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J Biol Chem, Vol. 274, Issue 38, 26661-26667, September 17, 1999
§,,,, and
From the Pathology and Physiology Research Branch,
Health Effect Laboratory Division, NIOSH, National Institutes of
Health, Morgantown, West Virginia 26505 and the ¶ Laboratory of
Experimental Immunology, NCI-Frederick Cancer Research and Development
Center, National Institutes of Health, Frederick, Maryland 21702
 |
ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Negative regulation of cytokine gene
transcription is an important mechanism in maintaining homeostasis of
immune function. In this study, we characterized a silencer element in
the human interleukin-3 gene promoter that is responsible for the
cell-specific expression of interleukin-3. This silencer activity was
proposed to be mediated by an unidentified nuclear inhibitory protein
(NIP). In this study, we have identified two nuclear factors that are responsible for the silencer activity in T cells. The NIP element forms
four specific DNA-protein complexes (designated as complexes A-D) with
the Jurkat nuclear proteins. Complex A contains a nuclear protein that
shares DNA-binding specificity with the transcription factor AP2
(designated as an AP2 sequence-recognizing factor (ASRF)). Formation of
this ASRF complex is required for the NIP silencer function, as
mutation of the ASRF-binding site abrogated the silencer activity.
Complex B contains the nuclear factor YY1
(Yin-Yang 1), whose function is to
down-regulate ASRF activity in the silencer. YY1 activity is supported
by data from mutation and cotransfection analyses. Complexes C and D
are formed by nonspecific binding proteins and do not express any
regulatory activity in the NIP element. These data indicate that a cell
type-specific silencer activity might be determined by a unique profile
of ubiquitous transcription factors.
Interleukin (IL)1-3 is a
potent growth factor that is involved in the regulation of
hematopoiesis (1, 2). Like the cytokine interferon- Negative regulation of cytokine transcription plays an important role
in regulating the response of the immune system to challenge (11).
Although there are several possibilities for down-regulating gene
transcription, silencer elements in the cytokine gene promoters constitute an important part of the gene regulation mechanism. A
silencer function has been observed in the IL-2 (12), IL-3 (8), IL-4
(13), TNF- In this study, we observed that the NIP element shares a striking
similarity with the IFN- Oligonucleotides and Antibodies--
Oligonucleotides were
synthesized by the phosphoramidite method on an Applied Biosystems
Model 392 DNA/RNA synthesizer. The synthesized oligonucleotides were
deprotected at 50 °C overnight. Complementary strands were denatured
at 80 °C for 5 min and annealed at room temperature. The
double-stranded probe was labeled with [32P]dCTP
(Amersham Pharmacia Biotech) using Klenow fragment (Life Technologies,
Inc.). The sequences of oligonucleotides used in this study are as
follow: a YY1-binding site in the Moloney murine leukemia virus gene
(4), 5'-TGCCTTGCAAAATGGCGTTACTGCAG-3'; and an AP2-binding site in the
SV40 virus (24), 5'-GGTGTGGAAAGTCCCCAGGCTCCCCAGCAC-3'. Antibodies
against nuclear proteins YY1 and AP2 were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA).
Cells--
Jurkat cells (human CD4+ T lymphoblast
cell line) were cultured in RPMI 1640 medium supplemented with 10%
fetal calf serum, 2 mM glutamine, and 100 units/ml
penicillin/streptomycin (complete medium). Fresh human peripheral blood
total T cells were purified by the nylon wool method, and purity was
monitored by CD3 staining (CD3+ > 95%). The cells were
cultured in RPMI 1640 medium supplemented with 2% fetal calf serum, 2 mM glutamine, and antibiotics.
mRNA Assay--
Jurkat cells (2 × 106)
were treated with PMA (10 ng/ml) plus ionomycin (1 µg/ml) for
different times as indicated in the figure legends. Total mRNA was
extracted using Trizol (Life Technologies, Inc.) according to the
manufacturer's instruction. IL-3 mRNA levels were determined by
the ribonuclease protection assay using a RiboQuant multiprobe
ribonuclease protection assay system (human cytokine/chemokine-4, Pharmingen, San Diego, CA).
Nuclear Extraction Procedure--
The nuclear extracts were
prepared as described before (22). Cells (5 × 107)
were treated with 500 µl of lysis buffer (50 mM KCl,
0.5% Nonidet P-40, 25 mM Hepes (pH 7.8), 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml
aprotinin, and 100 µM dithiothreitol) on ice for 4 min.
After 1 min of centrifugation at 14,000 rpm, the supernatant was saved
as a cytoplasmic extract. The nuclei were washed once with the same
volume of buffer without Nonidet P-40, put into a 300-µl volume of
extraction buffer (500 mM KCl and 10% glycerol with the
same concentrations of Hepes, phenylmethylsulfonyl fluoride, leupeptin,
aprotinin, and dithiothreitol as lysis buffer), and pipetted several
times. After centrifugation at 14,000 rpm for 5 min, the supernatant
was harvested as the nuclear protein extract and stored at Electrophoretic Mobility Shift Assay (EMSA)--
The DNA-protein
binding reaction was conducted in a 24-µl reaction mixture that
included 1 µg of poly(dI·dC) (Sigma), 3 µg of nuclear protein
extract, 3 µg of bovine serum albumin, 4 × 104 cpm
of 32P-labeled oligonucleotide probe, and 12 µl of 2×
reaction buffer (40 mM Tris (pH 7.4), 8% Ficoll 400, 4 mM EDTA, and 1 mM dithiothreitol). In some
cases, the indicated amount of double-stranded oligonucleotide was
added as an unlabeled competitor. This mixture was incubated on ice for
10 min without or for 20 min with antibody in the absence of
radiolabeled probe and then for 20 min at room temperature in the
presence of radiolabeled probe. After incubation, the reaction mixture
was resolved on a 5% acrylamide gel (National Diagnostics, Inc.,
Atlanta, GA) that had been prerun at 110 V for 1 h with 0.5× Tris
borate/EDTA buffer. The loaded gel was run at 210 V for 90 min, dried,
and placed on X-Omat film (Eastman Kodak Co.). The film was developed
after overnight exposure at Plasmid Vectors--
Five plasmid vectors were used in this
study: 1) The IL-3/pCAT plasmid (a generous gift from Dr. L. R. Gottschalk, Department of Medicine, University of Chicago, Chicago, IL
(7); in this reporter vector, the chloramphenicol acetyltransferase
(CAT) gene is controlled by the human IL-3 gene promoter ( Transfection Assays--
Jurkat T cells were grown in complete
medium as described above. Cells (5 × 106) were
transiently transfected with 5-10 µg of the reporter plasmid DNA
with DEAE-dextran (22). The pGL2 control luciferase expression vector
(0.5 µg) was used as an internal control. After transfection, the
cells were washed once in phosphate-buffered saline solution and
cultured in 10 ml of complete medium at 37 °C for 24 h. The cells were then stimulated with PMA (10 ng/ml) plus ionomycin (1 µg/ml) for 12 h, harvested, and disrupted by freezing-thawing three times. The cell lysate was used for the reporter gene assay. The
CAT, Functional Characterization of NIP--
In this study, Jurkat
cells (a human CD4+ T lymphoma) were used for
characterization of the IL-3 silencer NIP. This cell line is able to
express IL-3 mRNA upon activation by a combined stimulation of PMA
plus ionomycin, which mimics signals of the activated T cell receptor.
As shown in Fig. 1A, the
induced IL-3 mRNA was observed at 2 h following stimulation
(lane 4), and the mRNA level kept increasing up to
8 h (lanes 5 and 6). In the absence of
stimulation, the IL-3 mRNA was not detectable (lane 1).
This indicates that the IL-3 gene promoter was induced by a stimulation
of PMA plus ionomycin in Jurkat cells. In the rest of study when the
IL-3 promoter was assayed, the stimulation of PMA plus ionomycin was used to induce promoter activity.
Generally, silencer activities are dependent on promoter type and DNA
orientation. To examine whether NIP has these features, the function of
NIP was tested in different gene promoters and in the reverse
orientation. Three promoters were employed to test the promoter
specificity. They are the human IL-3 gene promoter (
The reporters with a reversed NIP element were used to study DNA
orientation dependence. The results show that NIP functioned only in
the proper orientation (Fig. 1D, NIPp). In the
reverse orientation (NIPr), NIP failed to inhibit the IL-3
promoter activity (Fig. 1D). This indicates that NIP is also
orientation-specific in its function. In the absence of stimulation,
the IL-3 gene promoter activity is too weak to be used for studying NIP
activity; thus, only the induced promoter activity was employed in this study.
Position effect on NIP activity in the IFN- DNA-Protein Complexes Formed by NIP and Nuclear Proteins--
To
explore the mechanism of NIP function, the DNA-protein interaction
between NIP and the nuclear proteins of Jurkat cells was investigated
with EMSA. A radiolabeled double-stranded NIP oligonucleotide was used
as a probe in the assay, and unlabeled NIP or Sp1 oligonucleotides (in
micrograms) were used as competitors. The results are shown in Fig.
2. The NIP DNA formed four major complexes with a Jurkat nuclear extract (complexes A-D)
(lane 1). All four complexes were reduced by the
specific competitor (unlabeled NIP) in a dose-dependent
pattern (lanes 2-5). Complexes C and D were reduced
significantly by the nonspecific competitor Sp1 probe (lane
6). These results suggest that among the four complexes, A and B
result from a specific interaction between the NIP probe and the Jurkat
nuclear proteins. Complexes C and D result from a nonspecific
interaction. It is interesting to note that when the level of complex A
was reduced by 20 µg of unlabeled NIP (lane 2),
the level of complex B was enhanced. When complexes C and D were
reduced by the Sp1 probe, complex B was also enhanced (lane
6). There was a fast migrating band close to the free probe. This
band also exhibited specific binding activity. Our later analysis
suggests that this band is a derivative of complex B since it can be
removed with unlabeled YY1 probe or antibody (Fig. 6). Complexes C and
D appear to be nonspecific complexes, and mutation analysis of
complexes C and D did not indicate any function of these two complexes
(data not shown). Therefore, the rest of this study was focused on
complexes A and B.
Protein Composition of Complex A--
A search into the
DNA-binding sequences of known transcription factors revealed that a
binding site for the nuclear factor AP2 was present in the NIP
oligonucleotide (boxed in Fig. 4A). To
investigate if any of the complexes contains AP2, we carried out both
an oligonucleotide competition assay and an antibody supershift assay
(Fig. 3). In this experiment, an
authentic SV40 AP2-binding oligonucleotide was used as a competitor
against the NIP probe. The formation of complex A was inhibited by the
AP2 oligonucleotide (Fig. 3A, lane 3). In the
assay, the AP2 oligonucleotide competed with the NIP probe as
efficiently as unlabeled NIP in complex A (lane 2). The AP2
oligonucleotide did not compete with the NIP probe in complexes B-D
(compare lanes 2 and 3). This suggests that the
protein in complex A, but not in complex B, shares DNA-binding specificity with the nuclear factor AP2. However, complex A was not
supershifted or removed by the anti-AP2 antibody (Fig. 3B, lane 2). This antibody was able to remove an authentic AP2
complex formed by the AP2 oligonucleotide probe and Jurkat nuclear
protein (Fig. 3C, lanes 1 and 2).
These results imply that the protein in complex A is not AP2 or that
the AP2 epitopes are masked by protein-protein interactions.
Additionally, the mobilities of complex A and the authentic AP2 complex
are totally different. Since the protein in complex A resembles AP2 in
its DNA-binding specificity, but does not appear to be AP2, we
designated it as ASRF.
Requirement of ASRF for the Silencer Function--
The DNA-binding
sequence of ASRF was mapped out by a series of base substitutions in
the NIP sequence (data not shown). A 3-base pair substitution in the
AP2-like binding site was sufficient to delete ASRF-binding activity in
the NIP element (oligonucleotide M1) (Fig.
4A). The protein-binding
activity of M1 was tested in the gel shift assay directly using
radiolabeled M1 as the oligonucleotide probe (Fig. 4B) or
indirectly using unlabeled M1 as a competitor (Fig. 4C). The
results show that M1 does not form complex A, but does form complexes
B-D (Fig. 4B, lane 2). In the absence of complex A, the intensity of complex B was enhanced compared with the control (lane 2 versus lane 1). These data again support the
hypothesis that there may be a competition between ASRF and complex B
in the NIP element. Complexes C and D were reduced after mutation of
the ASRF-binding site (Fig. 4B, lane 2). This
suggests that formation of complexes C and D may somehow be modulated
by the formation of the ASRF complex. In the competition experiment
(Fig. 4C), M1 did not compete with the NIP probe in forming
complex A, confirming that M1 cannot bind to ASRF (Fig. 4C,
compare lanes 1 and 2).
The function of M1 in the IL-3 promoter was examined by inserting one
copy of M1 into the IL-3/CAT reporter vector (Fig.
5, M1). In this experiment,
the IL-3/CAT vector and the NIP vector (containing one copy of
wild-type NIP in the IL-3/CAT reporter vector) were used as controls
(Fig. 5, Control and NIP, respectively). The
results demonstrate that in contrast to the NIP element, M1 has no
silencer function at all. Since the only difference between M1 and NIP
is the binding activity of ASRF, these data indicate that ASRF mediates
the silencer function of NIP.
Complex B Contains the Nuclear Factor YY1--
The search of
transcription factor-binding sequences in the NIP element also revealed
that there are two potential binding sites for the transcription factor
YY1. One site overlaps with the AP2-like binding site, and the other is
located at the 3'-end of NIP (under the YY1 consensus sequence in Fig.
7A). To investigate if YY1 is involved in the formation of
any NIP complexes, an authentic YY1-binding oligonucleotide and an
anti-YY1 antibody were utilized in the gel shift assay with the NIP
probe (Fig. 6). The results from the
oligonucleotide competition experiment show that complex B may contain
YY1 because the authentic YY1 oligonucleotide efficiently competed the
formation of complex B (Fig. 6A, lane 3 versus
lane 2). In the antibody supershift assay, part
of complex B was supershifted by the anti-YY1 antibody (Fig.
6B, lane 2), but was not affected by a control
antibody (lane 3). To confirm the activity of the anti-YY1
antibody, a supershift experiment was performed with a YY1 complex
formed by the radiolabeled YY1 oligonucleotide probe and Jurkat nuclear
protein (lanes 4-6). The results show that the authentic
YY1 complex migrated at the same position as complex B, and more
important, it was supershifted to an identical level (labeled as
Shifted Bands) as complex B by the anti-YY1 antibody (lane 5). These data strongly suggest that the transcription
factor YY1 is involved in the formation of complex B.
Functional Antagonism of YY1 and ASRF--
We next analyzed the
function of the YY1 complex by mutation and cotransfection in Jurkat
cells. Oligonucleotide M2 contains a 3-base pair substitution at the
second YY1 homology site located at the 3'-end of the NIP
oligonucleotide (Fig. 7A). The
protein-binding activity of M2 was examined in the same way as
described for M1. A radiolabeled M2 oligonucleotide was used as a probe
in EMSA with Jurkat nuclear protein. The results of this experiment
show that M2 lost YY1-binding activity (complex B) (Fig. 7B,
lane 2), but retained the ability to form the other three
complexes. Complexes C and D were reduced after mutation of the
YY1-binding site (lane 2), suggesting there are certain
interactions among these three complexes. However, this result
indicates that the second YY1 homology sequence (mutated in M2) is
responsible for the formation of the YY1 complex (complex B). The first
YY1 homology sequence, which overlaps with the ASRF site, is not
involved in the formation of the YY1 complex because M2 failed to
demonstrate YY1-binding activity. The same conclusion can be drawn from
the competition assay, in which unlabeled M2 was used as a competitor
against the NIP probe (Fig. 7C).
In Jurkat cells, modulation of YY1-binding activity by PMA plus
ionomycin is not significant. This might be due to a high basal level
of YY1 in Jurkat cells. To explore the question of inducibility of YY1,
purified human peripheral T cells were employed. The total T cells were
separated from the human peripheral blood by the nylon wool method and
treated with PMA plus ionomycin. This stimulation is sufficient to
induce activation of the IL-3 gene in the T cells. After a 2-h
treatment, the nuclear protein was extracted and examined in the gel
shift assay. The results show that the basal level of YY1 (band B) in
the T cells is low (Fig. 7C, lane 1), but its
binding activity is inducible by stimulation (lane 2). There
are four major complexes formed by the nuclear protein of unstimulated
fresh T cells (lane 1). After PMA/ionomycin stimulation,
three of the four complexes were induced, and one of them was reduced.
If the reduced band was used to normalize the induced bands, the
induction of the YY1 band would become even stronger. This suggests
that activation of the DNA-binding activity of YY1 correlates with
activation of IL-3 gene transcription in the T cells.
To test the functional activity of a change in the 3'-YY1 site, one
copy of M2 was linked to the IL-3 promoter, and the reporter vector was
transfected into Jurkat cells. The results of this experiment
demonstrate that mutation of the YY1 site resulted in a silencer
activity even stronger than that of wild-type NIP in the IL-3 promoter
(Fig. 8A). Instead of the 60%
inhibition of IL-3 promoter activity caused by NIP, >80% of the IL-3
promoter activity was lost in the presence of M2. This result suggests that the YY1 protein may play an opposite role to ASRF when binding to
the NIP element. To test this hypothesis, a YY1 expression vector was
cotransfected with the IL-3 promoter constructs. Overexpression of YY1
had no effect on the IL-3 promoter in the absence of the NIP element.
In the presence of NIP, overexpression of YY1 enhanced promoter
activity by 100%. The relative promoter activity was increased from
40% (NIP) to 80% (NIP + YY1) (Fig. 8B). These results provide functional evidence that YY1 inhibits ASRF in the IL-3 gene
promoter.
In previous studies, we identified a regulatory activity of the
transcription factor YY1 in the human IFN- The results from this study indicate that the NIP silencer is a
promoter-specific cis-acting element (Fig. 1). This
conclusion is different from a previous report in which that NIP
activity is claimed as a nonspecific silencer for the IL-3 promoter
(10). This difference might be due to the experimental systems. In that study, the human IL-3 silencer activity was characterized in the gibbon
T cell line MLA 144. The IL-3 cDNA was used as a reporter of the
IL-3 promoter, and RNase protection assay was used to monitor reporter
activity. The limitations of that system include the following.
(a) Gibbon T cells may be different from human T cells, and
this may result in a different activity of the NIP silencer. (b) In the reporter assay, reporter activity was not
normalized by an internal control. It is widely accepted that a
difference in transfection efficiency could result in a totally
distinct conclusion. (c) Additionally, a result from only
one reporter assay, not a mean value of multiple experiments, was shown
in that report. In our study, the IL-3 silencer activity was
characterized in the human Jurkat T cell line, and all the reporter
(CAT or This study demonstrates that YY1 regulates the human IL-3 gene promoter
through competition with ASRF activity in the NIP silencer element. We
confirmed that Jurkat cells expresses IL-3 mRNA upon stimulation of
PMA/ionomycin. Under the same condition, the IL-3 gene promoter
exhibited a dramatic inducible activity, indicating that our
experimental system is closely relevant to the physiological condition
under which IL-3 expression is regulated. In the four specific
DNA-protein complexes formed by the NIP oligonucleotide and Jurkat
nuclear extracts (Fig. 2), only complexes A and B appear to be
functional. Complex A is responsible for the silencer activity, and its
activity is regulated by complex B. Protein analysis revealed that
complex A contains a nuclear protein that shares DNA-binding specificity with AP2 (ASRF) (Fig. 3), and complex B contains the transcription factor YY1 (Fig. 6). It has been documented that a
consensus YY1-binding sequence contains 9 base pairs
((C/G/A)(G/T)(D/T/A)CATN(T/A)(T/G/C)) (23). In our study, we found that
the core sequence for YY1 binding can be reduced to 5 base pairs of
CCATT. This has been verified by mutation analysis of the YY1-binding
sites in the promoters of human IFN- This study provides evidence for a new model of cytokine gene
regulation by YY1. On the basis of previous data, two models by which
YY1 regulates cytokine gene transcription have been suggested. In the
first model, YY1 cooperates with an AP2-like protein in mediating
silencer activity (21, 22). In the second model, YY1 inhibits the
activity of an activator protein through competition for DNA binding
(22). This model has also been proposed for regulation of non-cytokine
genes. In the The AP2-like activity in the IFN- In summary, we report here that the transcription factor YY1 and ASRF
regulate the activity of the IL-3 NIP silencer element. ASRF activity
is required for the silencer function, including promoter specificity
and orientation dependence. In contrast, YY1 plays a role in the
positive regulation of the human IL-3 gene promoter. This activity may
be mediated by a direct competition with ASRF activity for DNA binding
in the NIP element. These data support that a cell type-specific
silencer activity might be determined by a unique profile of ubiquitous
transcription factors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(IFN-) (3,
4), IL-3 is produced by activated T cells and natural killer cells (5,
6), and its expression is primarily controlled at the transcriptional
level (7-9). Promoter deletion analysis revealed that enhancer and
silencer elements located within 300 base pairs of the transcription
start site control tissue-specific expression of the human IL-3 gene
(7-9). Interestingly, there is a cell type-specific silencer element at positions 
267 to 242 that binds a protein complex designated as
the nuclear inhibitory protein (NIP) (8). Although this silencer
element was partially characterized (10), the identification of the
proteins bound to this element and the mechanisms of the silencer
function remain unclear. In this study, we focused on the
characterization of transcription factors associated with this silencer element.
(14, 15), IFN- (16, 17), IFN-
(18, 19), and IFN-
(20, 21) gene promoters. A silencer element (BE element) in the human
IFN- gene promoter has been carefully investigated (21, 22). Two
transcription factors interact with the IFN- silencer: 1) YY1
(Yin-Yang 1), a ubiquitous nuclear
factor involved in the negative regulation of many mammalian genes
(23); and 2) an AP2-like protein that shares DNA-binding specificity,
but not antigenicity, with the transcription factor AP2. Simultaneous
binding of both nuclear factors is required for the inhibitory activity
of the IFN- BE element (22).
BE silencer in protein-binding activities.
Both YY1 and an AP2-like protein are found in DNA-protein complexes
formed by NIP and T cell nuclear protein. However, the function of YY1
is totally different in NIP compared with the BE element. In the NIP
element, the AP2 sequence-recognizing factor (ASRF) is required for the
silencer function, and interestingly, YY1 acts an inhibitor of ASRF
activity. This finding provides evidence that YY1 may act as a positive
regulator in the IL-3 gene promoter, and more important, this extends
the functional role of YY1 in regulating cytokine gene expression.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 °C.
The protein concentration was determined with the BCA protein assay
reagent (Pierce).
70 °C.
175)); 2)
the IFN-/-galactosidase expression vector (25), in which the
-galactosidase gene is controlled by the human IFN- gene promoter
(108); 3) the pBCTKp/CAT reporter vector (26), in which the CAT gene
is controlled by the herpes simplex virus thymidine kinase gene
promoter (105)); 4) the pGL2 control vector, a luciferase expression
vector containing the SV40 promoter (Promega, Madison, WI; this vector was used for monitoring transfection efficiency in transfection experiments); and 5) the CMV-YY1 expression vector and its control pCEP
vector (gifts from Dr. Keiko Ozato, Laboratory of Developmental and
Molecular Immunity, NICHD, National Institutes of Health, Bethesda, MD
(27)).
-galactosidase, and luciferase assays were carried out as
described previously (21, 22). The CAT and -galactosidase activities
were normalized by protein concentration and luciferase activity for
each transfection, and a mean value from three individual experiments
was analyzed by Student's t test with a confidence level of
p < 0.05-0.001.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Functional characterization of the NIP
silencer. A, shown is the induction of IL-3 mRNA by
PMA and ionomycin (Ion). Total cellular RNA was extracted
from Jurkat cells stimulated by PMA (10 ng/ml) and ionomycin (1 µg/ml). The IL-3 mRNA level was determined by the ribonuclease
protection assay as described under "Materials and Methods." Time
points of the treatment are indicated at the top of each lane.
B, shown is the inducibility of the IL-3 and IFN- gene
promoters. The IL-3 (175), IFN- (108), and thymidine kinase
(TK) promoters were examined in Jurkat cells the in
transient transfection assay (as indicated). Inducibility of the
promoters was tested following stimulation with PMA and ionomycin. The
relative activities of the reporters were used to represent responses
of the promoters. The asterisks indicate a significant
increase from the control (p < 0.001). C,
the activity of the NIP element was tested in the three promoters
(IL-3p, IFNp, and TKp, IL-3, IFN-,
and thymidine kinase promoters, respectively). One copy of NIP was
inserted at the HindIII site in the promoters. In the IL-3
reporter plasmid, the HindIII site is 16 base pairs upstream
of the IL-3 promoter (175). +NIP means a promoter with one
copy of NIP. The parental promoter serves as a control. The
asterisk indicates a significant decrease from the control
(p < 0.001). D, the activity of the NIP
element was investigated in reverse orientation in the IL-3 gene
promoter. NIPp, proper orientation; NIPr, reverse
orientation. The parental IL-3 promoter was utilized as a control. The
asterisk indicates a significant decrease from the control
(p < 0.001). GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
175), the human
IFN- gene promoter (108), and the thymidine kinase promoter. Both
the IL-3 (10-fold) and IFN- (6-fold) promoters were induced by
stimulation (Fig. 1B). Without stimulation, the percent
acetylation from the IL-3/pCAT reporter was 5%; with stimulation, the
percent acetylation was increased to 55%. Similarly, the
IFN-/-galactosidase reporter was induced from 64- to 384-fold
following stimulation. In contrast, the thymidine kinase/CAT reporter
was not induced upon stimulation. Its percent acetylation was ~15%
before or after stimulation. To analyze NIP activity, NIP was inserted
into the promoters. The results show that in the IL-3 promoter
(IL-3p), NIP reduced promoter activity by 60% (Fig.
1C), confirming the silencer function of NIP in the context
of the IL-3 promoter. This also demonstrates that this IL-3 promoter
construct is sufficient for characterization of the NIP function. In
contrast, NIP did not show a silencer activity in either the IFN-
promoter (IFNp) or the thymidine kinase promoter
(TKp) (Fig. 1C). These results demonstrate that
NIP activity appears to be specific for the human IL-3 promoter.
promoter was also
examined. This was done by inserting a small DNA fragment of different
lengths at the junction point between the NIP silencer and the IFN-
promoter. These inserts can make the silencer fragment into a half-turn
or full turn in the DNA -helices. We also tested the NIP silencer
activity in a longer IFN- promoter (560). All the results indicate
that the NIP silencer cannot repress a heterologous gene (IFN-)
promoter (data not shown).
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Fig. 2.
Specific DNA-protein complexes formed by the
NIP oligonucleotide probe and Jurkat nuclear protein. The gel
shift assay was conducted as described under "Materials and
Methods." Unlabeled oligonucleotides were used as specific or
nonspecific competitors as indicated at the tops of the lanes. The
numbers at the top of each lane refer to amount (in
micrograms) of the competitor oligonucleotide.
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Fig. 3.
Complex A is formed by an AP2-like nuclear
protein. Oligonucleotide competition and antibody supershift
experiments were conducted in gel shift assays to characterize complex
A. A, complex A is specifically competed by an authentic
AP2-binding oligonucleotide. 100 µg of competitor oligonucleotide was
used in the reaction. B, supershift analysis with an
antibody against the AP2 protein (sc-184, Santa Cruz Biotechnology,
Inc.). 4 µg of antibody was used in the reaction. C,
authentic AP2 complex formed by the radiolabeled AP2-binding
oligonucleotide and the PMA/ionomycin-activated Jurkat nuclear
proteins. The same anti-AP2 antibody (4 µg) was used to remove the
AP2 complex as in described for B.
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Fig. 4.
Mutation analysis of AP2-like binding
activity. A, DNA sequences of the NIP element and
oligonucleotide M1. The boxed sequence in the NIP element is
the binding sequence for the NIP protein as originally identified. The
box above the NIP element represents a consensus DNA-binding
sequence for the AP2 protein. The mutated sequence for the generation
of M1 is underlined. B, comparison of the
protein-binding patterns between NIP and M1. Radiolabeled NIP and M1
oligonucleotides were used as probes with the Jurkat nuclear protein in
the gel shift assay. C, competition analysis of the M1
complexes. 100 µg of oligonucleotide M1 was used as a competitor
against the NIP probe in lane 2.
View larger version (17K):
[in a new window]
Fig. 5.
Functional analysis of AP2-like binding
activity. The function of the M1 mutation that was generated by
deletion of the AP2-like binding activity from the NIP element was
examined in the IL-3 promoter. One copy of M1 was inserted at a
HindIII site 16 base pairs upstream of the IL-3 promoter
( 175) in the reporter plasmid. Transient transfection of Jurkat cells
and the reporter assay were carried out as described under "Materials
and Methods." Control, IL-3 parental vector;
NIP, IL-3 vector with the NIP element; M1, IL-3
promoter with one copy of the M1 element. The asterisk
indicates a significant decrease from the control (p < 0.001).
View larger version (54K):
[in a new window]
Fig. 6.
Complex B contains the nuclear protein
YY1. Oligonucleotide competition and antibody supershift
experiments were conducted as described under "Materials and
Methods." A, complex B is specifically competed by an
authentic YY1-binding oligonucleotide. 100 µg of competitor
oligonucleotide was used in the EMSA. B, supershift analysis
with anti-YY1 antibody (sc-281, Santa Cruz Biotechnology, Inc.).
Complex B was formed by incubating the NIP probe and Jurkat nuclear
protein (lanes 1-3). 4 µg of anti-YY1 antibody or
anti-AP2 antibody (as a control) was used as indicated at the top of
each lane. Authentic YY1 complex was formed by the radiolabeled
YY1-binding oligonucleotide and the Jurkat nuclear proteins
(lanes 4 and 5). The supershifted bands are
marked by an arrow in lanes 2 and
5.
View larger version (46K):
[in a new window]
Fig. 7.
Modulation of YY1-binding activity.
A, DNA sequences of the NIP element and oligonucleotide M2.
The boxed sequence in the NIP element is the binding
sequence for the NIP protein as originally identified. The
box above the NIP element identifies a consensus DNA-binding
sequence for the YY1 protein. The mutated sequence for the generation
of M2 is underlined. B, comparison of
protein-binding patterns between NIP and M2. Radiolabeled NIP and M2
oligonucleotides were used as probes with the Jurkat nuclear protein in
EMSA. C, competition analysis of the M2 DNA-protein
complexes. 100 µg of oligonucleotide M1 was used as a competitor
against the NIP probe in lane 2. D, induction of
YY1 activity in fresh human T cells. The nuclear protein was extracted
from fresh T cells after stimulation with PMA plus ionomycin
(Ion) for 2 h. The DNA-binding activity of the nuclear
protein was examined with the radiolabeled NIP probe.
View larger version (13K):
[in a new window]
Fig. 8.
Functional analysis of YY1-binding
activity. A, the function of the M2 mutation was
examined in the context of the IL-3 promoter. One copy of M2 was
inserted at the HindIII site 16 base pairs upstream of the
IL-3 promoter ( 175) in the reporter plasmid. Transient transfection
of Jurkat cells and the reporter assay were carried out as described
under "Materials and Methods." Control, IL-3 parental
vector; NIP, IL-3 vector with the NIP element;
M2, IL-3 promoter with one copy of the M2 element. The
asterisk indicates a significant decrease from NIP
(p < 0.001). B, shown are the effects of
overexpressed YY1 on the NIP function. A YY1 expression vector was
utilized in the cotransfection analysis of the NIP function. The ratio
of the YY1 expression vector to the IL-3 reporter vector was 1:1. The
parental vector of the YY1 expression plasmid was utilized as a control
DNA in the cotransfection. The control reporter (Control)
contains the IL-3 promoter only. The experimental reporter vector
contains one copy of NIP in the IL-3 promoter (NIP). The
asterisk indicates a significant increase from NIP
(p < 0.001).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
gene promoter (21, 22)
and the granulocyte/macrophage colony-stimulating factor gene promoter
(28, 29). In the IFN- promoter, YY1 acts as a repressor protein
through two mechanisms. In the silencer (BE) region, YY1 cooperates
with another protein, which also shares DNA-binding specificity with
AP2, to mediate the silencer function (21). In this case, binding of
both YY1 and the AP2-like protein is required for the silencer
function. We also observed that YY1 inhibited AP1 activity at an
AP1-YY1-binding site in the IFN- promoter. YY1 competed with AP1 for
the overlapping region in an AP1-YY1-binding site, leading to
inhibition of the AP1 enhancer function (22). In the human
granulocyte/macrophage colony-stimulating factor gene promoter, YY1
repressed AP1 activity and an Sp1-like activity through a similar
competition in the CAT enhancer element (28, 29). Thus, in the IFN-
and granulocyte/macrophage colony-stimulating factor gene promoters,
competition with an activator is the common mechanism by which YY1
inhibits cytokine gene promoter activity.
-galactosidase) activities were normalized by an internal
control. Moreover, a mean value of three experiments was used to show
reporter activity. Position effect was examined in the IFN- promoter
by adjusting the distance between the NIP element and the IFN-
promoter. The results did not support a possibility that position plays a role in the NIP silencer activity in the heterologous promoter.
(21, 22) and the
granulocyte/macrophage colony-stimulating factor (28, 29). An extra
flanking sequence is not required for YY1 protein binding. In the NIP
element, this was again verified. In the IL-3 gene promoter, YY1
functions by regulating ASRF activity through competition for DNA
binding. This conclusion is supported by the following evidence.
(a) In the competition assay, when the binding of ASRF was
reduced by the competition of an AP2 oligonucleotide, YY1 binding was
enhanced (Fig. 3A). (b) In the mutation analysis,
when ASRF-binding activity was abrogated in the NIP element by
mutation, YY1-binding activity was increased (Fig. 4B).
(c) In the functional analysis, loss of YY1 activity was
associated with an increased silencer activity, indicating that ASRF
activity was increased in the absence of YY1 (Fig. 8A).
(d) In a cotransfection assay, overexpression of YY1
resulted in reduction of the silencer activity (Fig. 8B), suggesting that YY1 inhibited the AP2-like activity.
-actin gene, YY1 inhibits the activity of the serum
response factor through competition (30). In the rat serum amyloid A1
gene, YY1 antagonizes NF-
B activity by competition (31). In these
two models, YY1 plays a negative role in gene regulation. Since the
DNA-binding activity of YY1 preexists in the resting cells, these two
models can explain the tight control of gene transcription of some
cytokines such as IFN- (22). Here, we suggest a third model in which
YY1 antagonizes the activity of a repressor protein through competition
for DNA binding. In this model, YY1 plays a positive role in cytokine gene transcription. This finding may explain why the Jurkat cells used
in this study can produce IL-3, but not IFN-. Jurkat T cells have a
strong endogenous YY1 activity compared with natural killer cell lines
(YT or NK3.3). In the IFN- promoter, YY1 acts as a repressor,
whereas in the IL-3 promoter, YY1 acts as a derepressor. It is
reasonable to suggest that in the Jurkat cells, the endogenous YY1
activity prevents activation of the IFN- promoter and facilitates activation of the IL-3 gene promoter.
silencer element (BE) and the ASRF
activity in the IL-3 silencer element (NIP) might be different members
of a novel transcription factor family. The common features of the two
AP2-like activities are as follows. (a) Both proteins
recognize a similar DNA sequence. (b) Both proteins mediate
the silencer activities in the respective promoters. (c) Their activities are promoter-dependent, and the DNA is
orientation-dependent. The differences in the two proteins
include the following. (a) The ASRF complex migrates slower
than the IFN- AP2-like protein-DNA complex (data not shown).
(b) ASRF is sufficient to mediate the silencer activity,
whereas the IFN- AP2-like protein needs cooperation with YY1 to
mediate the silencer activity. Interestingly, a protein with a similar
DNA-binding specificity may also play a role in the TNF- silencer. A
silencer element (254 to 230) in the TNF- promoter has been
identified (14), and it represses TNF- transcription in U937 cells.
This 25-base pair TNF- repressor site contains a 10-base pair
sequence homologous to the binding site of nuclear factor AP2, but it
does not bind the AP2 protein (32). Taken together, this information
suggests that these proteins may represent a novel family of
transcription factors that function as repressors in the
transcriptional regulation of cytokine gene expression.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Della Reynolds for carrying out the
mRNA assay. We appreciate the generous support of Dr. L. R. Gottschalk, who provided the IL-3 reporter plasmid (175).
| |
FOOTNOTES |
|---|
* 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: PPRB/NIOSH, 1095 Willowdale Rd., Morgantown, WV 26505. Tel.: 304-285-6286; Fax: 304-285-5938; E-mail: jby4@cdc.gov.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: IL, interleukin; IFN, interferon; NIP, nuclear inhibitory protein; TNF, tumor necrosis factor; BE, bifunctional element; ASRF, AP2 sequence-recognizing factor; PMA, phorbol 12-myristate 13-acetate; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol transferase.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Metcalf, D. (1992) Trends Biochem. Sci. 17, 286-289[CrossRef][Medline] [Order article via Infotrieve] |
| 2. | Spivak, J. L., Smith, R. R., and Ihle, J. N. (1985) J. Clin. Invest. 76, 1613-1621 |
| 3. | Young, H. A., and Hardy, K. J. (1990) Pharmacol. Ther. 45, 137-151[CrossRef][Medline] [Order article via Infotrieve] |
| 4. | Young, H. A., and Hardy, K. J. (1995) J. Leukocyte Biol. 58, 373-381[Abstract] |
| 5. |
Wimperis, J. Z.,
Niemeyer, C. M.,
Sieff, C. A.,
Mathey-Prevot, B.,
Nathan, D. G.,
and Arceci, R. J.
(1989)
Blood
74,
1525-1530 |
| 6. | Yang, Y. C., Ciarletta, A. B., Temple, P. A., Chung, M. P., Kovacic, S., Witek-Giannotti, J. S., Leary, A. C., Kriz, R., Donahue, R. E., Wong, G. G., and Clark, S. C. (1986) Cell 47, 3-10[CrossRef][Medline] [Order article via Infotrieve] |
| 7. |
Gottschalk, L. R.,
Giannola, D. M.,
and Emerson, S. G.
(1993)
J. Exp. Med.
178,
1681-1692 |
| 8. |
Mathey-Prevot, B.,
Andrews, N. C.,
Murphy, H. S.,
Kreissman, S. G.,
and Nathan, D. G.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
5046-5050 |
| 9. |
Shoemaker, S. G.,
Hromas, R.,
and Kaushansky, K.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
9650-9654 |
| 10. |
Engeland, K.,
Andrews, N. C.,
and Mathey-Prevot, B.
(1995)
J. Biol. Chem.
270,
24572-24579 |
| 11. | Ye, J., and Young, H. A. (1997) FASEB J. 11, 825-833[Abstract] |
| 12. |
Nabel, G. J.,
Gorka, C.,
and Baltimore, D.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
2934-2938 |
| 13. | Li-Weber, M., Eder, A., Krafft-Czepa, H., and Krammer, P. H. (1992) J. Immunol. 148, 1913-1981[Abstract] |
| 14. |
Fong, C. L.,
Siddiqui, A. H.,
and Mark, D. F.
(1994)
Nucleic Acids Res.
22,
1108-1114 |
| 15. |
Rhoades, K. L.,
Golub, S. H.,
and Economou, J. S.
(1992)
J. Biol. Chem.
267,
22102-22107 |
| 16. | Kuhl, D., de la Fuente, J., Chaturvedi, M., Parimoo, S., Ryals, J., Meyer, F., and Weissmann, C. (1987) Cell 50, 1057-1069[CrossRef][Medline] [Order article via Infotrieve] |
| 17. | Tanaka, N., and Taniguchi, T. (1992) Adv. Immunol. 52, 263-281[Medline] [Order article via Infotrieve] |
| 18. | Goodbourn, S., Burstein, H., and Maniatis, T. (1986) Cell 45, 601-610[CrossRef][Medline] [Order article via Infotrieve] |
| 19. | Nourbakhsh, M., Hoffmann, K., and Hauser, H. (1993) EMBO J. 12, 451-459[Medline] [Order article via Infotrieve] |
| 20. |
Chrivia, J. C.,
Wedrychowicz, T.,
Young, H. A.,
and Hardy, K. J.
(1990)
J. Exp. Med.
172,
661-664 |
| 21. |
Ye, J.,
Ghosh, P.,
Cippitelli, M.,
Subleski, J.,
Hardy, K. J.,
Ortaldo, J. R.,
and Young, H. A.
(1994)
J. Biol. Chem.
269,
25728-25734 |
| 22. | Ye, J., Cippitelli, M., Dorman, L., Ortaldo, J. R., and Young, H. A. (1996) Mol. Cell. Biol. 16, 4744-4753[Abstract] |
| 23. | Shi, Y., Lee, J. S., and Galvin, K. M. (1997) Biochim. Biophys. Acta 1332, F49-F66[Medline] [Order article via Infotrieve] |
| 24. | Imagawa, M., Chiu, R., and Karin, M. (1987) Cell 51, 251-260[CrossRef][Medline] [Order article via Infotrieve] |
| 25. |
Penix, L.,
Weaver, W. M.,
Pang, Y.,
Young, H. A.,
and Wilson, C. B.
(1993)
J. Exp. Med.
178,
1483-1496 |
| 26. |
Clark, A. R.,
Boam, D. S.,
and Docherty, K.
(1989)
Nucleic Acids Res.
17,
10130 |
| 27. |
Flanagan, J. R.,
Becker, K. G.,
Ennist, D. L.,
Gleason, S. L.,
Driggers, P. H.,
Levi, B. Z.,
Appella, E.,
and Ozato, K.
(1992)
Mol. Cell. Biol.
12,
38-44 |
| 28. |
Ye, J.,
Young, H. A.,
Ortaldo, J. R.,
and Ghosh, P.
(1994)
Nucleic Acids Res.
22,
5672-5678 |
| 29. | Ye, J., Zhang, X., and Dong, Z. (1996) Mol. Cell. Biol. 16, 157-167[Abstract] |
| 30. |
Lee, T. C.,
Shi, Y.,
and Schwartz, R. J.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
9814-9818 |
| 31. |
Lu, S. Y.,
Rodriguez, M.,
and Liao, W. S.
(1994)
Mol. Cell. Biol.
14,
6253-6263 |
| 32. | Fong, C. W., Siddiqui, A. H., and Mark, D. F. (1995) J. Interferon Cytokine Res. 15, 887-1114[Medline] [Order article via Infotrieve] |
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