From the H. Lee Moffitt Cancer Center, Interdisciplinary Oncology Program and the Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, Florida 33612
Received for publication, January 30, 2001
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ABSTRACT |
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The major histocompatibility complex (MHC)
class II transactivator (CIITA) acts as a master switch to activate
expression of the genes required for MHC-II antigen presentation.
During B-cell to plasma cell differentiation, MHC-II expression is
actively silenced, but the mechanism has been unknown. In plasma cell
tumors such as multiple myeloma the repression of MHC-II is associated with the loss of CIITA. We have identified that positive regulatory domain I binding factor 1 (PRDI-BF1), a transcriptional
repressor, inhibits CIITA expression in multiple myeloma cell lines.
Repression of CIITA depends on the DNA binding activity of PRDI-BF1 and
its specific binding site in the CIITA promoter. Deletion of a histone deacetylase recruitment domain in PRDI-BF1 does not inhibit repression of CIITA nor does blocking histone deacetylase activity. This is
in contrast to PRDI-BF1 repression of the c-myc promoter.
Repression of CIITA requires either the N-terminal acidic and conserved
PR motif or the proline-rich domain. PRDI-BF1 has been shown to
be a key regulator of B-cell and macrophage differentiation. These findings now indicate that PRDI-BF1 has at least two mechanisms of
repression whose function is dependent on the nature of the target
promoter. Importantly, PRDI-BF1 is defined as the key molecule in
silencing CIITA and thus MHC-II in multiple myeloma cells.
One of the essential factors required for the transcription of
MHC-II1 genes is the class II
transactivator, CIITA (1, 2). MHC-II expression correlates with CIITA
expression for both constitutive expression on B-cells and
cytokine-induced expression in other cell types (3-5). With only a few
exceptions to the rule CIITA functions as a "master regulator" for
MHC-II expression (2). During B-cell development MHC-II expression is
turned on at a very early stage and turned off when B-cells terminally
differentiate into plasma cells (6, 7). The process of extinguishing
MHC-II expression in plasma cells is poorly understood. Cell-cell
fusion between MHC-II-negative myeloma cells and MHC-II-positive
B-cells have indicated the presence of a dominant repressor in the
myeloma cell. (8-12). It was later shown that CIITA mRNA
transcripts are absent in myeloma cells, and introduction of CIITA
rescues MHC-II expression (13). Thus, the dominant repressor of MHC-II
expression appears to act by repressing CIITA expression.
CIITA has four distinct promoters, each transcribing a unique Exon 1, with three of the forms predominating (14). CIITA expression in B-cells
is predominantly from promoter 3 (14, 15). We have recently
characterized the CIITA promoter 3 (CIITAp3) in B-cells (16). In
vivo, two elements on the promoter, ARE-1 and ARE-2, are
occupied and essential for CIITA expression. However in the myeloma
cell line NCI-H929, the CIITAp3 is completely unoccupied suggesting
that CIITA transcription in myeloma cells may be mediated through
changes in promoter assembly (16).
One of the transcription factors induced when B-cells differentiate
into plasma cells is PRDI-BF1. It has been shown that introduction of
the murine homologue of PRDI-BF1 (Blimp-1) into B-cell lymphoma cell
lines leads to many phenotypic changes associated with differentiation
into early plasma cells, (17). Despite its dramatic effect on B-cells,
only two targets for PRDI-BF1 have been identified, c-myc
(18) and IFN- DNA Constructs and Cell Lines--
The CIITAp3 reporter
constructs (16), the c-myc PRF reporter construct
(18, 19), pc-PRDI-BF1, pcFLAG-PRDI-BF1(331-789), and
pcFLAG-PRDI-BF1(398-789) constructs (20) have been described previously. The pc-PRDI-BF1 vector was FLAG-tagged at the C-terminal end of PRDI-BF1. The three protein deletions, pcPRDI-BF1 Transient Transfection--
Cells were transfected by
electroporation as described previously (16). Transfections were
normalized by cotransfecting a constant amount of pRL-TK construct
(Promega). To make direct comparisons between the effect of
PRDI-BF1 on c-myc and CIITA promoters, the control
lanes were set at 100 for both TSA Generation of Antibodies to PRDI-BF1--
Two peptide sequences
N-terminal 1-17 residues and C-terminal 720-737 residues were
selected to raise polyclonal antibodies in rabbits (Research Genetics).
The antibodies were affinity purified from the serum.
Nuclear Extract Preparation and Electrophoretic Mobility Shift
Assays (EMSAs)--
Nuclear extracts were prepared according to Dignam
et al. (21). EMSA was performed as described previously (16)
using synthetic oligonucleotides, 2 µg of poly(dI·dC), and nuclear
extract or in vitro-translated proteins. The PRDI-BF1
oligonucleotide spans from RNA Analysis--
RNA was isolated using Trizol reagent (Life
Technologies, Inc.). Ribonuclease protection analysis was
performed using a 32P-labeled probe containing the
C-terminal (Kpn-I and Xho-I) portion of PRDI-BF1.
The RNA probe was hybridized with 15 µg of sample RNA, and digestion
was performed using RNase A/T1 (Ambion) according to manufacturer's specifications.
Western Blot and Immunoprecipitation--
Whole cell
extract was prepared in phosphate-buffered saline with 0.1% Nonidet
P-40, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, and Complete EDTA-free protease
inhibitor mixture (Roche Molecular Biochemicals). Anti-FLAG M2-agarose
affinity beads (Sigma) were used to immunoprecipitate overexpressed
FLAG-tagged proteins. Endogenous PRDI-BF1 was immunoprecipitated with
protein A-agarose beads and the N- or C-terminal PRDI-BF1 antibody at
4 °C for 1 h. The immunoprecipitated proteins were resolved by
a 10% SDS polyacrylamide gel electrophoresis, which was transferred to
a polyvinylidene difluoride membrane. The membrane was incubated with
the indicated primary antibody for 2 h followed by an anti-rabbit IgG horseradish peroxidase antibody. The secondary antibody was visualized by ECL plus (Amersham Pharmacia Biotech). For direct Western
blots or immunoprecipitations 1 × 106 or 5 × 106 cells were used per lane, respectively.
In Vitro Transcription/Translation
Reactions--
In vitro transcription/translation of
PRDI-BF1 and PRDI-BF PRDI-BF1 Represses the CIITA Promoter in Myeloma
Cells--
We have previously reported that endogenous CIITA mRNA
levels in myeloma cell lines are significantly lower compared with B-cells (16). Similarly, we observed that a CIITAp3 promoter construct with 1140 or 545 bp of promoter sequences had low activity in
myeloma cells, similar to an SV40 minimal promoter whereas in B-cell
lines the CIITA promoter was 2-4 times more active in comparison (data
not shown). This suggested that the repression of CIITA transcription
might be contained within the first 545 bp of the promoter. Progressive
5' deletions of CIITAp3 were transiently transfected into two myeloma
cell lines and tested for promoter activity. As shown in Fig.
1A deletion of the region from
PRDI-BF1 mRNA and Protein Levels are High in Human Myeloma
Cells--
The relative amount of PRDI-BF1 mRNA in myeloma cells
and B-cells was determined with a ribonuclease protection assay (Fig. 2A) and also confirmed by
Northern blotting (data not shown). Each of the myeloma cell lines
contained PRDI-BF1 mRNA whereas it was undetectable in B-cell
lines. We next examined the level of PRDI-BF1 protein present in
myeloma cells by immunoprecipitation. PRDI-BF1-specific antibodies were
raised against N- and C-terminal peptides. Both antibodies specifically
immunoprecipitated a band of ~100 kDa from myeloma cells. The band
comigrates with PRDI-BF1 protein expressed from the cloned cDNA
when transfected into a B-cell line (Fig. 2B, lanes
1 and 2). Importantly, we also tested for the presence
of PRDI-BF1 in bone marrow samples from myeloma patients. Although
these samples contain only 68 to 92% myeloma cells, PRDI-BF1 was
clearly visible in 2 of 3 patients (Fig. 2C). Thus PRDI-BF1
is present in myeloma cells concomitant with the loss of CIITA
expression.
PRDI-BF1 Binds to the CIITAp3 in Vitro--
Identification of
direct PRDI-BF1 binding to the region between Repressive Domains in PRDI-BF1--
The PRDI-BF1 protein had been
first identified by its ability to bind and repress the PRDI site
within the IFN-
Because deletion of the PR domain partially ablates the ability of the
protein to repress CIITA, it is essential to know whether this effect
is because of deletion of the PR domain or mis-folding of the
recombinant protein. One way to address the structure of the
recombinant protein is to determine whether it maintains DNA binding
activity. EMSA was performed using in vitro-translated PRDI-BF1 and PRDI-BF1 PRDI-BF1-mediated Repression of CIITAp3 Does Not Involve Histone
Deacetylase Activity--
It has been recently shown that the murine
homologue of PRDI-BF1 (Blimp-1) is able to repress c-myc
expression by recruitment of histone deacetylase (25). This recruitment
was reported to be through the proline-rich domain. Our data in Fig.
4A indicates that the deletion of the proline-rich domain
did not affect the repression of CIITA. Although the proline-rich
domain might have multiple functions, this suggests that histone
deacetylases might not be required for CIITA repression. To test the
importance of HDAC activity more directly we inhibited HDAC activity
and examined the effects on c-myc and CIITA transcription.
Addition of a histone deacetylase inhibitor, TSA, had no effect on
transcriptional repression of CIITA; however, consistent with the
previous study it completely abolished repression of c-myc
(Table I). A similar independence from
HDAC activity was also observed with endogenous PRDI-BF1. U266 myeloma
cells were transfected with the CIITA promoter construct only in the
presence or absence of TSA. If HDACs were involved in repression of
CIITA then a relief of repression would be observed upon addition of
TSA. However, no change in transcriptional activity was detected
between the TSA-treated and untreated cells. This clearly indicates
that PRDI-BF1-mediated repression of CIITAp3 does not require
HDACs.
With respect to PRDI-BF1-mediated repression of CIITA, our findings
demonstrate a role of the PR domain in transcription repression. The PR
domain of two related family members, RIZ and MDSI-EVI1, has been
reported (26) to significantly alter the function of the protein and
mediate protein-protein interaction (27). The PR domain shares
significant sequence identity to the yeast SET domain of
proteins that play an important role in determining chromosomal
structure and telomeric gene silencing. However the precise mechanism
by which PR domains bring about transcriptional repression has not yet
been characterized. It has been shown that RIZ1 has tumor suppressor
ability, and deletion of the PR domain promotes oncogenesis. A similar
observation was noted in the MDS1-EVI1 gene (28). The PR domain of
MDS1-EVI1 is a common target of viral insertions and chromosomal
translocations in leukemogenesis (29-31) suggesting it may have an
important biological function. Other regions within PRDI-BF1 can
partially compensate for loss of the PR domain. An N-terminal
truncation of the protein eliminating the acidic and PR domain but
retaining the proline-rich domain is still able to confer repressive
activity. This suggests that the proline-rich domain is a good
candidate for such a compensatory effect. In addition, deletion of the
N-terminal acidic domain of Blimp-1 significantly reduced its ability
to repress c-myc (25). Thus PRDI-BF1 (Blimp-1) appears to
utilize multiple repression domains whose function is dependent on the
target promoter.
In summary, we have identified that PRDI-BF1 binds to CIITA promoter
and silences CIITA expression in myeloma cells. This now defines the
mechanism of MHC-II silencing in plasmacytomas first observed over 14 years ago (9, 12). By use of histone deacetylase inhibitors and
specific deletions of PRDI-BF1 we have clearly demonstrated that the
proline-rich domain and histone deacetylase activity are not required
for PRDI-BF1-mediated repression of CIITA in contrast to their action
on c-myc. The findings also suggest that that the highly
conserved PR domain may have an important role in repression of
CIITA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(22). In this report we now show that PRDI-BF1
directly suppresses CIITA transcription. This results in the silencing
of MHC-II expression is long observed in plasma cells and multiple
myeloma and as such may be an attractive target for therapeutic
regulation of MHC-II.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
PR (deletes amino acids 36-166), pcPRDI-BF1
Pro (deletes amino acids
308-399), and pcPRDI-BF1
ZnF (deletes amino acids 508 to 691), were
generated in the pc-PRDI-BF1-FLAG construct. All constructs have been
confirmed by sequencing. Raji, IM-9, and CA46 are B-lymphoblastoid
cells. U266, 8266, SKO, and NCI-H929 cells are multiple myeloma cells. All cells were grown according to ATCC specifications.
and TSA+ samples.
190 to
158 base pairs of the promoter.
Unlabeled oligonucleotide competitors were used in a 50-100-fold molar excess.
PR proteins were performed using the TNT
quick-coupled transcription/translation system (Promega) according to
manufacturer's specifications. pCDNA blank vector was used as a
control. The 35S-labeled proteins were resolved by an SDS
polyacrylamide gel electrophoresis and visualized by autoradiography to
estimate the yield of full-length product prior to use in the DNA
binding assays.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
195 to
151 bp resulted in a 7-fold increase in promoter activity in
U266 cells and a 3.2-fold increase in NCI-H929 cells. Further deletion
down to position
113 bp removes the previously identified activator
element ARE-1 and severely diminished promoter activity. This indicates
that the factors necessary for activation of CIITAp3 are present in the
myeloma cells, but overall activity is silenced by elements between
195 and
151. Homology searches suggested that the region between
180 and
168 bp could be a putative binding site for the
transcriptional repressor, PRDI-BF1. To test whether PRDI-BF1 was able
to repress CIITAp3, the protein was transiently overexpressed in
B-cells. Introduction of PRDI-BF1 repressed transcription from the
CIITA promoter (CIITAp3.195) by 60% as compared with vector control
(Fig. 1B). Deletion of the potential PRDI-BF1 binding site
(CIITAp3.151) leads to the inability of PRDI-BF1 to significantly repress CIITA transcription. These findings and the observation that
the murine homologue of PRDI-BF1 is low or absent in B-cells and
increases in plasma cells (17) suggest that endogenous PRDI-BF1 in
human myeloma cells could be repressing CIITA transcription by binding
to this site on the promoter.
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Fig. 1.
PRDI-BF1 represses CIITAp3 through the 151
to
195 region. A, CIITAp3 deletion constructs (15 µg) were transiently transfected into the myeloma cells, and
luciferase activity (Luc) was measured. Deletion of the
PRDI-BF1 binding site (
195 to
151) results in a relief of
repression. The results are an average of three experiments for the
U266 cell line and one representative experiment for NCI-H929 cells.
B, CA46 B-cells were transfected with the promoter
constructs (15 µg) as indicated, along with PRDI-BF1 expression
vector (7 µg) or control vector. Deletion of the region between
195
and
151 markedly decreased the ability of PRDI-BF1 to repress CIITA
transcription. The results presented are an average of five
experiments.
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Fig. 2.
Expression pattern of PRDI-BF1 in multiple
cell types. A, PRDI-BF1 mRNA expression is detected
in each myeloma cell line by RNase protection assay. B, the
endogenous PRDI-BF1 protein detected in myeloma cells is of
approximately the same molecular weight as the cloned cDNA.
Lanes 1 and 2 are CA46 B-cells transfected with
blank vector or PRDI-BF1-FLAG expression vector, respectively.
Lanes 3 and 4 detect endogenous PRDI-BF1 in SKO
myeloma cells. IP, immunoprecipitation; F, FLAG;
N and C, N- or C-terminal PRDI-BF1 antibody,
respectively. C, detection of PRDI-BF1 protein in bone
marrow samples of multiple myeloma patients. Peripheral B-cells from a
healthy donor (lane 1) and U266 cells (lane 2)
and bone marrow samples from patient (Pt) 1 with 92% plasma
cells, patient 2 (68% plasma cells), and patient 3 (72% plasma cells)
are shown.
195 and
151 bp of the
promoter was done with a series of in vitro protein/DNA
binding studies utilizing the EMSA. Incubation of an
oligonucleotide spanning
190 to
158 bp with nuclear extracts from
myeloma cells revealed two specific complexes, which were competed by
an unlabeled self-oligonucleotide (Fig.
3A). Competition with
consensus binding sites such as an interferon response factor element (lane 3) that have similarity to the PRDI-BF1 site
did not alter complexes. To directly identify that PRDI-BF1 is part of
the complexes bound to DNA the U266 nuclear extracts were
immunodepleted of PRDI-BF1 by using either the N- or C-terminal
antibody. Western blots indicate that nearly all of the PRDI-BF1 was
removed (Fig. 3B). EMSAs performed using these depleted
extracts showed that the upper complex completely disappeared, whereas
the lower complex was reduced in intensity (Fig. 3C,
lanes 1-3). Confirmation that only PRDI-BF1 was depleted
and that the extract was not degraded was achieved using either a
B-cell specific activator protein (lanes 4-6) or
nuclear factor-1 (not shown) consensus binding site in the EMSA assay.
The difference between the two complexes containing PRDI-BF1 is not
known as yet, but it is possible that PRDI-BF1 is found in multiple
isoforms or that it can complex with other proteins.
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Fig. 3.
PRDI-BF1 binds to CIITAp3
in vitro. A, EMSA was performed using an
oligonucleotide containing the PRDI-BF1 binding site and U266 nuclear
extracts. NS, nonspecific band. B, depletion of
PRDI-BF1 from U266 nuclear extracts by immunoprecipitation with N- and
C-terminal PRDI-BF1 antibody (Ab). Western blot was
performed with the N-terminal PRDI-BF1 antibody. S, the
nuclear extract after immunoprecipitation; B,
immunocomplexed beads. C, EMSA using immunodepleted U266
nuclear extract indicates the upper complexes contain PRDI-BF1.
Lanes 1-3, PRDI-BF1 binding site is used as a probe.
Lanes 4-6, a control B-cell specific activator
protein transcription factor binding site is used as the probe
to demonstrate that PRDI-BF1 has been specifically depleted
(dep).
promoter, following viral induction (22). It is a
789-amino acid protein (depicted in Fig.
4A) bearing N- and C-terminal
acidic domains, a PR domain homologous to RIZ and MDSI-EVI
proteins (23), a proline-rich domain between amino acids 331 and 389, and a C-terminal zinc finger DNA binding motif. It has been
demonstrated that the proline-rich domain of PRDI-BF1 is able to
recruit the Groucho family of corepressors (20), which in turn can
interact with histone deacetylases (24). This suggested that the
proline-rich domain was critical for transcriptional
repression. To identify the domains responsible for PRDI-BF1-mediated
repression of CIITA we constructed three individual domain deletions in
the protein and obtained two N-terminal deletions of the protein that
maintain DNA binding activity (a kind gift from T. Maniatis). We
transiently overexpressed these proteins with the CIITAp3.195 reporter
in CA46 B-cells (Fig. 4, A and B). Several
observations have been made. First, deletion of the zinc finger DNA
binding motif abolishes PRDI-BF1 activity (lanes 7 versus 2). Second, deletion of the acidic and PR
domains does not affect repressive ability (lanes 3 versus 2), but deletion of the acidic, PR, and
proline-rich domains completely ablates PRDI-BF1 function (lanes
4 versus 2). Finally, internal deletion of
only the proline-rich domain does not affect repression (lanes
6 versus 2), whereas internal deletion of
the PR domain partially (30%) abrogates PRDI-BF1 function (lanes 5 versus 2). These findings indicate that a
complex interplay of multiple repression domains exists within the N
terminus of the protein. The proline-rich alone (lanes 3 versus 4) or the acidic and PR domains alone
(lanes 6 versus 2) are sufficient for
full activity. Because internal deletion of the PR domain maintains
only partial function, this suggests that PR domain is a key
contributor to repressive activity of PRDI-BF1. However, which domain
is most critical for CIITA repression remains unclear.
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Fig. 4.
Repressive domains of PRDI-BF1.
A, CA46 B-cells were transiently transfected with
CIITAp3.195 (15 µg) and cotransfected with control, wild type, or
various deletions of PRDI-BF1 (7 µg). Results are an average of four
experiments. B, CA46 cells were transfected with 15 µg of
the PRDI-BF1 deletion constructs and harvested 36 h later. In the
left panel whole cell extracts were subjected to
immunoprecipitation with anti-FLAG antibody followed by Western blot
using the same antibody. In the right panel a direct Western
blot was performed using antibody to the N terminus of PRDI-BF1.
Arrows indicate the wild type and mutant PRDI-BF1 proteins.
C, in vitro transcription/translation of PRDI-BF1
(lanes 3 and 4) and PRDI-BF1 PR (lanes
5 and 6) proteins. Lanes 1 and 2 are control (empty expression vector was used for the in
vitro transcription/translation reaction). Detection of the
35S-labeled proteins was performed by SDS polyacrylamide
gel electrophoresis. In the odd numbered lanes 2 µl of protein has
been loaded, and in the even numbered lanes 4 µl protein has been
loaded. D, PRDI-BF1
PR retains DNA binding activity. EMSA
was performed using a 32P oligonucleotide containing the
PRDI-BF1 binding site and the in vitro-translated proteins.
Lanes 1-3, control; lanes 4-6, PRDI-BF1;
lanes 7-9, PRDI-BF1
PR. SP, cold
specific competitor.
PR proteins to make a quantitative comparison between the DNA binding activity of the two. Similar levels of both
proteins of the expected molecular weight were expressed by in
vitro translation (Fig. 4C). As observed in Fig.
4D both proteins had comparable DNA binding activities. This
finding provides evidence that the loss of activity is not because of
catastrophic mis-folding although minor alterations are possible. In
addition, all the recombinant proteins were abundantly expressed when
transfected into CA46 B-cells. Detection of the protein expression
levels was performed by Western blot (Fig. 4B).
Cells were cotransfected with reporter plasmid and 5 µg of empty
expression vector (control) or PRDI-BF1 vector and divided into two
sets, one treated with 100 ng/ml TSA and the other without. The
luciferase activity for both promoters in the presence of empty
expression vectors was normalized at 100 relative luciferase units.
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ACKNOWLEDGEMENTS |
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We thank Dr. Maniatis (Harvard) for the PRDI-BF1 expression vectors and Dr. Dalton (Moffitt Cancer Center) for providing multiple myeloma patient bone marrow samples.
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FOOTNOTES |
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* This work is supported in part by National Institutes of Health Grant CA80990-R01 and by the Molecular Biology Core Facility at the H. Lee Moffitt Cancer Center. I. G. Y. is supported in part by the Institute of Biomolecular Science, University of South Florida.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: 12902 Magnolia
Dr., MRC 4072, H. Lee Moffitt Cancer Center, Tampa, FL 33612. Tel.: 813-979-3918; Fax: 813-979-7264; E-mail:
WRIGHTKL@moffitt.usf.edu.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M100862200
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ABBREVIATIONS |
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The abbreviations used are: MHC, major histocompatibility complex; CIITA, class II transactivator; CIITAp, CIITA promoter; PRDI-BF1, positive regulatory domain I binding factor 1; EMSA(s), electrophoretic mobility shift assay(s); bp, base pair HDAC, histone deacetylase; ARE, activator response element.
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