SmaI site upstream of the luciferase reporter gene in the
pGL2-Basic vector (Promega Corp., Madison, WI) to create the
p1300CIITA.Luc plasmid (Figs. 1A and 2). This
TfiI fragment was also cloned into the SmaI site
of pBluescript II (SK-) (Stratagene). Digestion with
KpnI and XhoI liberated a smaller fragment
containing 123 bp of 5'-untranslated and 545 bp of flanking sequence
that was cloned into the KpnI-XhoI site of the
pGL2-Basic vector to create p668CIITA.Luc.
The deletion construct p236CIITA.Luc was generated by PCR using
Taq polymerase and standard reaction conditions
(Perkin-Elmer Cetus). The 5' oligonucleotide primer for the PCR
corresponded to nucleotide -113 to -96 of the 5' flanking sequence of
CIITA shown in Figure 1B. This primer had a 12-nucleotide
extension at the 5' end with the sequence ACGTGGGGTACC, which generated
a KpnI site immediately adjacent to the sequence being
amplified. The 3' primer, 5'-ACGTACAAGCTTGATATCGAATTCCTG-3',
contained the HindIII site of the polylinker located
immediately adjacent to the 3' end of the CIITA fragment in
p668CIITA.Luc. The p668CIITA.Luc plasmid was used as template for the
PCR. The amplified DNA fragment was digested with
KpnI/HindIII and size fractionated by
electrophoresis. This fragment was substituted for the CIITA fragment
in p668CIITA.Luc by subcloning into vector sequences that had been
similarly digested and fractionated from p668CIITA.luc.
A two-step cloning procedure was used to construct a reporter
plasmid containing all of the flanking sequences present in the
original XbaI fragment. In the first step, the approximately
4-kb XbaI-KpnI fragment (Figs. 1A and
2) was removed as a SmaI-KpnI fragment from the
pBluescript II (SK-) (Stratagene) plasmid bearing the
entire 7-kb XbaI CIITA gene segment and cloned into the
SmaI-KpnI sites of p668CIITA.Luc to form the
p7000-2000CIITA.Luc plasmid. In the second step, the central
approximately 2-kb KpnI fragment was added to
p7000-2000CIITA.Luc, reconstituting in the plasmid, p7000CIITA.Luc, the
native approximately 7 kb of CIITA 5' flanking sequences found in the
original XbaI fragment. A very similar two-step cloning
procedure was used in the creation of p7000-668CIITA.SV40.Luc (Fig. 2),
a reporter plasmid that bears the SV40 promoter instead of the 668-bp
CIITA promoter-proximal fragment. For p7000-668CIITA.SV40.Luc, the 4-kb
XbaI-KpnI and 2-kb KpnI fragments were
cloned into the SmaI-KpnI sites upstream of the
enhancerless SV40 promoter in the pGL2-Promoter vector (Promega Corp.).
The CIITA DNA inserts and junctions in p236CIITA.Luc and p668CIITA.Luc,
and the junctions of plasmids p1300CIITA.Luc, p7000CIITA.Luc,
p7000-2000CIITA. Luc, and p7000-668CIITA.SV40.Luc have all been
confirmed by DNA sequencing.
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Transient transfections of Raji and U373-MG cells were performed
by electroporation using a Bio-Rad gene pulser (Bio-Rad, Richmond, CA).
Ten micrograms of plasmid DNA, purified using Qiagen columns (Qiagen,
Chatsworth, CA), were used in each transfection. Three million cells in
300 µl of culture medium were pulsed at 200 mV at a capacitance
setting of 960 µF. Raji cells were harvested for luciferase assays
48 h after transfection. U373-MG cells were allowed to adhere for
6 h after transfection, and then parallel cultures were treated
with 500 U/ml of human rIFN- (Genentech, South San Francisco, CA)
for 14 h before all cell cultures were harvested for luciferase
assays.
Transient transfection of 2fTGH and U3A cells was performed by the
calcium phosphate coprecipitation method (29). Cells were plated in
10-cm dishes at a density of 5 x 105 cells and
transfected 24 h later. Ten micrograms of reporter construct, or
10 µg of reporter construct in combination with 10 µg of either
pcDNA3 (Invitrogen Corp., Carlsbad, CA) or the STAT1 expression vector
were added to each dish of cells, and dishes were incubated at 37°C
in 5% CO2. After 6 h, the precipitates were removed,
and dishes were rinsed twice with PBS. For experiments without
TGF-ß1 pretreatment, culture medium was added with or
without 500 U/ml of IFN- and cells were harvested for luciferase
assays 14 h later. For experiments involving TGF-ß1
pretreatment of 2fTGH cells, culture medium was added with or without
10 ng/ml of TGF-ß1 (R&D Systems, Minneapolis, MN). After
12 h, culture medium was changed to medium with or without 500
U/ml of IFN-, and cells were harvested for luciferase assays 14
h later. The STAT1 expression vector (generously provided by Dr. James
Darnell, Jr., Rockefeller University, New York, NY) has been previously
described (32). The empty pcDNA3 vector (Invitrogen Corp.) was used as
a negative control.
Luciferase assays were performed using an LB 953 AutoLumat (EG&G,
Berthold, Germany), as previously described (33). The protein content
of cell extracts was determined by the Bradford assay (34). Luciferase
activity was measured as relative light units (RLU) per microgram of
protein. Transfections of the pGL2-Basic vector plasmid gave
essentially negligible luciferase activity for all cell types assayed.
Fold induction after IFN- treatment was calculated by dividing the
RLU of IFN--treated samples by the RLU of untreated samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study mechanisms for constitutive or IFN--inducible CIITA
mRNA expression, we isolated the 5' flanking sequences of the human
CIITA gene. Twelve genomic clones spanning 39 kb of human CIITA gene
flanking sequences were isolated as described in detail in
Materials and Methods (Fig. 1A). Two overlapping
clones that hybridize to an oligonucleotide probe containing
5'-untranslated sequence of the human CIITA gene (11) were subjected to
additional Southern blot analyses, allowing localization of the exon
that bears these sequences to a 973-bp KpnI-XbaI
genomic DNA fragment that is present in both clones. The nucleotide
sequence of this fragment is shown in Figure 1B. Primer
extension analyses of poly(A)+ RNA isolated from Raji cells
identified one predominant transcriptional start site located in this
fragment within 16 bp of 5' end of the human CIITA cDNA sequence (11).
The position of this start site is indicated as +1 in Figure 1B. An examination of the sequence directly upstream of this
site does not reveal the presence of a conventional TATA box or
initiator consensus (35). This region contains a consensus Sp1 binding
site at -351 and a consensus STAT1 protein-binding site at -239 (36).
There is an inverted octamer-like sequence at -45 (37), and PU.1 core
motif at -181 that is surrounded by a purine-rich sequence, as is
typical for PU.1 binding sites (38).
Localization of 5' flanking regions of the CIITA gene responsible
for constitutive and IFN--inducible CIITA expression
Fragments of the flanking region of the CIITA gene were used to
direct the expression of the luciferase reporter gene in various cell
types. Figure 2 shows the six constructs
that were used in these analyses. A genomic fragment containing 123 bp
of 5'-untranslated and 545 bp of flanking sequence (the region from
KpnI to TfiI in Fig. 1B) was
cloned into the pGL2-Basic vector to create p668CIITA.Luc. A shorter
construct, p236CIITA.Luc, which contains 123 bp of 5'-untranslated and
113 bp of flanking sequence, was derived from p668CIITA.Luc. A third
plasmid, p1300CIITA.Luc, which bears a TfiI fragment
containing 123 bp of 5'-untranslated and approximately 1200 bp of 5'
flanking sequences of the human CIITA, was also cloned into this
reporter vector. In addition, a two-step cloning procedure was used to
construct a plasmid containing all of the 5' flanking sequences present
in the original 7-kb CIITA XbaI fragment (Fig. 1A). In the first step, the approximately 4-kb
XbaI-KpnI fragment was added at the
KpnI site of p668CIITA.Luc to form p7000-2000CIITA.Luc, a
plasmid that bears an internal deletion of approximately 2 kb (Fig. 2).
In the second step, the central approximately 2-kb KpnI
fragment was added to p7000-2000CIITA.Luc to form p7000CIITA.Luc, a
plasmid that contains the native approximately 7 kb of CIITA 5'
flanking sequences found in the original XbaI fragment.
Raji Bcells express CIITA constitutively (13). Upon transient
transfection into Raji cells, p668CIITA.Luc, p1300CIITA.Luc,
p7000CIITA.Luc, and p7000-2000CIITA.Luc all exhibit high levels of
luciferase activity (Fig. 3). These
findings are consistent with p668CIITA.Luc containing promoter
sequences involved in constitutive expression of the CIITA gene in B
cells. A shorter construct, p236CIITA.Luc, reproducibly gives from 5-
to 16-fold less activity than p668CIITA.Luc. This finding suggests that
expression of CIITA in B cells is dependent on sequences that lie
between nucleotides -545 and -113 (marked with an asterisk in Fig. 1B) upstream of this transcriptional start site of
CIITA. In the experiment shown in Figure 3, the longer constructs
p1300CIITA.Luc and p7000-2000CIITA.Luc had slightly greater activity
than p668CIITA.Luc, while p7000CIITA.Luc had slightly less activity.
These results suggest that distinct sequences located upstream of
nucleotide -545 may be involved in the enhancement and the suppression
of the CIITA promoter in B cells. However, we cannot rule out that the
different lengths of CIITA flanking sequences present in these
constructs might result in these variations.
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induction of CIITA, transient transfections of the U373-MG human
glioblastoma cell line were performed. Treatment with IFN- has been
shown to induce both class II MHC and CIITA in these cells (7, 13).
Although p668CIITA.Luc and p1300CIITA.Luc have high constitutive
activity in Raji cells, these constructs do not exhibit
IFN--inducible activity in U373-MG cells (Fig. 4). A kinetic assay also was performed to
rule out the possibility that these constructs are transiently
activated in response to IFN- and the previous assay had missed the
peak of induction. Inducible luciferase activity is undetectable in
U373-MG cells treated with IFN- for intervals from 3 to 27 h
(data not shown). The lack of inducibility for p1300CIITA necessitated
the construction and testing of longer fragments. The plasmid
construct, p7000CIITA.Luc, is inducible by IFN- in U373-MG cells
(Fig. 4). The p7000-2000CIITA.Luc construct, which bears an internal
2-kb deletion, was also tested to further define the region within
p7000CIITA.Luc that is responsible for activity. As p7000-2000CIITA.Luc
retains IFN- inducibility, the activity is localized to the
approximately 4-kb XbaI-KpnI fragment of the
CIITA gene.
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-responsive region that was defined in
U373-MG cells is also functional in other cell types, the 2fTGH human
fibroblast cell line was tested. There were several reasons for
choosing this cell line. First, it is the parental line of a series of
mutant cell lines that are defective in specific molecules that are
important in IFN- responses (26). Second, IFN--inducible HLA-D
and CIITA expression by 2fTGH cells can be inhibited by TGF-ß (21).
These features were used in experiments presented later in this
work.
As shown in Figure 5A, both
p7000CIITA.Luc and p7000-2000CIITA.Luc demonstrate IFN--inducible
luciferase activity in 2fTGH cells. To further determine whether CIITA
5' flanking sequences that are inducible in the context of the CIITA
promoter can confer inducibility by IFN- on a heterologous promoter,
we constructed p7000-668CIITA.SV40.Luc, which contains the SV40
promoter instead of the 668-bp CIITA promoter fragment (Fig. 2).
Transient transfection experiments using 2fTGH cells show an average
fold induction for p7000-668CIITA.SV40.Luc of 4.7 (Fig. 5B). These results provide further evidence that the
5' flanking region included in this study plays a role in regulation of
the CIITA gene by IFN-.
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To investigate the role of STAT1 in activation of IFN-
inducibility of the 5' flanking sequences of the CIITA gene,
STAT1-defective U3A cells were transiently transfected with
p7000CIITA.Luc in combination with a STAT1 expression vector plasmid
(32). The empty expression vector, pcDNA3, was used as a negative
control. Neither the luciferase activity of p7000CIITA.Luc nor
p7000-2000CIITA.Luc is activated by IFN- treatment alone in U3A
cells (Fig. 6A). STAT1
reproducibly activates the IFN--induced luciferase activity of
p7000CIITA.Luc and p7000-2000CIITA.Luc with average inductions of 6.9-
and 5-fold, respectively (Fig. 6B). STAT1 does not
significantly activate induction of the pGL2-Basic vector, the negative
control reporter plasmid. IFN- is required for activation of the
STAT1 protein by phosphorylation. As expected, transfection of U3A
cells with p7000CIITA.Luc or p7000-2000CIITA.Luc in combination with
the STAT1 expression vector did not stimulate luciferase activity in
the absence of IFN- treatment (data not shown).
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of a heterologous promoter linked to the 5' flanking sequences
of CIITA. The reporter plasmid p7000-668CIITA.SV40.Luc, which contains
the SV40 promoter, instead of the 668-bp CIITA promoter fragment, is
stimulated by STAT1 expression an average of 5.4-fold (Fig. 6C). The pGL2-Promoter vector reporter construct that
contains an enhancerless SV40 promoter is not stimulated by STAT1
expression.
TGF-ß suppresses inducibility of the 5' flanking region of the
CIITA gene in response to IFN-
TGF-ß has been shown to inhibit the IFN--induced
transcription of class II MHC genes in a number of different cell types
(39, 40, 41). Recent data with several human cell lines including 2fTGH
indicate that pretreatment with TGF-ß reduces the induction of CIITA
mRNA in response to IFN- (21, 22). The mechanism of this inhibition
may involve suppression of the induction of the CIITA promoter by
IFN-. The luciferase activity of p7000CIITA.Luc when transiently
transfected into 2fTGH cells is activated by IFN- treatment (Fig. 7, top panel).
Pretreatment of the cells for 12 h with 10 ng/ml of
TGF-ß1 results in a complete suppression of this
IFN--induced luciferase activity. However, TGF-ß treatment in the
absence of IFN- also alters the basal luciferase activity of
p7000CIITA.Luc in these cells. This indicates that TGF-ß interferes
with the promoter activity of p7000CIITA.Luc even in the absence of
IFN-. Similar results are seen in transfections with the
p7000-2000CIITA.Luc plasmid (Fig. 7, bottom
panel).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is recognized as the primary cytokine that induces the
expression of these molecules, with CIITA serving as a master regulator
required for both constitutive and IFN--inducible expression.
Although extremely important, regulation of the CIITA gene is poorly
understood, and investigation into the mechanism of regulation of this
gene by IFN- has been fraught with difficulty. This difficulty has
been due in part to the levels of endogenous CIITA protein and mRNA,
which are low and can only be detected by the most sensitive
techniques. It is possible that such low levels might be indicative of
the tight controls that limit the expression of CIITA. A second reason
is the length of the regulatory DNA region that is required for
authentic regulation of the CIITA gene.
This study describes 5' flanking sequences of CIITA that control gene
expression in a pattern that parallels expression of the endogenous
CIITA gene. A 668-bp proximal fragment of the CIITA promoter contains
sequences sufficient for constitutive CIITA promoter activity in B
cells (Fig. 3). This fragment contains both an inverted imperfect
octamer-binding protein site (CTTTGCAT in CIITA vs consensus: ATGCAAAT)
(37) and a consensus binding site forPU.1 (Fig. 1B), a transcription factor that binds a purine-rich
sequence with a central core sequence of GGAA (38). Both of these sites
potentially bind transcription activators that are expressed in B cells
and as such, either or both sites may be involved in the constitutive
tissue-specific expression of CIITA in these cells. Deletion of the 5'
end of p668CIITA.Luc to 236 bp retains the octamer site, yet severely
attenuates the constitutive activity of this promoter in B cells. This
implies that B cell promoter activity either does not involve this site
or involves interactions between the octamer and other more distal
sites within the 668-bp fragment. Interestingly, although it contains a
region that resembles the IFN- activation sequence (Fig. 1B, consensus: TTNCNNNAA), which is a potential binding site
for IFN--activated STAT1 homodimers (42), this fragment is not
inducible by IFN- in the cell lines tested. While the 668-bp DNA
fragment is sufficient for constitutive CIITA promoter expression in B
cells, this study presents evidence that inducible activation of CIITA
by IFN- requires sequences that lie at least 2 kb 5' of this
fragment (Fig. 4). One possibility is that this distal activity
represents a second promoter region for CIITA that is only active in
inducible cell types in response to IFN-. However, when the 4-kb
XbaI-KpnI fragment that contains this activity
(Fig. 1A) is cloned into the pGL2-Basic vector and
transiently transfected into either U373-MG or 2fTGH cells, it has
extremely low basal activity much like that of pGL2-Basic, which is a
promoterless construct (data not shown). Therefore, it is more likely
that these sequences represent a distal IFN--inducible enhancer
activity. This premise is supported by data in this study that show
that this region can confer IFN- inducibility to a heterologous
promoter (Figs. 5B and 6C).
Although it has not been directly demonstrated previously, several
lines of evidence strongly implicate the involvement of STAT1 protein
in regulation of the CIITA gene by IFN-. CIITA mRNA expression is
induced upon IFN- treatment significantly earlier than mRNA for
class II MHC (12). Induction of CIITA mRNA by IFN- can be inhibited
by staurosporin (14), a kinase inhibitor that has been shown to block
phosphorylation of STAT1 in response to IFN- (43, 44). In addition,
antisense oligonucleotides complementary to STAT1 mRNA inhibit IFN-
induction of CIITA mRNA in astroglioma cells (45). Evidence is
presented in this study that induction of the CIITA 5' flanking
sequences by IFN- is deficient in the STAT1-defective cell line, U3A
(Fig. 6A), and that induction in these cells is
activated by IFN- treatment when there is concomitant overexpression
of the STAT1 protein (Fig. 6B). Therefore, this study
now presents strong evidence for the role of STAT1 in the regulation of
the IFN--inducible region of the CIITA promoter (see model, Fig. 8). The CIITA gene may be activated
either directly by STAT1 binding to sequences in the 5' flanking region
or indirectly by induction of another factor required for CIITA
transcription. Considering the short time period (less than 3 h)
and the lack of a requirement for new protein synthesis before
IFN--induced CIITA transcripts are first detectable (12, 14), it is
more likely that STAT1 directly regulates the CIITA promoter.
However, an indirect role for STAT1 cannot be excluded.
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-induced
transcription of class II MHC genes in many cell types, including
astrocytes and melanoma cells (39, 40, 41). The recent observation of
severe autoimmune disease, accompanied by increased expression of class
II MHC in TGF-ß1 null mice, emphasizes the important role
of TGF-ß in the suppression of class II MHC gene expression (24, 25).
The precise mechanism of suppression remains to be elucidated, but
current evidence suggests a role for TGF-ß in suppressing CIITA at
the level of transcription. Recently, sensitive reverse-transcriptase
PCR and ribonuclease protection assays have been used to demonstrate
decreases in IFN--induced CIITA mRNA levels in macrophage,
fibroblast, and astrocytoma cells that were pretreated with TGF-ß
(21, 22). This study represents the first report that identifies the
CIITA promoter as a target of TGF-ß-mediated gene suppression.
TGF-ß may act by interfering with the binding or transcriptional
activation activity of an IFN--activated factor such as STAT1.
However, the results shown in this study indicate that suppression by
TGF-ß of the CIITA promoter occurs even in the absence of IFN-.
Furthermore, enhancement of the CIITA promoter by IFN- occurs
despite the antagonistic effect of TGF-ß (Fig. 7). This finding
suggests that TGF-ß most likely interferes at a step of CIITA
promoter activation other than the IFN- induction pathway (Fig. 8, model). It is possible that TGF-ß suppression may be mediated by a
TGF-ß-inhibitory element (46). A TGF-ß-inhibitory element consensus
sequence, GNNTTGGtGA, has been found in a number of genes that are
inhibited by TGF-ß, and its identification in the CIITA
promoter will be of interest.
In summary, these findings indicate that the constitutive B cell
expression, as well as the TGF-ß and IFN--regulated control of
CIITA, are mediated by sequences found within a 7-kb fragment of the 5'
flanking DNA of the CIITA gene. They provide a basis for further
analyses to identify the mechanism for the constitutive expression of
CIITA, as well as induction of the gene by IFN- and suppression by
TGF-ß. This region of DNA can also be utilized to identify
potentially novel regulators of CIITA expression. Investigations into
the regulation of the CIITA gene could provide information crucial to
the development of new strategies for down-regulation of class II MHC
genes, which has broad implications in transplantation and the control
of autoimmune diseases.
Note added in proof. Since the submission of this manuscript,
two other groups have described a B-cell-specific CIITA promoter
activity that is concordant with the data presented in this study (47, 48). This current study limits the region required for constitutive
activity to nucleotides -545 to -113 upstream of this transcriptional
start site of CIITA. The report by Muhlethaler-Mottet et al. (48) also
described an IFN--inducible promoter for CIITA that is located
downstream of the region defined in this study. Together, these studies
demonstrate at least two regulatory regions that control the induction
of the human CIITA gene by IFN-.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jenny P.-Y. Ting, Department of Microbiology-Immunology, Lineberger Comprehensive Cancer Center, CB#7295, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. E-mail address:
3 Abbreviations used in this paper: Ii, invariant chain; CIITA, class II transactivator; RLU, relative light units.
Received for publication April 8, 1997. Accepted for publication September 18, 1997.
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