OX40 Gene Expression Is Up-Regulated by Chromatin
Remodeling in Its Promoter Region Containing Sp1/Sp3,
YY1, and NF-?B Binding Sites1
Yukiko Tone, Yoshitsugu Kojima, Keiji Furuuchi, Maya Brady, Yumi Yashiro-Ohtani,
Mark L. Tykocinski, and Masahide Tone2
OX40 is a member of the TNFR superfamily (CD134; TNFRSF4) that is expressed on activated T cells and regulates T cell-
mediated immune responses. In this study, we have examined the regulation of OX40 gene expression in T cells. Low-level OX40
mRNA expression was detected in both resting T cells and the nonactivated EL4 T cell line, and was up-regulated in both types
of T cells upon activation with anti-CD3 Ab. We have shown in this study that basal OX40 promoter activity is regulated by
constitutively expressed Sp1/Sp3 and YY1 transcription factors. NF-?B (p50 and p65) also binds to the OX40 promoter region, but
the level of direct enhancement of the OX40 promoter activity by this transcription factor is not sufficient to account for the
observed up-regulation of OX40 mRNA expression associated with activation. We have detected by chromatin immunoprecipi-
tation that histone H4 molecules in the OX40 promoter region are highly acetylated by activation and NF-?B binds to the OX40
promoter in vivo. These findings suggest that OX40 gene expression is regulated by chromatin remodeling, and that NF-?B might
be involved in initiation of chromatin remodeling in the OX40 promoter region in activated T cells. CD4?CD25?regulatory T
(Treg) cells also express OX40 at high levels, and signaling through this receptor can neutralize suppressive activity of this Treg
cell. In CD4?CD25?Treg cells, histone H4 molecules in the OX40 promoter region are also highly acetylated, even in the absence
of in vitro activation. The Journal of Immunology, 2007, 179: 1760–1767.
sion was determined to be restricted to T cells and up-regulated
upon signaling through the TCR (3). On the other hand, OX40
ligand (OX40L)3is expressed on a number of cell types, including
professional APCs (dentritic cells and macrophages) (1, 4). OX40/
OX40L interaction plays an important role in CD4?Th cell re-
sponses (1, 4) and the generation of memory T cells (5, 6). Al-
though OX40 can contribute to CD8?T cell responses, many
studies have suggested a preferential role for this receptor in CD4?
Th2 responses (1, 4). Indeed, OX40 signaling, in conjunction with
TCR and CD28 signaling, induces NFATc1 accumulation in the
nucleus to drive initial IL-4 transcription (7). Both OX40 (8)- and
OX40L (9)-deficient mice exhibit defects in CD4?T cell prolif-
erative responses, and in transgenic mice overexpressing OX40L
in dendritic cells, CD4?T cells accumulate in the B cell follicles
(10). Furthermore, OX40 is preferentially expressed on activated
X40 (CD134) is a member of the TNFR superfamily
(TNFRSF4) (1) that was originally identified as a rat T
cell activation marker (2). On one hand, OX40 expres-
CD4?T cells under certain conditions (11). Signaling through
OX40 can promote survival signals in effector T cells (12, 13) and
can also activate NF-?B through a TNFR-associated factor 2- and
5-mediated pathway (14).
OX40 is expressed at high levels on CD4?CD25?regulatory T
(Treg) cells (15, 16), suggesting that it might play an important
role in these cells. Indeed, signaling through this receptor can in-
hibit suppressive activity of these cells (17), paralleling the func-
tion of glucocorticoid-induced TNFR (TNFRSF18) (15, 18). This
molecule, like OX40, is also mainly expressed on T cells, and
functions as a costimulatory receptor on effecter T cells (19).
Why CD4?CD25?Treg cells have these two receptors with
similar functions, and how these cells can regulate high-level
expression of these two receptors are currently unknown. Thus,
although the role of OX40 in T cell-mediated immune responses
has been well documented, and even studied in autoimmunity
and immune responses to cancer (4), regulation of its gene ex-
pression in activated effector T cells, as well as in CD4?CD25?
Treg cells, is poorly understood.
In the present study, we show that OX40 mRNA expression is
up-regulated in the T cell line EL4 by activation with anti-CD3 Ab
(same as that in primary T cells). This suggests that we can use this
cell line as a model system to study OX40 gene expression. Using
the EL4 line, we show that basal OX40 promoter activity is reg-
ulated by the constitutively expressed transcription factors Sp1/
Sp3 and YY1. Significantly, we demonstrate drastic activation-
associated chromatin remodeling in the OX40 basal promoter
region. OX40 gene expression seems to be regulated by chromatin
remodeling upon activation, and NF-?B might be involved in ini-
tiation of this chromatin remodeling. Importantly, in CD4?CD25?
Treg cells (without any in vitro activation), chromatin structure in
the OX40 basal promoter region is relaxed by histone acetylation
similar to that in activated CD4?CD25?T cells.
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Phil-
adelphia, PA 19104
Received for publication February 24, 2007. Accepted for publication May 22, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by National Institutes of Health Grants R01
CA74958 (to M.L.T.) and R01 AI31044 (to M.L.T.).
2Address correspondence and reprint requests to Dr. Masahide Tone, Department of
Pathology and Laboratory Medicine, University of Pennsylvania, 412 Stellar Chance,
422 Curie Boulevard, Philadelphia, PA 19104-6100. E-mail address: mtone@mail.
3Abbreviations used in this paper: OX40L, OX40 ligand; Treg, regulatory T; ChIP,
chromatin immunoprecipitation; KO, knockout; UTR, untranslated region; HAT, hi-
stone acetyltransferase; HDAC, histone deacetylase.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
Materials and Methods
Cell culture and purification
EL4 cells were cultured in IMDM?GlutaMax (Invitrogen Life Technolo-
gies) with 5% FBS, and primary T cells were cultured in RPMI 1640 with
L-glutamine (Lonza) with 10% FBS. Penicillin (10 U/ml)-streptomycin (10
?g/ml) (Invitrogen Life Technologies) was also added into these media.
Total primary T cells (for RT-PCR and luciferase assays) and CD4?T cells
(for RT-PCR) were isolated from mouse spleen cells using a pan-T cell
isolation kit (Miltenyi Biotec) and a CD4?T cell isolation kit (Miltenyi
Biotec), respectively. For the chromatin immunoprecipitation (ChIP) as-
say, CD4?CD25?and CD4?CD25?Treg cells were purified by a cell
sorter using allophycocyanin-anti-CD4 Ab (L3T4; BD Pharmingen) and
bitotin-anti-CD25 Ab (7D4; BD Pharmingen) with streptavidin-PE (BD
Pharmingen). Purity of CD4?CD25?and CD4?CD25?T cells were 99
and 98%, respectively. If required, cells were activated by culturing with
anti-CD3 Ab (KT3) precoated (with 5 ?g/ml Ab in PBS) plates.
OX40 mRNA levels were analyzed by RT-PCR as described previously
(20). Briefly, cDNA was prepared using an oligo(dT) primer and 1 ?g of
total RNA from EL4 cells, primary total T cells, and CD4?T cells. To
perform semiquantitative RT-PCR, PCR was stopped at different cycle
numbers (OX40: 21, 24, 27, and 30 cycles; 18S rRNA: 13, 16, and 19
cycles). PCR products were analyzed by agarose gel with ethidium bro-
mide staining. Quantitative PCR was performed by real-time PCR using
TaqMan 7900HT (Applied Biosystems) with Power SYBR Green PCR
Master Mix (Applied Biosystems). The PCR primers used were as follows:
OX40 sense, GTAGACCAGGCACCCAACC; OX40 antisense, GGCCA
GACTGTGGTGGATTGG; 18S rRNA sense, CTTAGAGGGACAAGT
GGCG; and 18S rRNA antisense, ACGCTGAGCCAGTCAGTGTA.
Mapping of the transcription start site
The OX40 transcription start site was determined by RACE (21) with mi-
nor modifications as described previously (20). cDNA was prepared using
an antisense oligo primer (GTTGCACTGTGTACACTGCTTG) and total
RNA from nonactivated and activated CD4?CD25?T cells, CD4?CD25?
Treg cells, and activated EL4 cells. After adding a poly(G) tail at the
resulting cDNA end, OX40 cDNA were amplified using a poly(C) primer
and an antisense primer (ATTGTAGAAGCCAGTCTCACATG). To con-
centrate OX40 cDNA fragments, the resulting PCR products were ampli-
fied again using the second antisense primer (TGGCACTCACGACAG
CACTTGTG). The resulting PCR products were cloned and DNA
sequences were determined. To avoid PCR artifacts as a result of this
procedure, seven independent PCR products were analyzed.
Construction of OX40 promoter reporter plasmids and
OX40 promoter fragments (11 OX40 promoter deletion mutants, Sp1 site
knockout (KO), YY1 site KO, and NF-?B site KO) were cloned into the
pGL4 Basic Vector (Promega). All DNA sequences of inserted fragments
were determined to remove deformed fragments generated by PCR errors.
For the luciferase assay, 5 ? 106EL4 cells or 3 ? 106primary total T cells
were transfected with 5 ?g (for EL4) or 3 ?g (for primary T cells) of
luciferase reporter plasmids and 1 ?g (or 0.5 ?g for activated condition) of
pRL-CMV as an internal control plasmid, and cultured in six-well plates.
For EL4 and primary T cell transfection, Gene Pulser Xcell (Bio-Rad) and
Nucleofector with mouse T cell kit (Amaxa) were used, respectively. To
activate cells, transfected cells were transferred into anti-CD3 Ab (KT3)
precoated (with 5 ?g/ml Ab in PBS) six-well plates at 4 h posttransfection,
and cultured for additional 44 h. Cells were harvested, and promoter ac-
tivities were analyzed by Dual-Luciferase Reporter Assay System (Pro-
mega). These assays were repeated at least three times, and firefly lucif-
erase (pGL4) activities were normalized to Renilla luciferase (pRL-CMV,
internal control) activities.
Nuclear extracts were prepared using nonactivated EL4 and anti-CD3 ac-
tivated EL4 and total primary T cells (for 24 h) (for Fig. 3B) (for 2 h) (for
Fig. 5C). Harvested cells were homogenized in hypotonic lysis buffer (10
mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, and 1 mM DTT with
proteinase inhibitor mixture (Sigma-Aldrich)), and then nuclei were col-
lected by centrifugation. Nuclear proteins were extracted with extraction
buffer (20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM
EDTA, 25% glycerol, 1 mM DTT with protease inhibitor mixture (Sigma-
Aldrich)). EMSA was performed with 2 ?g of nuclear extract in 20 ?l of
EMSA reaction buffer (2 ?g of poly(dI-dC)poly(dI-dC), 20 mM HEPES
(pH 7.9), 1 mM MgCl2, 40 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 12%
glycerol). To perform the competition assay, a 100-fold excess of unla-
beled oligo competitor primer was added to the EMSA reaction mixture.
Samples were analyzed on a polyacrylamide gel containing 89 mM Tris
borate and 20 mM EDTA. For the supershift assay, nuclear extracts in
EMSA reaction buffer were incubated with anti-Sp1 (Santa Cruz; PEP2),
anti-Sp3 (Santa Cruz; D-20), anti-YY1 (Santa Cruz; H-10), NF-?B p65
(Santa Cruz; C-20), and NF-?B p50 (Santa Cruz; D-17) Abs for 15 min, at
which time probes were then added. Probe and competitor sequences
(sense strand) used were as follows: P1, ACGCCTGTGCCAAATACACA
GGAACACGTT; P2, GGAACACGTTCACATACCTTCTTGCCTGTC;
P3, CTTGCCTGTCCGCCTACTCTTCTTGCCCCA; P4, TCTTGCCCCA
CCTCCATAGTTCTTATAGCC; P5, ACCTCCATAGTTCTTATAGCCA
AAAACCCCAGACTCC; Sp1A KO, TCTGAATTCACCTCCATAGTTC
TTATAGCC; YY1 KO, TCTTGCCCCACCTCCGAATTCCTTATAG
CC; Sp1A/YY1 KO, TCTTGCCCCACGAATTCAGTTCTTATAGCC;
Sp1B KO, TTATAGCCACAGAATTCAAGGAAAA; and NF-?B KO, CC
TGCAAGGACTCGAGCCAGACTCC. DNA sequences of IL-10/Sp1
(20), CD40/Sp1 (22), and CD40/NF-?B (NF-?BA site) (22) competitors
were shown previously.
ChIP assay was performed using EL4 cells, CD4?CD25?T cells, and
CD4?CD25?Treg cells. These cells were fixed in fixation solution (1%
formaldehyde, 4.5 mM HEPES (pH 8.0), 9 mM NaCl, 0.09 mM EDTA,
and 0.045 mM EGTA) for 10 min at room temperature. The reaction was
stopped by adding glycine solution (final concentration, 0.1 M). Fixed cells
were then washed twice with cold PBS, and placed in lysis buffer (1% SDS,
10 mM EDTA, and 50 mM Tris-HCl (pH 8.0) with protease inhibitor
mixture (Sigma-Aldrich)). Chromatin was sheared by sonication on ice,
and the insoluble fraction was removed by centrifugation. This lysate was
precleared with control Ig plus salmon sperm DNA-protein A agarose (Up-
state) and Abs were added and incubated at 4°C overnight. Immunocom-
plexes were collected with salmon sperm DNA-protein A agarose, and
washed twice with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM
EDTA, 20 mM Tris-HCl (pH 8.0), and 150 mM NaCl), twice with high salt
buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH
8.0), and 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% IGEPAL-
CA630, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris-HCl (pH 8.0)),
and TE (10 mM Tris-Cl (pH 7.5) and 1 mM EDTA). Precipitated chro-
matin fragments were eluted with elution buffer (1% SDS, 0.1 M
NaHCO3), after adding NaCl (final concentration, 0.2 M), and samples
were incubated at 65°C overnight. After treatment with proteinase K, DNA
was extracted using PCR DNA purification kit (Qiagen). Quantitative real-
time PCR analysis with Power SYBR Green PCR Master Mix (Applied
Biosystems) was performed to determine the percentage of OX40 basal
promoter precipitated with Abs against input DNA. PCR primers used to
amplify the OX40 basal promoter and Abs were as follows: OX40 pro-
moter sense, TACGCCTGTGCCAAATACAC; OX40 promoter antisense,
acetyl histone H4 polyclonal Ab (Upstate); and rabbit IgG (Santa Cruz
OX40 mRNA expression is up-regulated by activation with
anti-CD3 Ab in primary T cells and the T cell line EL4
Because up-regulation of OX40 in activated T cells is key to phys-
iology of this receptor, we investigated how OX40 gene expression
is regulated. To determine whether OX40 expression is controlled
by transcriptional and/or posttranscriptional regulation in activated
T cells, RT-PCR for OX40 mRNA was performed using RNA
from the T cell line EL4 (Fig. 1), primary total T cells (A), and
CD4?T cells (B). To perform semiquantitative RT-PCR, the PCR
was stopped at different cycle numbers (Fig. 1A). OX40 mRNA
was detected in nonactivated total primary T cells and EL4 cells,
and up-regulated in cells activated with anti-CD3 Ab (Fig. 1A).
PCR products from OX40 mRNA were observed in earlier PCR
cycle numbers for primary T cells (both activated and nonacti-
vated), compared with EL4 cells, indicating that the expression
level of OX40 mRNA is higher in the former. To analyze the
induction of OX40 mRNA expression during activation with
1761 The Journal of Immunology
anti-CD3 Ab, real-time PCR was performed using RNA from
EL4 and CD4?T cells. OX40 mRNA levels were increased 18-
and 12-fold by activation in EL4 and CD4?T cells, respec-
tively (Fig. 1B). These findings suggest that OX40 expression is
at least partially controlled by transcriptional and/or posttran-
scriptional (e.g., stabilization of OX40 mRNA in activated
cells) regulation, and that we can use EL4 cells as a model
system to study the regulation of OX40 expression. In the 3?-
untranslated region (UTR) of OX40 mRNA, we could not iden-
tify a typical mRNA destabilization signal (AUUUA?AU-rich
sequence) (23, 24) (we have also previously detected in IL-10
mRNA (25)), we chose to focus on the transcriptional regula-
tion of OX40 gene expression.
The basal promoter of OX40 is located between positions
?4 and ?125
As a first step to investigate the transcriptional regulation of OX40
gene expression, we examined the OX40 promoter activity. To
determine the location of the OX40 promoter, we analyzed the
transcriptional start site by 5?-RACE, using RNA from CD4?T
cells, CD4?CD25?Treg cells, and EL4 cells. In total, we analyzed
71 5?-RACE clones to determine the major mRNA start site, and
to avoid artifacts resulting from the PCR procedure, seven inde-
pendent PCR products were used for this assay (two PCR products
from nonactivated CD4?T cells, two PCR products from activated
CD4?T cells, two PCR products from CD4?CD25?Treg cells
and a PCR product from activated EL4 cells). The same site was
mapped as a major mRNA start site in all reactions (55 clones of
total 71 clones) at 8 bp upstream of the first ATG of OX40 mRNA,
and this mRNA start site was defined as position ?1. The same
major transcription start site was determined in all T cells tested,
indicating that the OX40 promoter could be located upstream of
this start site in nonactivated and activated cells. No TATA box
sequence was found in the region 30 bp upstream of the major
transcription start site, suggesting that OX40 gene expression is
regulated by a TATA-less promoter. A 1.97-kb promoter fragment
(Pro 1) (Fig. 2) containing the major mRNA start site was PCR-
amplified from mouse genomic DNA, and 10 deletion mutants of
this promoter (all containing the major mRNA start site) (Pro 2 to
Pro 11) (Fig. 2) were generated by PCR and cloned into the pGL4
Basic Vector (Promega) (Fig. 2). The resulting luciferase reporter
plasmids were transfected into EL4 cells, and luciferase activities
were compared with that of the negative control plasmid (pGL4
Basic Vector, no promoter). A significant reduction of promoter
activity resulted from deletion of 69 bp (between Pro 9 and Pro
10) and 30 bp (between Pro 10 and Pro 11) regions in both
nonactivated EL4 cells and anti-CD3 Ab-activated cells. This
finding suggested that OX40 basal promoter activity is located
between ?125 (5? end of Pro 9 fragment) and ?4 (3? end of all
promoter fragments), and constitutively expressed transcription
factors might be binding to this basal promoter region. This
OX40 basal promoter activity was also analyzed in activated
primary T cells by a luciferase assay using selected promoter
constructs, and regulatory regions were mapped in the identical
positions (Fig. 2C).
Sp1/Sp3 and YY1 binding sites are present in the OX40
Having concluded via OX40 promoter deletion mutants that reg-
ulatory elements for OX40 promoter activity are located between
position ?125 (5? end of promoter 9 fragment) and ?26 (5? end of
promoter 11 fragment), we next sought to identify transcription
factors binding to this region. EMSA was performed using seven
oligo probes containing sequences in this region (Fig. 3). Four
performed using RNA from primary total T cells and EL4 cells, cul-
tured with (CD3) or without (Non) anti-CD3 Ab. Amplified OX40
mRNA (21, 24, 27, and 30 cycles) and 18S rRNA (13, 16, and 19
cycles; as control) PCR products were analyzed by agarose gel with
ethidium bromide staining. B, Real-time PCR was performed using
RNA from EL4 cells and CD4?T cells, cultured with (CD3) and with-
out (Non) anti-CD3 Ab. Expression levels were normalized by 18S
rRNA levels. The OX40 mRNA level in activated cells was then com-
pared with that in nonactivated cells.
Expression of OX40 mRNA in T cells. A, RT-PCR was
plasmids were constructed using pGL4 Basic Vector (Promega) and OX40
promoter fragments. All promoter fragments contained the same 3? end and
the position of the 5? end of each construct is indicated in B. B, Luciferase
activities generated using the reporter plasmids (Pro 1 to Pro 11) were
compared with that using negative control plasmid (no promoter, Basic) in
nonactivated (Non) EL4 cells and activated (CD3) EL4 cells with anti-CD3
Ab. Luciferase assays were repeated more than three times, and the activ-
ities were normalized using Renilla luciferase activity. C, Luciferase assay
was also performed (as described in B) using activated (CD3) primary total
T cells and indicated plasmids.
Promoter activity of the OX40 gene. A, Luciferase reporter
1762REGULATION OF OX40 GENE EXPRESSION IN T CELLS
slowly migrating complexes were detected by EMSA using the P4
probe, with both nuclear extracts from nonactivated and activated
EL4 cells (Fig. 3B). All four complexes (C1 to C4) with
labeled P4 probe disappeared with a 100-fold excess of unlabeled
P4 competitor, but not with P2 (Fig. 4A), indicating sequence-
specific complex formation. To determine the transcription factor
binding regions in this P4 probe, EMSA was performed using
seven mutant P4 oligo probes (results using three mutant probes
are shown in Fig. 6, B and C, but results using other mutants are
not shown). This result suggests that these factors bind within a
TGCCACCTCC sequence. The potential Sp1 family binding
sequence was identified in this sequence by the transcription
factor database (TESS). These results suggest that complexes
C1, C2, and C3 could form with the transcription factor Sp1
family on a GCCCCACCTC sequence (position ?70 to ?61).
We therefore performed competition assays using P4 probe with
CD40 (22) and IL-10 (20) Sp1 oligo competitors that we used
previously for analysis of CD40 and IL-10 promoter activity.
Indeed, these competitors inhibited C1, C2, and C3 complex
formation with32P-labeled P4, but not C4 formation (Fig. 4A).
Complex formation with Sp1 (C1) and Sp3 (C2 and C3) was
also confirmed by supershift EMSA using anti-Sp1 and anti-Sp3
Abs (Fig. 5A). Two complexes with Sp3 were presumably
formed with two different-sized Sp3 molecules as described
EMSA using mutant P4 probes (data not shown) also sug-
gests that the factor in complex C4 binds within a CCACCTC
CATAG sequence. The transcription factor database suggests
that complex C4 might form with transcription factor YY1 on
ACCTCCATA (position ?65 to ?58) in the identified se-
quence, which was confirmed by supershift EMSA using an
anti-YY1 Ab (Fig. 5A).
Four slowly migrating complexes (C5 to C8) were also detected
by EMSA using P6 probe, with nuclear extracts from nonactivated
and activated EL4 cells (Figs. 3B and 4B). C5, C6, and C7 com-
plex formation with32P-labeled P6 was inhibited with a 100-fold
excess of unlabeled P6, but not with P5 (Fig. 4B), indicating se-
quence-specific complex formation. However, C8 complex forma-
tion was not inhibited by either unlabeled P6 competitor (contain-
ing exactly the same sequence as in P6 probe) or P5, suggesting
that the factor in complex C8 might recognize the 5? end sequence
with phosphate (32P) in the P6 probe. Indeed, a 2-bp mutation at
the 5? end of this probe inhibited C8 complex formation (data not
shown). These results suggest that C8 complex formation is an
artifact due to this procedure using oligo probes. EMSA using
mutant probes (data not shown) suggests that these factors bind
within a TAGCCACACCCT sequence; the transcription factor
database also suggests that complexes C5, C6, and C7 might
?26). A, Seven oligo probes for EMSA were designed and prepared.
Positions of these probes are indicated by solid lines (P1 to P7). B,
EMSA was performed using oligo probes shown in A and nuclear ex-
tracts from nonactivated (Non) EL4 cells and activated (CD3) EL4 cells
with anti-CD3 Ab.
Binding of nuclear factors to the OX40 promoter (?125 to
petition assays performed using the P4 probe and a nuclear extract from
EL4 cells with the indicated competitors. Complex formation with probe
P4 (?) was inhibited with the indicated competitors. B, Competition assays
performed using the P6 probe and a nuclear extract from EL4 cells with the
Binding of Sp1/Sp3 to the OX40 basal promoter. A, Com-
Supershift EMSA was performed with a nuclear extract from EL4 cells
(A–C) and indicated Abs or no Ab (?). A, With P4 probe. B, With P6
probe. C, With P7 probe. D, Sp1, Sp3, YY1, and NF-?B binding to probes
P4, P6, and P7 was confirmed by EMSA using a nuclear extract from
activated (CD3) primary total T cells. Competition assays were also per-
formed using P4, P6, P7, IL-10 Sp1, and CD40 NF-?B (indicated as CD40
Sp1, Sp3, YY1, and NF-?B bind to the OX40 promoter. A,
1763The Journal of Immunology
form with transcription factor Sp1 family on GCCACACCCT
sequence (position ?48 to ?39) in the identified sequence; and
this was confirmed by competition assay (Fig. 4B) and super-
shift EMSA using anti-Sp1 and anti-Sp3 Abs (Fig. 5B). Two
Sp1/Sp3 binding regions are present in the OX40 promoter,
which we have named Sp1A (the Sp1 site in P4) and Sp1B (the
Sp1 site in P6), respectively (Fig. 6A). These transcription fac-
tors binding to the OX40 promoter was confirmed by competi-
tion assays using a nuclear extract from activated primary T
cells (Fig. 5D).
Basal OX40 promoter activity is regulated by Sp1/Sp3 and YY1
as positive regulators
To examine the contribution of Sp1, Sp3, and YY1 to OX40 pro-
moter activity, each transcription factor binding site was mutated
(Fig. 6) in the OX40 promoter constructs Pro 2 (1.35 kb) (Figs.
2 and 6) and Pro 9 (0.13 kb) (Figs. 2 and 6), and luciferase
reporter assays were performed. The structures of the resulting
plasmids are illustrated in Fig. 6A, and mutated sequences are
shown with wild-type sequences in Fig. 6B. No binding of Sp1/
Sp3 or YY1 to the mutated sequences was detected by EMSA
(Fig. 6C). A significant reduction of promoter activity with the
mutation of each of the transcription binding regions was ob-
served, in both nonactivated and activated EL4 cells. No pro-
moter activity (or only very weak promoter activity in activated
cells) was detected with the triple mutation (Sp1A, YY1, and
Sp1B) in this promoter, indicating OX40 basal promoter activ-
ity is regulated by constitutively expressed transcription factors,
Sp1/Sp3 and YY1.
Although luciferase activity generated using Pro 1 to Pro 9 re-
porter constructs in activated cells was ?1.5- to 2-fold higher than
that in nonactivated cells (Fig. 2), we could not confirm the pres-
ence of a CD3 activation response element in this promoter region
for the following reasons: 1) taking the variability of luciferase
assays into consideration, a 1.5-fold difference might be not sig-
nificant; 2) the transcriptional environments in between nonacti-
vated and activated cells are not the same (e.g., amount of poly-
merase), and any observed enhancement of promoter activity could
simply be a consequence of the different environments. Thus, CD3
response elements must be identified with other assay systems,
such as EMSA and luciferase assay using point mutated promoter
NF-?B p50 and p65 bind to the OX40 basal promoter region
By EMSA with probe P7 (Fig. 3B), we detected a transcription
factor bound to this probe in nuclear extracts from activated EL4
cells, but not nonactivated cells. The transcription factor database
suggested that NFAT might bind to the AGGAAAAA (?35 to
?28) sequence in the P7 probe. Indeed, this sequence is similar to
NFAT consensus sequences in the IL-2 promoter (GAGGAAAA).
We therefore performed a supershift assay using anti-NFAT Ab;
however, NFAT binding to the promoter region was not detected.
Because the structural similarity and heterodimer formation of
NFAT and NF-?B p50 has been recently suggested (27–31), we
also performed a supershift assay using anti-NF-?B p50, p52, p65,
rel-B, and c-rel Abs. The complex formation was further shifted
with both p50 and p65 Abs (Fig. 5C), indicating that NF-?B p50
and p65 bind to the OX40 basal promoter region. NF-?B binding
was also confirmed by EMSA and a competition assay using a
nuclear extract from activated primary T cells (Fig. 5D). However,
we could not detect any CD3 activation response elements in the
OX40 promoter region using the deletion mutants by luciferase
reporter assay (Fig. 2). We therefore mutated the NF-?B binding
region in the OX40 Pro 9 construct and the luciferase activity gen-
erated using the resulting plasmid was compared with that using
wild-type Pro 9 in nonactivated and activated EL4 cells (Fig. 7).
The NF-?B binding sequence and the mutated sequence are shown
in Fig. 7A, and the lack of NF-?B binding to the mutant sequence
was also confirmed by EMSA (Fig. 7B). OX40 promoter activity
was reduced to 60% by mutation in activated cells (Fig. 7C), but
this promoter activity with the mutated sequence was also
slightly reduced in nonactivated cells (90%). Considering the
variability of the luciferase assay and the result in Fig. 2 (no
CD3 response elements were detected by luciferase assay with
by Sp1/Sp3 and YY1. A, Sp1 and YY1 binding
sequences were mutated in Pro 2 and Pro 9 con-
structs, and luciferase assay was performed. The
structures of the mutant luciferase reporter plas-
mids are illustrated. Mutated sites are indicated
with an X. Luciferase activities generated using
mutant plasmids in nonactivated (Non) and ac-
tivated (CD3) EL4 cells with anti-CD3 Ab were
compared with those using the wild-type plas-
mid. Luciferase assays were repeated more than
three times, and the activities were normalized
using Renilla luciferase activity. B, Wild-type
sequences and mutated sequences of Sp1A,
YY1, and Sp1B sites. Mutated sequences are in-
dicated in bold. C, EMSA was performed with
probes P4 (P4 Wt), P6 (P6 Wt), Sp1A mutant
(Sp1A KO), YY1 mutant (YY1 KO), Sp1A/
YY1 double mutant (Sp1A/YY1 KO), and mu-
tant Sp1B (Sp1B KO). Complexes with Sp1,
Sp3, and YY1 are indicated.
The OX40 promoter is regulated
1764REGULATION OF OX40 GENE EXPRESSION IN T CELLS
a series of deletion mutants of the OX40 promoter), we cannot
conclude any contribution of NF-?B in the direct enhancement
of OX40 promoter activity. The up-regulation of OX40 mRNA
observed in activated EL4 cells and primary T cells (Fig. 1) is
Chromatin remodeling occurs at the OX40 basal promoter after
We hypothesized that NF-?B may contribute to chromatin remod-
eling in the basal promoter in vivo, rather than direct enhancement
of OX40 promoter activity. OX40 gene expression may be up-
regulated by chromatin remodeling in activated T cells. To inves-
tigate this possibility, induction of OX40 mRNA expression (by
RT-PCR) and histone H4 acetylation in the basal OX40 promoter
region containing Sp1, YY1, and NF-?B binding regions (by ChIP
assay) were monitored at different time points after activation with
anti-CD3 Ab in EL4 cells (Fig. 8A). Induction of histone H4 acet-
ylation was increased gradually after activation, and induction of
OX40 mRNA expression was found to reflect the ratio of histone
H4 acetylation in the basal promoter region (Fig. 8A).
OX40 gene expression seems to be up-regulated by chromatin
remodeling in the OX40 basal promoter region, and NF-?B may
contribute to this chromatin remodeling event. To investigate this
possibility, we performed ChIP assay using anti-NF-?B p50 Ab in
EL4 and CD4?CD25?T cells. Binding of NF-?B p50 to the basal
OX40 promoter region was detected in activated EL4 and
CD4?CD25?T cells in vivo (Fig. 8B). We have also detected
binding of NF-?B p50 and p65 to the basal OX40 promoter
region by EMSA (Fig. 5, C and D). Taken together, these find-
ings indicate that NF-?B might contribute to the up-regulation
of OX40 gene expression through chromatin remodeling in ac-
tivated T cells.
To examine whether histone H4 is acetylated in the basal OX40
promoterregion in activated
CD4?CD25?Treg cells (which expresses OX40 at high levels),
ChIP assay was performed. Acetylation of histone H4 in the basal
OX40 promoter region in CD4?CD25?T cells is increased by
activation, and interestingly, in CD4?CD25?Treg cells, histone
H4 molecules in this promoter region are highly acetylated. We
isolated these Treg cells from mouse spleen cells and immediately
used them for ChIP assay, indicating that histone H4 molecules in
the OX40 promoter region are highly acetylated in these cells with-
out activation in vitro. The chromatin structure of the OX40 pro-
moter region is configured to express OX40 at high levels in
In this study, we have studied the up-regulation of OX40 gene
expression in T cells, on the premise that up-regulation of OX40
on T cell surfaces is critical for its physiological function. We have
focused on transcriptional control of the OX40 gene, and in this
first analysis of the OX40 promoter, we report that OX40 basal
promoter activity is regulated by constitutively expressed tran-
scription factors, Sp1/Sp3 and YY1. However, given that OX40
mRNA levels are low in nonactivated cells, the OX40 promoter
seems to function fully with activation-induced transcription fac-
tors in activated T cells. Although we identified NF-?B binding to
the OX40 promoter region, there was only a 1.5-fold direct en-
hancement of OX40 promoter activity by this transcription factor
(as determined by a luciferase reporter assay using KO promoter
constructs (Fig. 7)). Thus, NF-?B-driven enhancement of OX40
promoter activity does not on its own account for the large up-
regulation of OX40 mRNA expression in activated cells. We con-
sidered three possibilities: 1) OX40 gene expression may be con-
trolled by posttranscriptional mechanisms (i.e., OX40 mRNA
might be unstable in resting T cells and stabilized via activation);
2) OX40 gene expression may be up-regulated by chromatin re-
modeling in activated cells; and/or 3) OX40 gene expression may
ity. A, Structure of the mutant luciferase reporter plasmids. Mutated sites
are indicated with an X. NF-?B consensus sequence and mutant sequence
(bold) are indicated. B, EMSA was performed with the wild-type probe
(Wt) and the mutant probe (NF-?B KO). C, Luciferase assays were per-
formed using Wt and NF-?B mutant constructs in nonactivated (Non) and
activated (CD3) EL4 cells. Luciferase activities generated with the mutant
construct were compared with that using the Wt construct. Luciferase assay
were repeated more than three times, and the activities were normalized
using Renilla luciferase activity.
Contribution of NF-?B in regulating OX40 promoter activ-
ing in its promoter region. A, RT-PCR and ChIP assays were performed
using nonactivated (0) and activated (2, 12, and 24 h) EL4 cells with
anti-CD3 Ab. Chromatin fragments were immunoprecipitated with anti-
acetyl histone H4 Ab (Ac-H4) or control Ab (Ig), and the precipitated
OX40 promoter region was analyzed by real-time PCR. B, ChIP assay
performed using anti-NF-?B p50 (p50) and control Ab (Ig) using nonac-
tivated (Non) and activated (CD3) EL4 and CD4?CD25?T cells. C, ChIP
assay performed using anti-acetyl histone H4 Ab (Ac-H4) and control Ab
(Ig) using nonactivated (Non) and activated (CD3) CD4?CD25?T cells
and CD4?CD25?Treg cells.
OX40 gene expression is regulated by chromatin remodel-
1765The Journal of Immunology
be up-regulated by an activation-specific enhancer. Several con-
siderations steered us away from the first possibility, namely, post-
transcriptional control. We have previously shown that IL-10
mRNA levels in EL4 cells are regulated by posttranscriptional reg-
ulation, through AUUUA?AU-rich mRNA destabilization signals
located in the 3?-UTR (25). Although there is an AUUUUA se-
quence in the 3?-UTR of OX40 mRNA, it is not accompanied by
AU-rich sequences, and therefore, OX40 mRNA seems to lack the
mRNA destabilizing sequence configuration of IL-10 mRNA (25)
and other mRNAs (23, 24). Additionally, no potential micro-RNA
binding regions can be identified in OX40 mRNA, using the micro-
RNA database (PicTar).
Consequently, we turned our attention to the second possibility,
namely, chromatin remodeling of OX40 gene. Using a ChIP assay,
we strikingly found that histone H4 molecules in this region are
highly acetylated upon T cell activation, and that NF-?B binds to
the OX40 basal promoter region in vivo. Providing a potential link
between these two findings, a recent report describes that NF-?B
can recruit a number of different coactivators, including p300/
CBP, p/CAF, and SRC-1 (32, 33), which have histone acetyltrans-
ferase (HAT) activity. Taken together, these findings suggest that
OX40 gene expression is up-regulated by chromatin remodeling in
activated T cells, and NF-?B might be involved in the initiation of
this chromatin remodeling.
Although we cannot rule out involvement of an activation-spe-
cific enhancer in regulating OX40 gene expression, our OX40 pro-
moter chromatin remodeling findings prompt us to posit the fol-
lowing sequence of events accounting for OX40 up-regulation: 1)
in resting T cells, chromatin in the OX40 promoter region is in the
closed form, albeit some Sp1, Sp3, and YY1 can access this pro-
moter and yield small amounts of OX40 mRNA; 2) NF-?B is nu-
clear translocalized by activation, binds to the OX40 basal pro-
moter, and recruits HAT and subsequently acetylate histone
molecules to this promoter region; and 3) once OX40 promoter
chromatin structure is relaxed by histone acetylation, Sp1, Sp3,
and YY1 transcriptional factors can more easily access the binding
regions, leading to the up-regulation of OX40 mRNA production.
Although this study has focused on the up-regulation of OX40
gene expression, subsequent down-regulation is also of interest.
OX40 up-regulation peaks at 48 h, and then declines again (3). The
chromatin remodeling mechanism we have proposed in this study
to account for OX40 up-regulation might be extended to also ex-
plain its down-regulation as well. Specifically, it is possible that
down-regulation is mediated by deacetylation of histone molecules
in the OX40 promoter region. Not only can NF-?B recruit HATs,
but NF-?B p50 can additionally recruit histone deacetylase 1
(HDAC1) (34). Thus, in the late activation stage, NF-?B p50
might recruit HDACs and deacetylate histone molecules to the
OX40 promoter region, closing the chromatin structure and thereby
down-modulating OX40 gene expression.
In this study, we focused on regulation of OX40 expression by
signaling through the TCR/CD3 complex. Enhancement of OX40
expression was also observed with costimulatory signals through
CD28 (12, 35). Involvement of activated STAT6 on OX40 gene
expression by CD28 signaling was excluded by a study using
STAT6-deficient mice (36). CD28 signaling might up-regulate
OX40 gene expression by indirect pathways. Indeed, we could not
identify a CD28 response element by luciferase assay in the OX40
promoter region (data not shown).
There is an interesting possibility that the constitutive Sp1 and
YY1 transcription factors might contribute to OX40 up-regulation
in more than one way. Beyond directly regulating OX40 basal
promoter activity, they could potentially contribute on the chro-
matin remodeling side as well. Recently, Sp1 interactions with
HAT (p300) and HDAC1 (37, 38) have been reported, as well as
ability of YY1 to recruit HAT (39) and HDACs (40, 41). Thus,
histone acetylation in the OX40 promoter region might be regu-
lated by cooperation between HATs and HDACs that are associ-
ated with both induced (NF-?B) and constitutively expressed (Sp1
and YY1) transcription factors.
YY1 can reportedly regulate transcription as both a positive and
a negative regulator (42). Because OX40 promoter activity was
reduced to about one-half by mutation of the YY1 site in this
promoter region, YY1 seems to function largely as a positive reg-
ulator in the case of OX40 gene expression. This YY1 site actually
overlaps the Sp1A site (Fig. 6B), which is of interest given that
interaction between Sp1 and YY1 has been suggested in the case
of other genes (43, 44). This could be the case for OX40 gene
expression as well.
Approximately 5% of CD4?T cells are CD4?CD25?Treg cells
that express OX40 at high levels, and signaling through this receptor
can neutralize the suppressive activity of these Treg cells (17). Sig-
nificantly, we have found that histone molecules in this promoter
region are highly acetylated in CD4?CD25?Treg cells (absence
of activation in vitro). Thus, chromatin structure of OX40 promoter
might be always in the relaxed form in Treg cells. Building upon
our description of chromatin remodeling of the OX40 promoter
region in the course of T cell activation, we further posit that the
differentiation of Treg cells from precursors (presumably driven by
some self-Ag) may also be associated with relaxing of chromatin
structure in the OX40 promoter region. This region would then be
kept open in mature CD4?CD25?Treg cells by some unknown
molecular mechanism. Detailed study of chromatin remodeling in
the OX40 promoter region in CD4?CD25?Treg cells could thus
potentially contribute to an understanding of how transcriptional
regulation is controlled in CD4?CD25?Treg cells and how this
category of cells develops.
The authors have no financial conflict of interest.
1. Watts, T. H. 2005. TNF/TNFR family members in costimulation of T cell re-
sponses. Annu. Rev. Immunol. 23: 23–68.
2. Paterson, D. J., W. A. Jefferies, J. R. Green, M. R. Brandon, P. Corthesy,
M. Puklavec, and A. F. Williams. 1987. Antigens of activated rat T lymphocytes
including a molecule of 50,000 Mrdetected only on CD4 positive T blasts. Mol.
Immunol. 24: 1281–1290.
3. Gramaglia, I., A. D. Weinberg, M. Lemon, and M. Croft. 1998. Ox-40 ligand: a
potent costimulatory molecule for sustaining primary CD4 T cell responses.
J. Immunol. 161: 6510–6517.
4. Sugamura, K., N. Ishii, and A. D. Weinberg. 2004. Therapeutic targeting of the
effector T-cell co-stimulatory molecule OX40. Nat. Rev. Immunol. 4: 420–431.
5. Gramaglia, I., A. Jember, S. D. Pippig, A. D. Weinberg, N. Killeen, and M. Croft.
2000. The OX40 costimulatory receptor determines the development of CD4
memory by regulating primary clonal expansion. J. Immunol. 165: 3043–3050.
6. Maxwell, J. R., A. Weinberg, R. A. Prell, and A. T. Vella. 2000. Danger and
OX40 receptor signaling synergize to enhance memory T cell survival by inhib-
iting peripheral deletion. J. Immunol. 164: 107–112.
7. So, T., J. Song, K. Sugie, A. Altman, and M. Croft. 2006. Signals from OX40
regulate nuclear factor of activated T cells c1 and T cell helper 2 lineage com-
mitment. Proc. Natl. Acad. Sci. USA 103: 3740–3745.
8. Kopf, M., C. Ruedl, N. Schmitz, A. Gallimore, K. Lefrang, B. Ecabert,
B. Odermatt, and M. F. Bachmann. 1999. OX40-deficient mice are defective in
Th cell proliferation but are competent in generating B cell and CTL responses
after virus infection. Immunity 11: 699–708.
9. Chen, A. I., A. J. McAdam, J. E. Buhlmann, S. Scott, M. L. Lupher, Jr.,
E. A. Greenfield, P. R. Baum, W. C. Fanslow, D. M. Calderhead, G. J. Freeman,
and A. H. Sharpe. 1999. Ox40-ligand has a critical costimulatory role in dendritic
cell:T cell interactions. Immunity 11: 689–698.
10. Brocker, T., A. Gulbranson-Judge, S. Flynn, M. Riedinger, C. Raykundalia, and
P. Lane. 1999. CD4 T cell traffic control: in vivo evidence that ligation of OX40
on CD4 T cells by OX40-ligand expressed on dendritic cells leads to the accu-
mulation of CD4 T cells in B follicles. Eur. J. Immunol. 29: 1610–1616.
11. Roos, A., E. J. Schilder-Tol, J. J. Weening, and J. Aten. 1998. Strong expression
of CD134 (OX40), a member of the TNF receptor family, in a T helper 2-type
cytokine environment. J. Leukocyte Biol. 64: 503–510.
1766 REGULATION OF OX40 GENE EXPRESSION IN T CELLS
12. Rogers, P. R., J. Song, I. Gramaglia, N. Killeen, and M. Croft. 2001. OX40 Download full-text
promotes Bcl-xLand Bcl-2 expression and is essential for long-term survival of
CD4 T cells. Immunity 15: 445–455.
13. Song, J., S. Salek-Ardakani, P. R. Rogers, M. Cheng, L. Van Parijs, and M. Croft.
2004. The costimulation-regulated duration of PKB activation controls T cell
longevity. Nat. Immunol. 5: 150–158.
14. Kawamata, S., T. Hori, A. Imura, A. Takaori-Kondo, and T. Uchiyama. 1998.
Activation of OX40 signal transduction pathways leads to tumor necrosis factor
receptor-associated factor (TRAF) 2- and TRAF5-mediated NF-?B activation.
J. Biol. Chem. 273: 5808–5814.
15. McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach,
M. Collins, and M. C. Byrne. 2002. CD4?CD25?immunoregulatory T cells:
gene expression analysis reveals a functional role for the glucocorticoid-induced
TNF receptor. Immunity 16: 311–323.
16. Takeda, I., S. Ine, N. Killeen, L. C. Ndhlovu, K. Murata, S. Satomi, K. Sugamura,
and N. Ishii. 2004. Distinct roles for the OX40-OX40 ligand interaction in reg-
ulatory and nonregulatory T cells. J. Immunol. 172: 3580–3589.
17. Valzasina, B., C. Guiducci, H. Dislich, N. Killeen, A. D. Weinberg, and
M. P. Colombo. 2005. Triggering of OX40 (CD134) on CD4?CD25?T cells
blocks their inhibitory activity: a novel regulatory role for OX40 and its com-
parison with GITR. Blood 105: 2845–2851.
18. Shimizu, J., S. Yamazaki, T. Takahashi, Y. Ishida, and S. Sakaguchi. 2002. Stim-
ulation of CD25?CD4?regulatory T cells through GITR breaks immunological
self-tolerance. Nat. Immunol. 3: 135–142.
19. Tone, M., Y. Tone, E. Adams, S. F. Yates, M. R. Frewin, S. P. Cobbold, and
H. Waldmann. 2003. Mouse glucocorticoid-induced tumor necrosis factor recep-
tor ligand is costimulatory for T cells. Proc. Natl. Acad. Sci. USA 100:
20. Tone, M., M. J. Powell, Y. Tone, S. A. Thompson, and H. Waldmann. 2000.
IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3.
J. Immunol. 165: 286–291.
21. Frohman, M. A., M. K. Dush, and G. R. Martin. 1988. Rapid production of
full-length cDNAs from rare transcripts: amplification using a single gene-spe-
cific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85: 8998–9002.
22. Tone, M., Y. Tone, J. M. Babik, C. Y. Lin, and H. Waldmann. 2002. The role of
Sp1 and NF-?B in regulating CD40 gene expression. J. Biol. Chem. 277:
23. Barreau, C., L. Paillard, and H. B. Osborne. 2005. AU-rich elements and asso-
ciated factors: are there unifying principles? Nucleic Acids Res. 33: 7138–7150.
24. Chen, C. Y., and A. B. Shyu. 1995. AU-rich elements: characterization and im-
portance in mRNA degradation. Trends Biochem. Sci. 20: 465–470.
25. Powell, M. J., S. A. Thompson, Y. Tone, H. Waldmann, and M. Tone. 2000.
Posttranscriptional regulation of IL-10 gene expression through sequences in the
3?-untranslated region. J. Immunol. 165: 292–296.
26. Hagen, G., S. Muller, M. Beato, and G. Suske. 1994. Sp1-mediated transcrip-
tional activation is repressed by Sp3. EMBO J. 13: 3843–3851.
27. Badran, B. M., S. M. Wolinsky, A. Burny, and K. E. Willard-Gallo. 2002. Iden-
tification of three NFAT binding motifs in the 5?-upstream region of the human
CD3? gene that differentially bind NFATc1, NFATc2, and NF-?B p50. J. Biol.
Chem. 277: 47136–47148.
28. de Lumley, M., D. J. Hart, M. A. Cooper, S. Symeonides, and J. M. Blackburn.
2004. A biophysical characterisation of factors controlling dimerisation and se-
lectivity in the NF-?B and NFAT families. J. Mol. Biol. 339: 1059–1075.
29. Giffin, M. J., J. C. Stroud, D. L. Bates, K. D. von Koenig, J. Hardin, and L. Chen.
2003. Structure of NFAT1 bound as a dimer to the HIV-1 LTR ?B element. Nat.
Struct. Biol. 10: 800–806.
30. Jin, L., P. Sliz, L. Chen, F. Macian, A. Rao, P. G. Hogan, and S. C. Harrison.
2003. An asymmetric NFAT1 dimer on a pseudo-palindromic ?B-like DNA site.
Nat. Struct. Biol. 10: 807–811.
31. Wolfe, S. A., P. Zhou, V. Dotsch, L. Chen, A. You, S. N. Ho, G. R. Crabtree,
G. Wagner, and G. L. Verdine. 1997. Unusual Rel-like architecture in the DNA-
binding domain of the transcription factor NFATc. Nature 385: 172–176.
32. Sheppard, K. A., D. W. Rose, Z. K. Haque, R. Kurokawa, E. McInerney,
S. Westin, D. Thanos, M. G. Rosenfeld, C. K. Glass, and T. Collins. 1999.
Transcriptional activation by NF-?B requires multiple coactivators. Mol. Cell
Biol. 19: 6367–6378.
33. Zhong, H., R. E. Voll, and S. Ghosh. 1998. Phosphorylation of NF-?B p65 by
PKA stimulates transcriptional activity by promoting a novel bivalent interaction
with the coactivator CBP/p300. Mol. Cell 1: 661–671.
34. Zhong, H., M. J. May, E. Jimi, and S. Ghosh. 2002. The phosphorylation status
of nuclear NF-?B determines its association with CBP/p300 or HDAC-1. Mol.
Cell 9: 625–636.
35. Walker, L. S., A. Gulbranson-Judge, S. Flynn, T. Brocker, C. Raykundalia,
M. Goodall, R. Forster, M. Lipp, and P. Lane. 1999. Compromised OX40 func-
tion in CD28-deficient mice is linked with failure to develop CXC chemokine
receptor 5-positive CD4 cells and germinal centers. J. Exp. Med. 190:
36. Toennies, H. M., J. M. Green, and R. H. Arch. 2004. Expression of CD30 and
Ox40 on T lymphocyte subsets is controlled by distinct regulatory mechanisms.
J. Leukocyte Biol. 75: 350–357.
37. Hilton, T. L., Y. Li, E. L. Dunphy, and E. H. Wang. 2005. TAF1 histone acetyl-
transferase activity in Sp1 activation of the cyclin D1 promoter. Mol. Cell Biol.
38. Hung, J. J., Y. T. Wang, and W. C. Chang. 2006. Sp1 deacetylation induced by
phorbol ester recruits p300 to activate 12(S)-lipoxygenase gene transcription.
Mol. Cell Biol. 26: 1770–1785.
39. Lee, J. S., K. M. Galvin, R. H. See, R. Eckner, D. Livingston, E. Moran, and
Y. Shi. 1995. Relief of YY1 transcriptional repression by adenovirus E1A is
mediated by E1A-associated protein p300. Genes Dev. 9: 1188–1198.
40. Yang, W. M., C. Inouye, Y. Zeng, D. Bearss, and E. Seto. 1996. Transcriptional
repression by YY1 is mediated by interaction with a mammalian homolog of the
yeast global regulator RPD3. Proc. Natl. Acad. Sci. USA 93: 12845–12850.
41. Yang, W. M., Y. L. Yao, J. M. Sun, J. R. Davie, and E. Seto. 1997. Isolation and
characterization of cDNAs corresponding to an additional member of the human
histone deacetylase gene family. J. Biol. Chem. 272: 28001–28007.
42. Gordon, S., G. Akopyan, H. Garban, and B. Bonavida. 2006. Transcription factor
YY1: structure, function, and therapeutic implications in cancer biology. Onco-
gene 25: 1125–1142.
43. Lee, J. S., K. M. Galvin, and Y. Shi. 1993. Evidence for physical interaction
between the zinc-finger transcription factors YY1 and Sp1. Proc. Natl. Acad. Sci.
USA 90: 6145–6149.
44. Seto, E., B. Lewis, and T. Shenk. 1993. Interaction between transcription factors
Sp1 and YY1. Nature 365: 462–464.
1767 The Journal of Immunology