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Promoter Confers Th1 Selective Expression1
* Division of Rheumatology, Departments of Medicine, and
Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or IL-4, respectively. To identify
transcriptional paths leading to activation and silencing of the
IFN- gene, we analyzed transgenic mice that express a reporter gene
under the control of the 5' IFN- promoter. We found that as the
length of the promoter is increased, -110 to -225 to -565 bp, the
activity of the promoter undergoes a transition from Th1 nonselective
to Th1 selective. This is due, at least in part, to a T box expressed
in T cells-responsive unit within the -565 to -410 region of the
IFN- promoter. The -225 promoter is silent when compared with the
-110 promoter and silencing correlates with Yin Yang 1 binding to the
promoter. The p38 mitogen-activated protein kinase signaling pathway,
which also regulates IFN- gene transcription, regulates the -70- to
-44-bp promoter element. Together, the results demonstrate that a
minimal IFN- promoter contains a T box expressed in T cells
responsive unit and is sufficient to confer Th1 selective expression
upon a reporter. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and participate in
cell-mediated immunity that controls infection by intracellular
pathogens. The predominant factors that control Th1 differentiation are
cytokines. IL-12 stimulates Th1 differentiation and IL-4 inhibits Th1
differentiation.
Control of IFN- gene expression in Th1 and Th2 effector cells is not
completely understood. Activation of Stat4 by IL-12 is necessary to
achieve Th1 differentiation (5, 6). The transcription
factor, T box expressed in T cells
(T-bet),3 has recently
been described as a Th1-specific factor that plays a central role in
Th1 differentiation and IFN- gene expression (7, 8).
Whether T-bet acts directly or indirectly to regulate IFN- promoter
activity is not known. In addition, p38 mitogen-activated protein (MAP)
kinase is selectively active in Th1 vs Th2 effector cells and the p38
MAP kinase signaling pathway is required for efficient IFN- gene
expression by Th1 cells (9). The small G-protein, RAC-1,
is also selectively expressed in Th1 vs Th2 effector cells and is also
necessary for efficient IFN- gene expression by Th1 effector cells
(10). RAC-1 is necessary for activation of both the p38
MAP kinase and NF-
B signaling pathways following stimulation of the
TCR. These two pathways coordinately contribute to efficient IFN-
gene expression in Th1 cells. It is not completely clear how these
signaling pathways target the IFN- promoter. In addition, the 5'
IFN- promoter contains binding sites for a number of other
transcription factors that regulate transcriptional activity
(11, 12, 13, 14, 15, 16, 17, 18, 19, 20). The proximal promoter contains imperfect
AP-1/CREB binding sites and these factors bind to this region of the
promoter and can regulate transcription. Additional upstream binding
sites for AP-1, Yin Yang 1 (YY1), NF-AT, Stats, and NF-B also
regulate transcriptional activity.
With the goal of identifying IFN- promoter regions that confer
Th1-specific expression upon a reporter gene, we previously analyzed
transgenic mice that express the luciferase gene under the control of
multimerized proximal (2x: -70 to -44 bp) or distal (4x:
-98 to -78 bp) elements of the IFN- promoter
(21, 22, 23). The 4x: -98 to -78 bp construct displays
greater activity in effector Th1 than Th2 cells, while the 2x: -70 to
-44 bp construct displays equal activity in effector Th1 and Th2
cells. In addition, activity of the 2x: -70- to -44-bp element is
responsive to the combination of TCR and IL-12R signaling, while the
4x: -98- to -78-bp element is only responsive to TCR signaling.
Neither of these transcriptional elements is responsive to cytokine
signaling (IL-2, IL-12, IL-18).
In this study, using a similar approach, we have investigated
regulation of the native IFN- promoter. We find that the -110- to
+64-bp or -225- to +64-bp IFN- promoters are not selectively active
in effector Th1 cells. In contrast to these two promoters, the -565 to
+64-bp IFN- promoter is selectively active in effector Th1 cells. In
addition, the p38 MAP kinase signaling pathway targets the 2x: -70-
to -44-bp transcriptional element of the IFN- promoter.
Comparison of the -225- to +64-bp promoter to the -110 to +64-bp
promoter indicates that elements between -225 and -110 bp confer
extreme silencing upon promoter activity. Given the increased activity
of the -565 to +64-bp promoter in Th1 cells and the known role of
T-bet in mediating IFN- gene expression in Th1 cells, we tested if
this construct was more active in Jurkat cells or was transactivated by
T-bet. The results show that T-bet potently transactivates the -565-
to +64-bp IFN- promoter in Jurkat cells. This may explain the
selective activity of this promoter compared with the -225- to +64-bp
IFN- promoter in Th1 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The -565 to +64, -225 to +64, and -110 to +64 regions from
the human IFN- gene were amplified by PCR from a plasmid containing
the 8.6-kb complete human IFN- gene. PCR primers were designed to
create 5' XhoI and 3' HindIII sites in each PCR
product. These fragments were purified and subcloned into the
XhoI and HindIII sites of the luciferase plasmid.
After digestion with HpaI, fragments were purified and
injected into pronuclei of B6D2 fertilized eggs. Transgenic mice were
generated as previously described (24). Transgene-positive
mice were screened by PCR with tail DNA using primers that are
selective for the luciferase gene (23). The dominant
negative (dn) p38 transgenic mice have been previously described
(9).
Cell purification, in vitro cultures, and analyses
Spleen cells were harvested from wild-type or transgenic mice
between 4 and 6 wk of age. RBCs were removed by hypotonic lysis.
CD4+ and CD8+ T cells were
purified by negative selection, respectively. Ia+
cells and NK cells were removed by incubation with an anti-IE, IA
mAb (m5/115; American Type Culture Collection, Manassas, VA), and an
anti-NK cell mAb (NK 1.1; ATCC), respectively. An anti-CD8 mAb
(TIB 105; ATCC) was used to deplete CD8+ T cells,
and an anti-CD4 mAb (GK1.5; ATCC) was used to deplete
CD4+ T cells. Cells were incubated for 30 min at
4°C, washed, and further incubated with goat anti-mouse and
anti-rat IgG bound to magnetic beads (Genome Therapeutics, Waltham,
MA) for 30 min at 4°C with rocking. Cells bound to beads were removed
with a magnet. Average purity of CD4+ or
CD8+ T cells was 
90–95% as determined by
flow cytometry. RBC-depleted splenocytes from C57BL10 mice were
depleted of CD4+ and CD8+ T
cells by negative selection with anti-CD4 and anti-CD8 mAb, and
were irradiated at 3000 rad from a cesium 137 source and used as
APCs.
Reagents used to stimulate T cells were anti-CD3 mAb (145-2C11 clone; ATCC), 5 ng/ml IL-4, or 5 ng/ml IL-12. Recombinant human IL-2 was a gift from Hoffmann-LaRoche (Nutley, NJ), and IL-4 and IL-12 were purchased from BD PharMingen (San Diego, CA). Immobilized anti-CD3 mAb was prepared by adding 0.5 ml of 10 µg/ml 2C11 mAb in 0.1 M sodium bicarbonate (pH 9.6) to a 24-well tissue culture plate for 3–6 h at 37°C or overnight at 0–4°C. Culture plates were washed thoroughly before use.
Cells were cultured in complete RPMI 1640 medium with 10% FCS, 100
U/ml penicillin, 100 U/ml streptomycin, 2 mM L-glutamine,
and 5 x 10-5 M 2-ME in 24-well tissue
culture plates in a volume of 1 ml at a density of 1 x
106/ml with stimuli as described in the text at
37°C in 5% CO2 in air. Syngeneic irradiated
APCs were used at a density of 1 x 106/ml
of culture fluid. CD4+ T cells were stimulated
with plate-bound anti-CD3 mAb and APC for 5 days under neutral
conditions (no further additions), Th1 conditions (5 ng/ml IL-12 and 10
µg/ml anti-IL-4 mAb), or Th2 conditions (5 ng/ml IL-4 and 10
µg/ml anti-IFN- mAb). Cultures were harvested and restimulated
with plate-bound anti-CD3 mAb for an additional 48 h before
analysis.
Cultures were harvested, washed twice in PBS, and suspended in 50 µl of lysis buffer (Promega, Madison, WI) for 30 min at 20°C. The supernatant fluid was harvested and aliquots were assayed for luciferase activity with 100 µl of Luciferase Reagent (Promega) in a luminometer (Turner TD20/20; Promega) for 15 s. Cultures were performed in duplicate. Duplicate analyses of two aliquots from each cell lysate were performed and the results were averaged. Results are expressed as the average of these readings per 106 cells with the SE. The background measurement with Luciferase Reagent alone was subtracted from each reading. Results are expressed in relative light units. Experiments were performed a minimum of three times.
Plasmids and transient transfections
Luciferase reporter plasmids were prepared using a similar
strategy as outlined above. The human IFN- gene-luciferase plasmid
was obtained from T. Hoey (Tularik, South San Francisco, CA); the YY1
and T-bet expression vectors were obtained from H. Young (National
Cancer Institute, Frederick, MD) and L. Glimcher (Harvard University,
Boston, MA), respectively. The dn p38 and MAP kinase kinase 6 (MKK6)
expression vectors were obtained from M. Rincon (University of Vermont,
Burlington, VT). Jurkat cells, in log phase growth, were
harvested and transfected with various plasmids as outlined in the text
using lipofectamine or electroporation. After a rest period of 24
h, cultures were left unstimulated or were stimulated with PMA (50 nM)
and ionomycin (1 µg/ml). After overnight culture (peak of the
response), cells were harvested and equivalent numbers of viable cells
were analyzed for luciferase activity.
EMSA and chromatin immunoprecipitation (ChIP) assay
Nuclear extracts were prepared by established procedures.
Binding reactions were conducted essentially as previously described
(25, 26) using 5–15 µg of nuclear proteins and 1.5
x 104 cpm of 32P-end
labeled double-stranded probes. Probes were prepared from a plasmid
containing the IFN- gene by RT-PCR and purified by excising bands
from agarose gels. Anti-YY1 and NF-AT Abs were from Santa Cruz
Biotechnology (Santa Cruz, CA). ChIP assays were performed essentially
as previously described (27). Briefly, cultured cells were
treated with paraformaldehyde (1%) for 10 min at room temperature.
Glycine was added to a final concentration of 0.125 M to stop the
reaction. Chromatin was prepared by sequential suspension of cells in
cell lysis buffer and protease inhibitors and nuclei in nuclei lysis
buffer and protease inhibitors. After suspension in nuclei lysis
buffer, samples were sonicated to achieve an average chromatin length
of 600 bp. Chromatin samples were precleared with blocked Staph A
cells (Sigma-Aldrich, St. Louis, MO). After washing, blocked Staph A
cells were added to clarified chromatin samples. Three conditions were
routinely used: 1) no Ab; 2) no chromatin; or 3) anti-YY1 Ab. After
reversal of cross-links and deproteination, the presence of selected
transgenic DNA sequences was analyzed by PCR. Primers used were 5'
-225 to -200 and 3' -46 to -25 (human IFN- gene).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promoter
Previously, we produced and characterized murine transgenic
reporter lines that contained the luciferase gene under the control of
multimerized versions of the proximal (2x: -70 to -44 bp) or distal
(4x: -98 to -78 bp) elements of the IFN- promoter
(21, 22, 23). In this study, we wanted to examine the activity
of the natural promoter. To do so, we generated a series of reporter
constructs containing the luciferase gene under the control of various
lengths of the native IFN- promoter. Promoter lengths examined
included -110 to +64 bp, -225 to +64 bp, and -565 to +64 bp.
Transgenic mice were prepared containing these constructs to determine
whether a minimal promoter was sufficient to achieve selective
expression in effector Th1 cells. To determine IFN- promoter
activity under neutral/Th1/Th2 differentiation conditions, we purified
CD4+ splenic T cells from transgenic mice and
stimulated them with anti-CD3, APC, and either no additional
treatments, IL-12, and anti-IL-4 mAb (Th1 conditions), or IL-4 and
anti-IFN- (Th2 conditions). After 5 days, cultures were
harvested and restimulated with anti-CD3. Cell extracts were
prepared and luciferase activity was measured. Murine lines generated
from multiple founders were examined to determine the extent that
integration effects may contribute to the results. Luciferase
measurements were performed in multiple cultures and were found to be
highly reproducible. IFN- levels in cultures were also determined
for each experiment by ELISA and served as an internal control. Mean
IFN- values in secondary cultures after differentiation under
neutral, Th1, or Th2 conditions were 22 ± 4, 255 ± 20, and
34 ± 5 ng/ml, respectively. We did not observe substantial
intersample variation in IFN- levels among different experiments
using different transgenic lines.
We measured luciferase activity in secondary cultures of T cells that
had differentiated under neutral, Th1, or Th2 conditions. The -110- to
+64-bp promoter was not selectively active in effector Th1 cells (Fig. 1
). Lines generated from three
independent founders were examined and each showed similar
characteristics. In fact, somewhat more luciferase activity was found
in cultures that had differentiated under Th2, rather than Th1, culture
conditions. The -225- to +64-bp promoter was also not selectively
active in T cells that had differentiated under Th1, rather than
neutral or Th2 conditions (Fig. 1). Lines from two independent founders
were examined and both showed similar characteristics. Quantitative
comparison between different reporter lines is not absolute because of
the possibility of integration effects and variable copy number.
However, comparison of luciferase activity between the -110- to +64-bp
reporter lines and the -225- to +64-bp reporter lines demonstrated a
marked reduction in total transcriptional activity in the -225- to
+64-bp promoter.
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).
This argues that additional information is contained in this upstream
region that contributes to Th1 selective control of the IFN-
promoter. T cells from one -565- to +64-bp line expressed 30-fold
greater luciferase activity than the other two lines. However,
selective Th1 expression was observed in all three lines. This increase
in overall transcriptional activity in the -565- to +64-bp transgenic
line 3 may result from integration effects.
Under similar culture conditions, CD8+ T cells
also differentiate into effector cells that selectively produce either
IFN- (Tc1) or IL-4 (Tc2). Therefore, we wanted to determine whether
the -565- to +64-bp promoter also displayed differential activity in
effector Tc1 and Tc2 cells as compared with CD8+
T cells that had differentiated under neutral conditions. To address
this question, we used line 3. In contrast to effector Th1 cells, the
-565- to +64-bp IFN- promoter was not selectively active in
effector Tc1 cells (Fig. 1). Rather, CD8+ T cells
cultured under neutral conditions, with IL-12 and anti-IL4, or with
IL-4 and anti-IFN-, exhibited similar levels of luciferase
activity upon restimulation with anti-CD3. This indicates that to
achieve selective IFN- expression in effector Tc1 cells, additional
information is required than provided by the -565- to +64-bp IFN-
promoter.
Regulation of IFN- promoter activity by the p38 MAP kinase
signaling pathway
IFN- transcription by effector Th1 cells is mediated, at least
in part, by the p38 MAP kinase signaling pathway (9). To
identify sites within the IFN- promoter regulated by this pathway,
we determined the ability of a dn p38 MAP kinase mutation to inhibit
IFN- promoter activity in both transient transfection assays as well
as in transgenic reporter lines. To initiate these studies, we
performed transient transfection assays in Jurkat cells and tested the
ability of the dn p38 mutant to inhibit transcriptional activity
directed by various lengths of the IFN- promoter. Jurkat cells were
transfected with reporter constructs alone or with equivalent amounts
of either an empty vector or dn p38 mutant vector, allowed to rest for
24 h, and were stimulated with PMA and ionomycin. The following
reporter constructs were tested: -565 to +64 bp, -410 to +64 bp,
-225 to +64 bp, -110 to +64 bp, 4x: -98 to -78 bp, and 2x: -70
to -44 bp. Transcriptional activity directed by each of these reporter
constructs, except for the 4x: -98- to -78-bp construct, was
inhibited by the dn p38 mutant construct (Fig. 2A). MKK6 selectively
activates p38 (9), so we wanted to compare the ability of
MKK6 to stimulate promoter activity. The 4x: -98- to -78-bp and 2x:
-70- to -44-bp promoter constructs were compared since 2x: -70 to
-44 bp is inhibited by the dn p38, but 4x: -98 to -78 bp is not
inhibited by dn p38. MKK6 stimulated activity of the 2x: -70- to
-44-bp promoter by 4-fold, but had no effect on the activity of the
4x: -98- to -78-bp promoter (Fig. 2B).
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promoter constructs. To do this, we intercrossed transgenic
mice containing the dn p38 mutation with transgenic mice containing
either the -565- to +64-bp reporter, the -110- to +64-bp reporter, or
the 4x: -98- to -78-bp reporter (unfortunately, the 2x: -70- to
-44-bp reporter line no longer exists). T cells were purified from
these transgenic mice and stimulated as outlined above. The presence of
the dn p38 mutation resulted in inhibition of activity of both the
-565- to +64-bp and the -110- to +64-bp IFN- promoters (Fig. 2C). This inhibition was greater in Th1 effector cells than
in effector cells that had differentiated under Th2 conditions. In
contrast, the dn p38 mutation did not inhibit promoter activity of the
4x: -98- to -78-bp distal element. These differences are comparable
to those obtained from the transient transfection experiments. Taken
together, these results suggest that the dn p38 mutation interferes
with the activity of the proximal -70- to -44-bp element contained
within the -110 IFN- promoter rather than the distal element (-98
to -78 bp).
YY-1 binds to the IFN- promoter in vitro and in vivo, and
regulates its activity
The next step in our investigation was to focus on differences in
the activity of the -110- to +64-bp promoter and the -225- to +64-bp
promoter. To do so, we performed EMSA using a labeled -225 to -110
probe and nuclear extracts prepared from T cells that had been allowed
to differentiate under neutral, Th1, or Th2 conditions after secondary
anti-CD3 stimulation. We found a single protein-DNA complex that
was formed with extracts from T cells that were differentiated under
these conditions (Fig. 3A).
The intensity of this complex was approximately equivalent when
extracts were compared from T cells differentiated under neutral, Th1,
or Th2 conditions. The -225 to -110 region contains known binding
sites for AP-1, NF-AT, and YY1 transcription factors
(11, 12, 13, 14, 15). We performed competition experiments to further
characterize this complex. Oligonucleotides containing either
"self" or specific AP-1, NF-AT, IFN- activation site,
NF-B, or YY1 binding sites were tested for their ability to block
formation of the specific complex formed with the -225- to
-110-labeled probe and nuclear extracts from effector Th1 cells (Fig. 3B). Both the unlabeled -225 to -110 and the
oligonucleotide containing a YY1 binding site served as effective
competitors. In contrast, oligonucleotides containing an IFN-
activation site, NF-B, or AP-1 or NF-AT binding site were less
effective at blocking formation of the -225 to -110 protein-DNA
complex (Fig. 3B). We also determined if either
anti-NF-AT Ab or anti-YY1 Ab could prevent formation of the
protein-DNA complex formed with the -225- to -110-labeled probe (Fig. 3C). Anti-YY1 Ab reduced the intensity of the -225 to
-110-bp protein-DNA complex while the anti-NF-AT Ab did not
prevent complex formation. Under these conditions, we were unable to
detect formation of a supershifted complex with the anti-YY1 Ab. In
control experiments, we tested the anti-YY1 Ab using nuclear
extracts from Jurkat cells transfected with a YY1 expression vector and
the labeled -225 to -110 probe. These extracts formed a protein-DNA
complex with similar electrophoretic mobility and much greater
intensity than observed with nuclear extracts from activated T cells.
Under these conditions, the anti-YY1 Ab completely prevented
complex formation and produced a very minor supershifted complex (data
not shown). This suggests that this anti-YY1 Ab does not form
supershifted complexes very efficiently but rather either prevents
binding of YY1 to its recognition sequences or destabilizes these
complexes.
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promoter (12). We prepared additional -215 to
-110 oligonucleotide probes that contained a mutation in each or both
of the YY1 binding sites. Mutation of either or both of the YY1 binding
sites resulted in severely diminished formation of protein-DNA
complexes with the labeled -215 to -110 oligonucleotide (Fig. 3D). In contrast, mutation of either the AP-1 site or the
NF-AT site within this region did not affect complex formation. This
raises the possibility that YY1 binds to these two sites in a
cooperative manner to stabilize protein-DNA binding.
The above results indicate that nuclear extracts from effector T cells
form complexes with the -225 to -110 region of the IFN- promoter
in vitro that contain YY1. However, they do not demonstrate that YY1
actually complexes with this same region of the IFN- promoter in
vivo. To test this possibility, we performed ChIP assays. Splenic
CD4+ T cells were purified from transgenic
-225-luc reporter mice and cultured under neutral, Th1, or Th2
conditions. After restimulation with anti-CD3, cells were treated
with paraformaldehyde and processed for ChIP assays. Chromatin was
immunoprecipitated with no Ab, anti-YY1 Ab, or isotype-control Ab.
DNA was purified and amplified by PCR with specific primers to the
human IFN- promoter. ChIP assays showed that YY1 bound to the -225
to +64 transgenic IFN- promoter in effector T cells in vivo (Fig. 3E). We also performed similar assays with T cells from the
-565- to +64-bp reporter transgenic line and obtained similar results.
YY1 also formed complexes with the -565- to +64-bp IFN- promoter in
effector Th1 cells (Fig. 3E, right panel). In
transient transfection assays, we confirmed that YY1 inhibited
transcriptional activity of the -565 (data not shown) and -225
IFN- promoters, but not the -110 IFN- promoter (Fig. 4). In addition, mutation of either of
the two YY1 binding sites (Fig. 3D) blocked the inhibitory
effects of YY1 in transient transfection assays. These transient
transfection assays essentially confirm previous studies demonstrating
that YY1 inhibits IFN- promoter activity in Jurkat T cells
(11).
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promoter
The nuclear factor, T-bet, is expressed in Th1 cells, but not Th2
cells, and is required for efficient IFN- gene expression by Th1
cells (7, 8). The -565- to +64-bp IFN- promoter also
displayed Th1 selective activity, but the -225- to +64-bp or -110- to
+64-bp promoters were not selectively active in effector Th1 cells.
Potential T-bet binding sites have not been found in this region of the
murine or human promoters. Rather, potential T-bet binding sites have
been identified at about -2 kb in the murine IFN- gene and in the
third intron of the human IFN- gene (7). Therefore, we
wanted to determine whether T-bet regulated the activity of the -565-
to +64-bp IFN- promoter region. We performed transient transfection
assays as an initial test of this hypothesis. T-bet or empty vector
plasmids were transfected into Jurkat cells along with reporter
plasmids containing the luciferase gene either under control of human
IFN- "mini-gene" (7) or under the control of the
-565-, -525-, -445-, -410-, -225-, or -110- to +64-bp IFN-
promoters. We found that T-bet transactivated both the human
IFN--luciferase mini-gene construct (as previously reported, Ref.
7) and the -565- to +64-bp IFN- promoter-luciferase
construct (Fig. 5). Overall, the
mini-gene construct was routinely expressed at much lower levels than
the 5' IFN- promoter-luciferase constructs. Deletion analysis of the
-565- to +64-bp IFN- promoter indicated that sequences between
-445 and -410 bp made a critical contribution to the activity of this
T-bet responsive unit and the overall strength of the promoter.
Transactivation was observed in unstimulated cells as well as cells
stimulated with PMA and ionomycin. Taken together, these data argue
that the region of the human IFN- promoter between -565 and -415
bp contains a T-bet response unit whose activity is strongly influenced
by sequences between -445 and -415 bp.
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promoter may be through indirect,
rather than direct, mechanisms.
Important regulatory regions within promoters are usually conserved
among species. Therefore, we compared the human sequence between -450
and -400 bp to the corresponding murine sequence. There was marked
identity (>90%) between human and murine sequences (Fig. 6). We were also able to identify a
series of potential transcription factor binding sites using the
TRANSFAC transcription factor database (28). This program
identified a series of sequences that matched exactly with a core 4-bp
sequence for each of the following transcription factors: RORA1, AP1,
TCF11, OCT1, CEBP
, and NF-AT. In contrast, we were unable to find
core sequences that matched a T-box consensus site.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Th2 (or Tc2) cells produce IL-4. Although activation of high level
IFN- transcription is the hallmark of Th1 differentiation, little is
known about the cis-acting sequences required to achieve
specificity (Th null vs Th1 vs Th2) or high levels of rates of
transcription. To probe regulation of IFN- gene transcription during
Th/Tc differentiation, we analyzed transgenic mice that express the
luciferase gene under the control of varying lengths of the native
IFN- promoter. We prepared three different transgenic lines. One
contained the -110 to +64 IFN- promoter-luciferase reporter gene.
This construct contains both the -98- to -78-bp and -70- to -44-bp
elements that we had previously analyzed as multimers
(21, 22, 23). We also prepared transgenic lines that contained
the -225 to +64 IFN- promoter-luciferase and -565 to +64 IFN-
promoter-luciferase reporter constructs. The -110- to +64-bp IFN-
promoter was not selectively expressed in effector Th1 cells. Similar
levels of activity were observed in T cells that had differentiated
under neutral, Th1, or Th2 conditions. Similarly, the -225- to +64-bp
IFN- promoter was not selectively expressed in effector Th1 cells.
Rather, these lines all showed silencing of IFN- promoter activity
(>10-fold) when compared with the activity of lines containing the
-110- to +64-bp IFN- promoter. The -565- to +64-bp IFN-
promoter displayed both selective activity in effector Th1 cells when
compared with T cells that differentiated under either neutral or Th2
conditions as well as increased overall activity in effector Th1 cells.
In contrast, the -565- to +64-bp IFN- promoter was expressed in
effector CD8+ T cells that had differentiated
under neutral conditions, with IL-12 and anti-IL-4 mAb (Tc1), or
IL-4 and anti-IFN- mAb (Tc2). Taken together, these data
illustrate that a minimal IFN- promoter is able to confer Th1
selective expression upon a reporter gene in CD4+
T cells, but not Tc1 selective expression in CD8+
T cells.
The small G-protein, Rac-1, regulates IFN- gene expression in
CD4+ T cells (10). Stimulation of
IFN- gene expression by Rac-1 appears to be regulated via two
distinct intracellular signaling pathways, the NF-B signaling
pathway and the p38 MAP kinase signaling pathway. We have previously
shown that an intact NF-B signaling pathway is required to generate
full activation of the 4x: -98- to -78-bp element in the IFN-
promoter via TCR signaling (29). This appears to be due at
least in part to the requirement for NF-B to induce c-Jun expression
via TCR signaling. In this study, we also show that the p38 MAP kinase
signaling pathway is necessary for activation of the 2x: -70- to
-44-bp element via TCR signaling. These results do not rule out the
possibility that these two pathways may contribute to IFN- gene
transcription via additional mechanisms. However, they demonstrate that
two distinct intracellular signaling pathways regulated by Rac-1
converge to regulate the activity of two distinct transcriptional
elements found proximal to one another in the IFN- promoter.
Additional regulatory features of IFN- promoter activation are found
when the -225- to +64-bp promoter is analyzed. When compared with the
activity of the -110- to +64-bp IFN- promoter, the -225- to +64-bp
IFN- promoter exhibits extreme silencing. Silencing is associated
with binding of YY1 to this region of the promoter determined by both
EMSA and ChIP assays. Three potential YY1 binding sites have been
previously identified in this region of the IFN- promoter, two of
which are contained within the -225 to +64-bp IFN- promoter
(11, 12). These regions have also been previously
associated with silencing of promoter activity in transient
transfection assays of transformed cell lines. Our studies indicate
that the silencing activity of this region of the IFN- promoter is
also operational in native T cells in vivo and is probably regulated by
YY1. YY1 also exerts positive effects on transcription and this is
context-dependent (30, 31). Other studies suggest that
YY1, in cooperation with NF-AT, exerts positive effects on
transcriptional activation of the IFN- promoter (13).
This difference may be attributed to the context of the YY1 binding
sites in the IFN- promoter. Clearly, in the absence of upstream
regulatory regions, the -225- to +64-bp IFN- promoter is silenced
compared with the -110- to +64-bp promoter. However, in the presence
of additional upstream regulatory regions -565 to +64 bp, silencing is
alleviated, especially in effector Th1 cells. At this point, we do not
know if cooperativity between these two regions may influence the
selective expression of -565- to +64-bp IFN- promoter activity in
effector Th1 cells or if the strength of the upstream signal is
sufficiently strong to overcome silencing activity. Nevertheless, these
results argue that silencing activity of the -225- to -110-bp region
of the IFN- promoter contributes to overall promoter activity.
T-bet is a Th1-specific transcription factor that controls the
expression of IFN- (7, 8). How T-bet controls IFN-
gene expression is not clear. Activity of the -565 to +64 IFN-
promoter is markedly higher in effector Th1 cells compared with T cells
differentiated under either neutral or Th2 conditions, whereas shorter
lengths of the IFN- promoter (-225, -110) are not selectively
active in effector Th1 cells. This raises the possibility that upstream
regions of the -565- to +64-bp IFN- promoter may contain
T-bet-responsive elements. The combination of results from reporter
transgenic mice and transient transfection assays establishes that the
5' IFN- promoter can function as a T-bet responsive unit. Our
deletion analysis indicates that sequences between -445 and -410 bp
are critical for this function. T box-like elements are also found at
about -2 kb from the murine IFN- gene start site and in the third
intron of the human IFN- gene. These sites may also be
T-bet-responsive units and additional T-bet response elements may
reside elsewhere in the IFN- gene.
Using the -565- to -415-IFN- gene sequence or shorter
oligonucleotides as probes, we were unable to detect Th1-specific
protein-DNA complexes by EMSA. We were also unable to detect formation
of T-bet-specific complexes by comparing extracts from Jurkat cells or
Jurkat cells transfected with T-bet. This raises the possibility that
T-bet does not bind directly to this region of promoter to stimulate
transcription. Alternatively, experimental conditions may not have been
optimal to detect T-bet-specific protein-DNA complexes. Additional
approaches will be necessary to provide further insight into the
molecular mechanisms by which T-bet stimulates transcription through
the -565 to -415 T-bet-responsive unit in the IFN- promoter.
Within the -565- to -415-bp region of the IFN- promoter, our
deletion analysis suggests that the region between -445 and -415 bp
is key to the strength of promoter activity induced by T-bet. This
region is highly conserved between human and murine species (Fig. 6).
In addition, this region contains consensus core-binding sequences for
several transcription factors, RORA1, AP-1, TCF11, Oct1, CEBP, and
NF-AT. However, it does not contain a consensus binding site for T-bet
(Ref. 7 and Fig. 6), again suggesting that T-bet may act
indirectly to regulate IFN- promoter activity.
Both the small G protein, Rac-1, and the nuclear transcription factor,
T-bet, regulate IFN- gene transcription. Rac-1 appears to impact
both NF-B and p38 MAP kinase signaling pathways. Our data indicate
that these two pathways are required for efficient activation of the
-98- to -78-bp and -70- to -44-bp elements of the IFN- promoter,
respectively. A T-bet-responsive unit is localized upstream of these
sites between -565- and -415 bp of the IFN- promoter, and the
complete -565- to +64-bp IFN- promoter is selectively expressed in
effector Th1 cells compared with effector T cells that have
differentiated under either neutral or Th2 conditions.
A similar approach has been used to investigate IL-4 gene transcription
(32, 33). Similar to Th1 selective activity of the -565
IFN- promoter, an 800-bp 5' IL-4 promoter is sufficient to confer
Th2 selective upon a reporter gene. When compared with the native
cytokine gene, reporter gene expression levels are relatively low in
the case of both the IL-4 promoter and the IFN- promoter. Locus
control regions, as well as multiple upstream and intronic regions, act
synergistically to confer both high level and Th2-selective expression
upon the IL-4 gene. This suggests that additional regulatory regions
may also be required to achieve full IFN- gene transcriptional
activity and proper activation and repression of gene transcription in
T cells that have differentiated under neutral, Th1, or Th2 conditions.
We are currently addressing this possibility.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas M. Aune, Division of Rheumatology, Department of Medicine, Vanderbilt University School of Medicine, Medical Center North T3219, 21st and Garland, Nashville, TN 37232. E-mail address: Thomas.M.Aune{at}Vanderbilt.edu
3 Abbreviations used in this paper: T-bet, T box expressed in T cells; MAP, mitogen-activated protein; YY1, Yin Yang 1; ChIP, chromatin immunoprecipitation; MKK6, MAP kinase kinase 6; dn, dominant negative.
Received for publication May 21, 2002. Accepted for publication August 9, 2002.
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