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J. Biol. Chem., Vol. 277, Issue 9, 7386-7395, March 1, 2002
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§,
,,,,**, and
From the Department of Immunology, University of
Washington School of Medicine, Seattle, Washington 98195, ¶ Division of Rheumatology, Children's Hospital of Philadelphia,
Philadelphia, Pennsylvania 19104, and the Program in Immunology
and Department of Pediatrics, Stanford University School of Medicine,
Stanford, California 94305
Received for publication, October 28, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We observed that the human CD40 ligand (CD40L)
gene 5'-flanking region conferred weak promoter activity in activated
CD4 T cells, suggesting that additional regions are required for
optimal CD40L gene transcription. We therefore examined a 3'-flanking segment of the CD40L gene, which contained a putative NF- CD40 ligand (CD40L),1
also known as CD154, is a member of the tumor necrosis factor (TNF)
ligand superfamily that is transiently expressed on the cell surface of
activated CD4-positive T cells (1). It binds CD40, a TNF receptor
superfamily molecule expressed on B cells, dendritic cells, and
mononuclear phagocytes (2). The phenotype of humans with a genetic
CD40L deficiency reveals that the CD40L/CD40 interaction is critical
for T cell- and B cell-mediated immunity to protein antigens and for
enhanced mononuclear phagocyte activation to certain opportunistic
pathogens, such as Pneumocystis carinii (3).
The molecular events precipitating CD40L expression on activated CD4 T
cells have similarities to those eliciting expression of the cytokines
interleukin (IL)-2 and TNF. Cognate mRNAs for all three proteins
are rapidly expressed de novo upon activation of
antigenically naive human CD4 T cells (4-7). Full T cell activation requires coordinate engagement of the Although the molecular events leading to CD40L expression appear to be
similar to those eliciting expression of other cytokines (TNF and
IL-2), transcriptional regulation of the gene encoding CD40L remains
poorly understood. Previous studies suggest that both the IL-2 and TNF
genes contain enhancer-like segments that include functional
NF- A transcriptional enhancer active in mononuclear phagocyte cells has
also been identified in the 3'-flanking region of the human TNF gene
(18). This region includes a NF- Like the TNF and IL-2 genes, CD40L gene transcription in activated T
cells requires the cooperative binding of NFAT and leucine zipper
proteins to the 5'-flanking region (22-24). A putative NF- Here, we show that the human CD40L gene contains a T cell-specific,
NF- Cells--
Human CD4 T cells isolated from the peripheral blood
of normal adult donors were either used directly (freshly isolated
cells) or primed by in vitro treatment with mitogen
(concanavalin A (Amersham Biosciences, Inc.)) and recombinant human
IL-2 (Proleukin; Promega, Madison, WI) as previously described (28).
Jurkat thymoma cells (J.DNAX subline (29)), Jurkat D1.1 (gift of Dr.
Seth Lederman, Columbia University, New York), 8.1.6 Epstein-Barr
virus-transformed human B cells (30), and U937 human monocytoid cells
(31) were maintained in RPMI 1640 complete medium containing 10% fetal
calf serum (Hyclone Laboratories, Logan UT), 100 units/ml penicillin, 100 µg/ml streptomycin, 20 µg/ml gentamicin, 25 mM
Hepes, and 2 mM L-glutamine.
Real Time PCR Analysis of mRNA--
Total cellular RNA was
isolated (32) from CD4 T cells after 3 h of culture with or
without activating stimuli (1.5 µM ionomycin (Calbiochem)
plus 50 ng/ml PMA (Sigma)). Random hexamer-primed reverse transcription
(2 µg of total RNA/sample) was performed using the reagents contained
in a TaqMan Gold RT-PCR kit (PerkinElmer Life Sciences) following the
manufacturer's instructions. Two µl of each reverse transcription
reaction was submitted to real time quantitative PCR in an ABI Prism
5700 Sequence Detection System (PerkinElmer Life Sciences).
Amplifications were performed in 96-well plates using
oligonucleotide primers for transcript amplification of human
CD40L (sense, 5'-CCAGGTGCTTCGGTGTTTGT-3'; antisense,
5'-ATGGCTCACTTGGCTTGGAT-3') (6), human IL-2 (sense, 5'-AAACTCACCAGGATGC-TCACATT-3'; antisense, 5-'-TGTGGCCTTCTTGGGCA-3') (33), and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (proprietary primers purchased from PerkinElmer Life Sciences). For all
primer sets, the reverse-transcribed samples were amplified in
triplicate using the reagents contained in a SYBR Green PCR Core
Reagents Kit (PerkinElmer Life Sciences). Fluorescence signals were
detected during each of 40 cycles (denaturing 15 s at 95 °C,
annealing/extension 1 min at 60 °C), as determined by binding of
SYBR green to double-stranded DNA products (34). Denaturing curves of
CD40L, IL-2, and GAPDH products, performed at the end of the PCR,
confirmed the homogeneity of the DNA products. A standard curve was
included in each plate, consisting of serial dilutions of a
reverse-transcribed sample of activated adult CD4 T cell RNA amplified
for CD40L, IL-2, and GAPDH transcripts. The cycle threshold (Ct) value
is the calculated PCR cycle at which products first become detectable
and is inversely related to the abundance of transcripts in the initial
RNA sample (34). This value was determined using software included as
part of the ABI Prism 5700 Sequence Detector System, following the
manufacturer's instructions. The Ct values for GAPDH transcripts in
the stimulated and unstimulated RNA samples were used as a control, and
results were normalized to GAPDH. Comparable GAPDH cycle threshold
values indicated that the amount of mRNA reverse transcribed and
potentially amplifiable by PCR was similar in unstimulated and
stimulated samples.
Reporter Gene Plasmids--
The human CD40L gene sequence has
been previously published (35). The 1284-bp 3'-flanking region of the
CD40L gene analyzed in this study is shown in Fig. 1A. Maps
of the firefly luciferase reporter gene constructs, all derived from
the pGL2 basic vector (Promega), are shown in Fig. 1B. To
simplify the nomenclature, these constructs are named based on their
CD40L promoter and 3'-flanking segments, omitting reference to the
intervening luciferase reporter gene. pCD40L was created by subcloning
a 1.3-kb HindIII/HindIII fragment containing the
human CD40L promoter 5'-flanking segment from the construct
gp39-luc (22) immediately upstream of the luciferase
cDNA segment of pGL2-Basic. The pIL-2, pIL-4, and pIL-13 constructs
directed by the 5'-flanking segments of the human IL-2 (0.6-kb), IL-4
(0.6-kb), and IL-13 (1.1-kb) genes, respectively, were similarly
derived from pGL2-Basic, as previously described (22, 28, 36). pSV40,
in which the luciferase cDNA is directed by a 0.2-kb segment of the
SV40 large T antigen promoter, was purchased (pGL2 Promoter Vector;
Promega). The 1284-bp segment of the human CD40L gene's 3'-flanking
region (3'FL) was subcloned into pCD40L at adjacent BamHI
and SalI cloning sites, located 1 kb 3' of the luciferase
gene, creating pCD40L.3'FL (Fig. 2B). Subcloning used a
naturally occurring 5' BamHI site and a 3' SalI site generated by PCR using Vent polymerase (New England
Biolabs) and a human CD40L genomic Site-directed Mutagenesis of the pCD40L.3'FL
Plasmid--
Site-directed mutagenesis was performed using a
commercial kit (QuikChange; Stratagene) to introduce dinucleotide
substitution mutations into the NF- Transient Transfection and Reporter Gene Analysis--
Ten µg
of firefly luciferase reporter gene plasmid DNA in complete RPMI medium
was used for transient transfection by electroporation. The conditions
used for the electroporation of primary CD4 T cells have been described
(22, 28, 37). Jurkat cells (1.0 × 107
cells/condition) were transiently transfected by electroporation at 260 V and 960 microfarads using a Genepulser electroporator (Bio-Rad).
Electroporation conditions for 8.1.6 Epstein-Barr virus-transformed human B cells (2.5 × 107 cells/condition) were 210 V
and 960 microfarads, and for U937 human monocyte-like cells (2.5 × 107 cells/condition), conditions were 250 V and 960 microfarads. Following electroporation, cells were incubated for 20-24
h at 37 °C, harvested, and plated at a density of 1.0 × 106 viable cells/well, in 96-well U bottom microtiter
plates (Corning Glass). Cells were left untreated or stimulated with
ionomycin (1.5 µM; Calbiochem) plus PMA (25 ng/ml; Sigma)
or with the combination of monoclonal antibodies (mAbs) directed
against CD3- Nuclear Protein Extract Preparation and Electromobility Shift
Assays (EMSAs)--
Nuclear protein extracts were prepared as
previously described (29) from CD4 T cells activated by incubation for
2 h with CD3 and CD28 mAb, and their protein concentration was
determined by the Bradford method using a commercial kit (Pierce). For
EMSAs, SDS-PAGE-purified double-stranded oligonucleotides (purchased from Invitrogen) were used as either 32P-labeled probes or
as unlabeled competitors. For each oligonucleotide, only the coding
strand sequence is shown, with the NF- DNase I-hypersensitive Site Mapping--
Techniques used for
purification of nuclei and DNase I digestions were modifications from
Siebenlist et al. (46). Briefly, human thymoma CD4 T-lineage
cells of the D1.1 Jurkat cell line, which constitutively expresses
CD40L (47), were grown in RPMI medium containing 10% fetal bovine
serum. Fifty million cells were harvested and centrifuged three times
in ice-cold Hanks' balanced salts solution. The final pellet was
resuspended in ice-cold nuclear isolation buffer (60 mM
KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl (pH 7.4), 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, and 0.3 M sucrose). Nuclei were then prepared by
lysis with 0.1% Nonidet P-40 and layering onto an equal volume of
nuclear isolation buffer, containing 1.7 M sucrose as a
cushion. Nuclei were centrifuged for 30 min at 30,000 × g in an ultracentrifuge using a SW-40.1 rotor, and the
nuclear pellet was resuspended in 2.5 ml of nuclear isolation buffer, containing 5% glycerol, and aliquoted into 0.5-ml samples. Graded amounts of DNase I (Worthington) were added to each nuclear sample for
3 min at room temperature. Reactions were stopped by the addition of 50 µl of 5% SDS, 100 mM EDTA. Samples were then digested
with proteinase K at 37 °C overnight, and DNA was subsequently
isolated by repeated organic extraction and ethanol precipitation.
DNase I-treated or -untreated DNA samples were digested to completion with BamHI, electrophoresed on 1% agarose gels (10 µg of
sample/lane), and blotted onto Hybond Plus nylon membrane (Amersham
Biosciences) using 20× SSC. A 289-bp 32P-labeled probe,
corresponding to the extreme 3'-end of the 2.2-kb BamHI
fragment that contains the 3'-untranslated region of the human
CD40L gene and downstream sequences, was generated by PCR using primers
5'-ATTTCATCTCCTGCTGCCACC-3' and 5'-GGGCCAATTCAATGCTA-3' derived
from publicly available human genome sequence. Hybridization was
performed in roller bottles using QuickHyb (Stratagene, La Jolla, CA)
solution with 1.25 × 107 cpm of spun column-purified
probe per 10 ml of hybridization solution at 68 °C. Blots were
washed, with the last wash of high stringency (0.05× SSC with 0.1%
SDS at 68 °C for 30 min).
Real Time PCR-based Chromatin Immunoprecipitation (ChIP)
Assays--
ChIP assays were carried out using a commercial kit
following the manufacturer's instructions (Upstate Biotechnology,
Inc., Lake Placid, NY). Briefly, 20 × 106 CD4 T cells
were isolated from the peripheral blood of healthy adult donors and
stimulated for 2 h at 37 °C with CD3 and CD28 mAbs.
Cross-linking with 1% formaldehyde for 15 min at 37 °C was followed
by cell lysis and sonication of DNA into 200-800-base pair fragments.
Proteins linked to DNA were immunoprecipitated with preimmune sera or
antiserum 1141 (reactive with the N terminus of NF- CD40L 5'-Flanking Promoter Activity Is Lower than That of the IL-2
Promoter in Polyclonally Activated CD4 T Cells--
The promoter
activity of the 1.3-kb 5'-flanking segment of the CD40L gene (pCD40L)
or a 0.6-kb 5'-flanking segment of the IL-2 gene (pIL-2) was assessed
using luciferase reporter constructs. After transfection of these
constructs into peripheral blood CD4 T cells, cells were either treated
with medium alone (unstimulated) or polyclonally activated using
ionomycin and PMA at concentrations that yield maximal levels of CD40L
and IL-2 gene transcription and mRNA accumulation (5, 14, 15).
Transfection with the promoterless parent plasmid, pGL2, served as a
negative control and produced barely detectable luciferase expression
(Fig. 2A). Transfection of pCD40L or pIL-2 resulted in
reporter gene activity that was markedly enhanced by CD4 T cell
stimulation with ionomycin and PMA. We consistently observed that
pCD40L-mediated luciferase expression was considerably lower than
reporter gene expression driven by pIL-2 in both freshly isolated T
cells (Fig. 2A) and in CD4 T cells that had previously been
primed in vitro (data not shown). The relatively low
promoter activity of the 1.3-kb 5'-flanking CD40L gene segment does not
appear to be due to strong negative regulatory elements, since we have
previously shown that truncation of the 5' region of this segment does
not increase promoter activity (22).
Polyclonally Activated CD4 T Cells Accumulate Similar or Higher
Levels of CD40L Transcripts than IL-2 Transcripts--
The observation
that pCD40L-mediated reporter gene expression was lower than
pIL-2-driven reporter expression suggested that the level of CD40L
transcripts would also be reduced in comparison with IL-2 mRNA
levels. This assumed that the half-lives of CD40L and IL-2 mRNA are
similar in ionomycin and PMA-stimulated CD4 T cells, which has been
verified.2 To address this,
total RNA was isolated from peripheral blood CD4 T cells 3 h after
culture, with or without ionomycin and PMA stimulation, and
reverse-transcribed RNA was analyzed for CD40L, IL-2, and GAPDH
mRNA levels using real time PCR. A lower Ct value for CD40L and
IL-2 transcripts in stimulated versus unstimulated CD4T
cells indicated that activation increased CD40L and IL-2 gene
transcription in agreement with previous results (Fig. 2B and Refs. 5 and 15). The high Ct value and low level of CD40L and IL-2
transcripts in total RNA from unstimulated cells was not due to
degraded template, since similar levels of GAPDH transcripts were
detected in unstimulated and stimulated T cells (data not shown). In
contrast to the low level of activity of the CD40L promoter in
transient transfection experiments, the Ct values in activated CD4 T
cells indicated that CD40L transcripts were present in equal or greater
amounts than IL-2 transcripts (Fig. 2B). This is unlikely to
be an artifact resulting from more efficient amplification of the CD40L
product, since both CD40L and IL-2 PCR products were 50 bp in size and
were amplified using identical reaction conditions. Together, these
findings suggest that regions of the CD40L gene other than the 1.3-kb
5'-flanking region are necessary for optimal transcription in
polyclonally activated CD4 T cells.
A 1284-bp Segment of the CD40L 3'-Flanking Region Acts as an
Enhancer of CD40L Promoter Activity in CD4 T Cells--
Because the
3'-flanking region of many genes expressed by T lymphocytes, such as
the genes encoding CD2 and IL-4 (49), contain transcriptional
enhancers, we investigated whether the 3'-flanking region of the CD40L
gene might contribute to its transcriptional activation in CD4
T-lineage cells. Examination of the 3'-flanking region DNA sequence
revealed a potential NF-
The 3'-flanking segment enhanced basal CD40L promoter activity.
However, in Jurkat CD4 cells stimulated with ionomycin and PMA, or with
CD3 mAb alone or in combination with CD28 mAb (51), the 3'-flanking
segment greatly increased the CD40L promoter independent of the
orientation of the 3' segment (Fig. 3A). These data suggest that this 3'-flanking segment acted as a classical
(orientation-independent) enhancer (52).
Transformed human CD4 T-lineage cell lines, such as Jurkat,
have been reported to differ from primary human CD4 T cells in their
transcriptional regulation of activation-dependent genes, such as IL-2 (37). Therefore, we tested whether the 3'-flanking segment
could enhance CD40L promoter activity in freshly isolated CD4 T cells.
In the absence of stimulation, luciferase expression was not detected
for any of the CD40L promoter-directed constructs or for the
promoterless pGL2 parent vector in CD4 T cells (Fig. 2A, and data not shown).
However, similar to Jurkat T cells, the 3'-flanking segment increased
CD40L promoter activity in stimulated CD4 T cells in an
orientation-independent manner (Fig.
3B).
The CD40L 3'-Flanking Region Enhances the Activity of Heterologous
Cytokine Promoters in a T Cell-specific Manner--
We next assessed
the specificity of the transcriptional enhancer activity for CD40L
versus other activation-dependent genes in CD4
T-lineage cells by inclusion of the CD40L 3' segment in firefly
luciferase reporter constructs directed by the promoters for cytokine
genes IL-2 and IL-13 and for the large T antigen of the SV40 virus. The
3'-flanking segment of the CD40L gene augmented ionomycin- and
PMA-stimulated activity of the IL-2 and IL-13 promoters in both Jurkat
cells and in freshly isolated peripheral blood mononuclear cells, used
as a source of T cells in this experiment (Fig.
4). In contrast, this segment failed to
enhance SV40 promoter activity in either source of T cells, suggesting
that the 3'-flanking segment is not a generic enhancer of promoters
that are active in CD4 T-lineage cells.
To evaluate the tissue specificity of the transcriptional enhancer
activity of the 3'-flanking region of the CD40L gene, a human
Epstein-Barr virus-transformed B cell line, 8.1.6, or the monocyte-like
cell line, U937, was transfected with pCD40L or pCD40L.3'FL and
evaluated for reporter gene activity. Low amounts of CD40L promoter
activity were detectable in both of these cell lines in the absence of
stimulation, but this increased ~1.5-2-fold in response to ionomycin
and PMA treatment (data not shown). However, there was no significant
increase in ionomycin- and PMA-stimulated transcriptional activity
mediated by the 3'-flanking segment (Fig. 4) in the context of either
the CD40L promoter or the heterologous cytokine promoters. Similar to
Jurkat and nontransformed T cells, the CD40L 3'-segment did not augment
SV40-promoter activity in 8.1.6 or U937 cells. These results suggest
that activation-induced enhancer activity of the 3'-flanking region of
the CD40L gene is relatively T cell-specific.
A DNase I-hypersensitive Site Is Present in the 3'-Flanking Region
of CD40L--
For many genes, key transcriptional regulatory regions
are identified based on their hypersensitivity to digestion with DNase I (53). To determine whether this applied to the CD40L 3'-flanking region, we isolated nuclei from a CD40L-expressing CD4 T-lineage cell
line, subjected these to DNase I treatment, and evaluated the
3'-flanking region for hypersensitive sites using Southern blotting and
an appropriate probe. In the absence of DNase I treatment, a
full-length 2.2-kb BamHI fragment was detected (Fig.
5). Treatment of nuclei with increasing
concentrations of DNase I revealed a 1.4-kb band (Fig. 5,
lane 4), which mapped to the 3'-flanking region
that contained the putative NF- A Functional NF-
We next tested whether the putative NF-
To distinguish the role of these two transcription factor families we
used site-directed mutagenesis. Two full-length mutant enhancers were
created. We compared the enhancement of CD40L promoter activity by the
wild type 1284-bp segment with that conferred by either single
(GTTAATTTCC) or double dinucleotide mutations (GTTAATTTAAC). The single dinucleotide mutation was expected to effectively abrogate binding by most NF- The NF-
The inclusion of specific antiserum reactive with the p50 form of the
NF-
To address whether binding of NF- CD40L gene expression is crucial in both the initiation and
progression of various immune responses, particularly for those involving T cells and B cells, as demonstrated by the severe
immunological consequences of CD40L genetic deficiency. Ligation of
CD40 is an important step for regulation of expression of cytokines,
adhesion molecules, apoptotic mediators, and microbicidal activities by a number of cell types. In addition, CD40L plays a key role in pathogenesis of chronic inflammatory diseases such as autoimmune disorders, graft versus host disease, atherosclerosis, and
neurodegenerative disorders (1, 58-61). Definition of the critical
regulatory regions of the CD40L gene is therefore not only of great
importance in understanding the events invoked in the generation of the
adaptive immune response, but may allow the development of novel
therapies that target this important protein in a variety of diseases.
We found that the 3'-flanking region of the CD40L gene substantially
enhanced the activity of the CD40L promoter in transformed CD4
T-lineage cell lines as well as in primary peripheral blood CD4 T
cells. This DNA segment enhanced transcription in either orientation, indicating that it acted as a classic enhancer (52). The
enhancement of CD40L promoter activity by the 3'-flanking region was
observed with activation using CD3 mAb alone or a combination of CD3
and CD28 mAbs, indicating that it was not unique to pharmacological stimulation. These results suggest that such enhancement is likely to
apply to physiological activation of CD4 T cells following engagement
of the The core 475-bp CD40L enhancer segment contained a decameric sequence,
GGAATTTTCC, that is similar to well characterized and functional
NF- The active DNA-binding forms of NF- Analysis of enhancer activity in peripheral blood CD4 T cells revealed
that most of the activity of the 3'-flanking segment was localized to a
475-bp subregion. This truncation, in which only 75 bp of the
3'-untranslated region remained, actually further increased enhancer
activity compared with the initial 1248-bp segment, suggesting the
presence of inhibitory regions in the 3'-untranslated region (see Figs.
1B and 5A). Determination of whether the
3'-untranslated region also contains transcriptional activation regions
that contribute to enhancer activity will require additional study, but
our preliminary results with additional truncation constructs are
consistent with this
possibility.3
Our results contrast with several well described transcriptional
contexts in which p50 homodimer binding correlates with the inhibition
rather than the activation of transcription (e.g. in the
5'-flanking promoters of the class I MHC (67) and IL-2 genes (68)).
However, p50 homodimers have been shown in other contexts to act as
transcriptional activators, such as in the induction of transcription
of the long terminal repeat promoter of the human immunodeficiency
virus, type 1 (69). Although p50 lacks the Rel homology domain found in
p65, c-Rel, and RelB (65), which associates with co-activator proteins
(70), p50 has been shown to contain other domains that mediate
transcriptional activation (71, 72). p50 can also associate with other
non-NF- Both basal and activation-induced transcription were increased by the
3'-flanking enhancer in Jurkat cells, while in primary cells, such as
purified T cells or unfractionated peripheral blood mononuclear cells,
enhancer function was strictly activation-dependent. Since
CD40L mRNA and transcription by primary T cells are usually undetectable in the absence of activation (6, 7, 14, 15), these results
suggest that the transcriptional regulatory environment of Jurkat cells
may not fully replicate the normal inhibition of CD40L gene
transcription that occurs in the absence of T cell activation. Further,
although selected Jurkat cell lines have been identified that
constitutively express CD40L at high levels (58), we are unaware of any
Jurkat or other transformed T-lineage cell lines in which most CD40L
gene expression is regulated in a physiological,
activation-dependent manner. For these reasons, as well as
previous studies documenting substantial differences between Jurkat
cells and primary T cells in the transcriptional regulation of the IL-2
gene (37), we relied on primary circulating leukocyte populations
containing T cells to further define regions of the CD40L 3' flanking
segment that were necessary for enhancer activity.
Studies of the IL-2 gene as well as the genes encoding other proteins,
such as granulocyte-macrophage colony-stimulating factor and TNF, have
shown that CD28 engagement increases both the rate of cytokine gene
transcription and cytokine mRNA stability, resulting in marked
increases in cytokine production (75, 76). Our data using reporter gene
constructs is consistent with CD28 engagement having a modest effect on
CD40L gene transcription but does not exclude the possibility that
CD40L mRNA stability may also be enhanced in this context. The
modest increase in CD40L promoter activity that occurred with the
combination of CD3 and CD28 mAbs compared with CD3 mAb alone is also
consistent with previous reports finding only a significant but modest
increase in CD40L surface expression by CD4 T cells achieved by the
combination of CD3 and CD28 engagement (77). These results also suggest
that the 5'-flanking region of the CD40L gene contains a functional
CD28 response element, as recently reported (27), but does not rule out
the possibility that the 3'-flanking enhancer may also contain such a
cis-element with such responsiveness.
There is precedence both for 3'-flanking enhancers of genes expressed
by T lymphocytes, such as the CD2 protein (45) and IL-4 (49), and for
3'-flanking region enhancers utilizing NF- Our results suggest that the 3'-flanking region of other
activation-dependent genes expressed by T cells, such as other
members of the TNF ligand superfamily, could play a role in
transcriptional activation. Interestingly, TNF has been reported (18)
to contain a 3'-flanking transcriptional enhancer that is active in
monocyte lineage cells stimulated with lipopolysaccharide or the
exotoxin, toxic shock syndrome toxin-1. Like the CD40L enhancer, this
TNF enhancer segment includes a NF- Finally, the observation that a relatively small region of the CD40L
gene 3'-flanking region acts in a relatively T cell-specific and
activation-specific manner may have usefulness as a means to augment
activation-dependent transcription by T cells in a number
of contexts. Such an approach might be considered for gene therapy
treatment of inherited CD40L deficiency (3, 81) or other immune
deficient states in which expression of TNF ligands or hematopoietin
cytokines by T cells is reduced, such as during early postnatal life
(7). However, the importance of maintaining the normal physiologic
pattern of CD40L gene expression in such therapy is suggested by the
development of T cell lymphomas in mice following the administration of
retroviruses in which CD40L expression is directed by a constitutively
active promoter (62). Therefore, it will be of interest to determine
whether including authentic transcriptional regulatory elements, such
as the CD40L 3'-flanking enhancer region, in gene expression vectors
results in higher levels of T cell activation-dependent
expression of CD40L in vivo.
B/Rel cis-element, for its ability to enhance CD40L promoter
function. This segment augmented CD40L promoter activity in an
orientation-independent manner in CD4 T-lineage cells but not in human
B cell or monocyte cell lines. Mapping of CD4 T-lineage cell nuclei
identified a DNase I-hypersensitive site in the flanking region near
the NF-B/Rel sequence, suggesting a transcriptional regulatory role.
This was further supported by truncation analysis and site-directed
mutagenesis, which indicated that the CD40L 3'-flanking NF-B/Rel
cis-element was critical for enhancer function.
Electrophoretic mobility shift assays showed that the
cis-element preferentially bound the p50 form of the
NF-B1 gene contained in human T cell nuclear protein extracts. This
binding also appeared to occur in vivo in CD4 T cells based
on chromatin immunoprecipitation assays using NF-B p50-specific
antiserum. Together, these results suggest that the CD40L gene
3'-flanking region acts as a T cell-specific classical transcriptional
enhancer by a NF-B p50-dependent mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-T cell receptor-CD3
complex and the costimulatory molecule CD28, leading to an increased
concentration of free intracellular calcium,
[Ca2+]i, and the activation of small GTP-binding
proteins, such as Ras, and protein kinase C isoenzymes (8, 9).
These events lead to an increase in the activity of transcription
factors implicated in cytokine and TNF ligand gene transcription
including NF-B/Rel, nuclear factor of activated T cells (NFAT), and
leucine zipper (Fos/Jun, CREB/ATF, and Maf) families of proteins
(9-12). T cell activation has been mimicked through direct
intracellular activation of [Ca2+]i- and
Ras/protein kinase C-dependent signaling pathways by the
combination of calcium ionophore, such as ionomycin, and phorbol
esters, such as phorbol-12-myristate 13-acetate (PMA) (13). These
combined stimuli potently induce de novo transcription of
the genes encoding IL-2, TNF, and CD40L (4-7, 14-16).
B/Rel cis-activating elements important for their
transcriptional activation. The 5'-flanking region of the human IL-2
gene consists of an inner core promoter immediately upstream of the 5'
transcription start site and an adjacent 275-bp segment, located
between bp 
52 and 326 with respect to the transcription start site.
This adjacent region behaves as a classic enhancer (i.e. it
increases the activity of the core IL-2 promoter and does so in an
orientation-independent manner) (17). This enhancer contains most of
the cis-activating elements known to be required for optimal
IL-2 gene transcription, including binding sites for NF-B/Rel, NFAT,
and leucine zipper proteins (11, 12).
B/Rel binding site that may
contribute to increased TNF promoter activity (19). Currently, it is
not known if this TNF 3'-flanking enhancer segment is active in
T-lineage cells. Multiple putative NF-B/Rel cis-elements have been identified in the TNF gene's 5'-flanking region and appear
to be important for TNF expression by mononuclear phagocytes (20).
However, in contrast to the NF-B site of the IL-2 gene, the
NF-B/Rel elements in the TNF promoter do not appear to regulate gene
expression in T cells (21). Rather, the concerted binding of NFAT and
leucine zipper proteins to the 5'-flanking region of the TNF gene
appears to be essential for expression in activated CD4 T cells
(21).
B/Rel cis-element, GGGATTTCCA, has been identified in the
5'-flanking region of the CD40L gene based on sequence analysis (25)
and has recently been reported to enhance promoter activity, possibly by binding NF-B p65 (26). Interestingly, a CD28 response element has
also been identified in the CD40L promoter, identifying a role for CD28
engagement in the regulation of the CD40L promoter in CD4 T-lineage
cells (27). However, no enhancer elements contributing to CD40L gene
transcription by T cells or other cells have been described.
B/Rel protein-dependent, classical enhancer in its 3'-flanking region that may be important in the regulation of CD40L transcription.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage clone as template.
This 1284-bp segment begins 103 bp 3' of the CD40L termination codon
and ends 401 bp after the final residue of exon 5 (Fig. 1, A
and B (35)). Constructs containing the entire 1284-bp
3'-flanking segment or truncations of this region are numbered using
the first residue of the BamHI site as position 1. A similar
approach was used to produce a construct containing the 3' segment in
the reverse orientation (pCD40L.R3'FL). An endogenous PstI
site was utilized to generate truncations of the full-length segment.
Both the 5' (BamHI/PstI site (bp 1-808)) and the
3' (PstI/SalI site (bp 809-1284)) fragments were
subcloned into the pBS/KS cloning vector (Stratagene). This was
followed by secondary subcloning into pCD40L using BamHI and a polylinker-derived SalI site (5' fragment) or a
polylinker-derived BamHI and SalI site. The
construct containing the 5' fragment (pCD40L.3'FL:1-808) or the 3'
fragment (pCD40L.3'FL.809-1284) retained their original orientation
with respect to the CD40L promoter. The 1284-bp CD40L 3'-flanking
segment was also subcloned at the BamHI/SalI site
3' of the luciferase cDNA segment in pIL-2, pIL-13, and pSV40
constructs to create pIL-2.3'FL, pIL-13.3'FL, and pSV40.3'FL,
respectively. All PCR generated segments were sequenced to ensure that
no polymerase-induced mutations occurred. In some experiments,
transfection efficiency was assessed by co-transfection of a
Renilla luciferase (Promega) reporter gene construct, in which expression was driven by either the human -actin promoter (p-actin-RL, a gift of M. Sweetser, University of Washington) or by
a minimal T7 bacteriophage promoter (Promega).
B/Rel site of the
3'-flanking segment of the pCD40L.3'FL from the wild type sequence of
GGGAATTTTCCA to GTTAATTTCCA (pCD40L.3'FL.mut1) or to
GTTAATTTAAC (pCD40L.3'FL.mut2). All mutated
constructs were sequenced on both strands to verify these mutations and
to confirm that no other alterations were introduced.
(mAb 64.1 (38)), 1:500 dilution of sterile ascites and
CD28 (mAb 9.3 (39)), 1:200 dilution of sterile ascites, for 6 h
prior to harvesting. CD3 and CD28 mAbs were purchased from
Bristol-Myers Squibb Co. Cells were harvested and analyzed for firefly
luciferase activity as previously described (28). For Jurkat cell
lysates, a Monolight 1500 luminometer (Analytical Luminescence
Laboratories, Ann Arbor, MI) was used. Primary human CD4 T cells have a
relatively low transfection efficiency following electroporation (40),
so a more sensitive luminometer, Berthold model LB9507, was used. For the data presented, at least one experiment was carried out in which
either 0.1 µg of p-actin-RL was included with transfections of
Jurkat thymoma cells or 0.5 µg of pRL-null was included with transfections of primary human CD4 T cells. Cell lysates isolated from
these cells were assayed sequentially for firefly and
Renilla luciferase activity using the reagents of the Dual
Luciferase Assay Kit (Promega). Renilla activity between
samples varied less than 10% in these experiments. Therefore, the data
for firefly luciferase activity are presented without correction for
transfection efficiency.
B binding sites underlined and
mutated residues indicated in boldface type (see Table I). The
CD40L 3'-flanking NF-B oligonucleotide,
5'-TGGAGGGAATTTTCCCAACC-3' (+941 to +960 bp of the 1284-bp
3'-flanking segment (Fig. 1A)), was radiolabeled using T4
polynucleotide kinase and [
-32P]ATP and then annealed.
Competitors consisted of either the wild type CD40L 3'-flanking NF-B
oligonucleotide and derivatives containing either a single
dinucleotide substitution mutation,
5'-TGGAGTTAATTTTCCCAACC-3' (single mutant), or
two dinucleotide mutations,
5'-TGGAGTTAATTTTAACAACC-3' (double
mutant), or a 5'-flanking CD40L gene oligonucleotide containing a
putative NF-B element, 5'-GGTAGGGATTTCCACAGCTG-3' (bp
1203 to 1187 bp with respect to the transcription start site (22, 25)). As a positive control, an oligonucleotide competitor, 5'-GAGGGGACTTTCCGAG-3', containing the NF-B site
of the enhancer of the murine immunoglobulin (Ig) chain gene
(41), was used. EMSAs were performed following the method of Ortiz
et al. (42), using 1 µg of nuclear protein and 1.0 × 104 cpm of probe per reaction. Where indicated, unlabeled
double-stranded oligonucleotides were preincubated with nuclear protein
prior to the addition of probe. In some experiments, nuclear protein was preincubated with 1.0 µl of supershifting antisera specific for
individual NF-B/Rel family proteins or with a commercial supershifting mAb reactive with NFATc2 (NFAT1, NFATp) (Affinity Bioreagents, Golden, CO). The panel of NF-B/Rel-specific antisera (generously provided by Dr. N. R. Rice, NCI-Frederick Cancer
Research and Development Center) included specific antiserum 1141 (N-terminal region of p50), 1267 (N-terminal region of p52), 1226 (C-terminal region of p65), and 1266 (C-terminal region c-Rel) as well
as preimmune serum. The generation and characterization of these antisera have been described previously (43-45). In preliminary EMSAs,
1.0 µl of antiserum resulted in maximal supershifting of DNA-protein
complexes formed between a probe containing the Ig chain enhancer
NF-B site and protein present in activated T cell nuclear extracts.
B p50) (both
generously provided by Dr. Nancy Rice) using salmon sperm DNA-protein
A-agarose beads. Immunoprecipitated protein-DNA complexes were washed,
eluted, and unlinked, and proteins were then degraded by proteinase K
digestion for 1 h at 45 °C. DNA was
phenol/chloroform-extracted, precipitated with glycogen carrier, and
used as a template for real time PCRs following an approach similar to
that of Christenson et al. (48). Genomic sequence primers encompassing the NF-B/Rel cis-element (902F,
5'-CCTAAAGCTCCCAGCCAGGT-3'; 972R, 5'-AAACATCACAAAGGTTGGGAAAAT-3')
were used to amplify immunoprecipitated DNA as template. Samples
were done in triplicate as described above for the real time PCR
analysis of mRNA levels using the reagents contained in a SYBR
Green PCR Core Reagents kit (PerkinElmer Life Sciences). Fluorescence
signals were detected during each of 40 cycles (denaturing for 15 s at 95 °C, annealing/extension for 1 min at 60 °C) as determined
by binding of SYBR green to double-stranded DNA products.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B/Rel cis-element, GGAATTTTCCC,
at bp 946-956 with respect to the BamHI site of exon 5, which matched 9 of 10 residues of the ideal NF-B/Rel consensus sequence, GGGRNYYYCC (50) (see Table I).
Since NF-B/Rel family proteins have been implicated in the
activation-dependent expression of several T cell-derived
cytokines, including IL-2 and IL-3 (50), we tested the ability of a
1284-bp segment containing this putative NF-B/Rel binding site to
modulate the activity of the CD40L promoter. This segment was subcloned
downstream (~1.0 kb) of a luciferase reporter gene cDNA segment
driven by the human CD40L proximal promoter (pCD40L), maintaining the
normal orientation between the 3'-flanking segment and pCD40L to yield
pCD40L.3'FL (Fig. 1B). In
order to evaluate orientation dependence, a reporter gene construct
with the 3' segment subcloned in the reverse direction (pCD40L.R3'FL)
was generated.
Comparison of NF-B/Rel sites described in this study
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Fig. 1.
Reporter gene constructs used to define
enhancer activity of the human CD40L gene 3'-flanking region.
A, sequence of the entire 1284-bp segment of the human CD40L
3'-flanking region (36) used in constructs, with the natural
BamHI site (bp 1-6) in italic type, the
NF- B/Rel cis-element (bp 946-954) underlined,
and the 3'-untranslated region of the fifth exon indicated in
boldface type. B, design of the
reporter gene constructs. The entire 1284-bp segment was included in
CD40L.3'FL and in reverse orientation in the CD40L.R3'FL construct.
pCD40L.3'FL/1-808 and pCD40L.3'FL/809-1284 are CD40L promoter-directed
luciferase constructs that contain either bp 1-808 or 809-1284,
respectively, of the 3'-flanking CD40L segment.
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Fig. 2.
CD40L promoter activity but not transcript
abundance is substantially lower than that for the IL-2 gene in
activated CD4 T cells. A, luciferase activity in CD4 T
cells transiently transfected with pGL2-based reporter gene constructs
directed by either no promoter segment (pGL2), the 1.3 kb of the
5'-flanking region of the human CD40L gene (pCD40L), or 0.6 kb of the
human IL-2 gene 5'-flanking region (pIL-2). Following transfection, CD4
T cells were either stimulated with ionomycin and PMA
(Ionomycin + PMA) or with medium alone
(Unstimulated) for 6 h prior to assaying for luciferase
activity. B, CD40L and IL-2 mRNA levels, normalized
against GAPDH, in CD4 T cells after 3 h of stimulation with
ionomycin and PMA as determined by real time PCR. For each transcript
type, the accumulation of product with each cycle of PCR and the Ct
value are shown.
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Fig. 3.
The 1.3-kb CD40L 3'-flanking segment enhances
CD40L promoter activity in CD4 T-lineage cells in an
orientation-independent manner. Cells were transiently transfected
with luciferase reporter gene constructs driven either by the CD40L
5'-flanking promoter alone (pCD40L) or in conjunction with the 1284-bp
3'-flanking segment, in either its normal (pCD40L.3'FL) or reverse
orientation (pCD40L.R3'FL) with respect to the promoter. A,
luciferase activity in transfected Jurkat thymoma cells after 6 h
of either no stimulation or stimulation with either ionomycin and PMA
or with the combination of CD3- and CD28 mAbs. B,
luciferase activity in transfected primed CD4 T cells after stimulation
with ionomycin and PMA for 6 h. The results shown were normalized
for transfection efficiency based on the activity of co-transfected
Renilla luciferase reporter gene constructs as described
under "Experimental Procedures" and are representative of at least
two independent experiments.
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Fig. 4.
The CD40L 3'-flanking segment enhances the
activity of the human CD40L, IL-2, and IL-13 promoters but not the SV40
promoter in a T cell-specific manner. Reporter gene constructs
directed by the 5'-flanking promoters for the CD40L, IL-2, or IL-13
gene, alone, or in conjunction with the 1284-bp 3'-flanking region of
the CD40L gene, were electroporated into Jurkat thymoma cells, adult
peripheral blood mononuclear cells (PBMC), a human B cell
line, or human monocyte-like U937 cells and were activated with
ionomycin and PMA for 6 h. For each promoter, the results are
expressed as the -fold induction in luciferase activity obtained in the
presence of the CD40L 3'-flanking region relative to that obtained with
the promoter alone. The results shown are representative of three
independent experiments.
B/Rel site. These findings indicated
that the NF-B/Rel site was contained within a region having an open
chromatin configuration, consistent with this region playing a role in
transcriptional regulation of the CD40L gene.
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Fig. 5.
CD4 T-lineage cells contain a DNase
I-hypersensitive site in the 3'-flanking region of the CD40L gene that
is in proximity to the NF- B/Rel element.
The Jurkat D1.1 cell line, which constitutively expresses CD40L, was
used in DNase I hypersensitivity site assays. Molecular weight markers
are indicated to the left of the gel. Lanes
1-4 correspond to 0, 0.86, 1.6, and 3.5 µg/ml DNase I,
respectively. A map depicting the BamHI sites, probe site,
the 2.2-kb band corresponding to full-length BamHI fragment,
and the 1.4-kb band that corresponds to the expected size of cleavage
in or near the NF-B/Rel site are shown.
B/Rel Site in the 3'-Flanking Region Is Required
for Enhancement of CD40L Promoter Activity in CD4 T Cells--
To
determine whether the putative NF-B/Rel site was important for
3'-flanking region enhancement of CD40L transcription, we compared the
enhancement of the CD40L promoter by truncations of the 1284-bp segment
that either lacked (bp 1-809 segment) or contained (bp 809-1284) this
site (Fig. 1B). While the presence of the 1-809 bp segment
did not consistently increase CD40L promoter activity by CD4 T cells
(Fig. 6A), additional
truncations within this region suggested the presence of both positive
and negative regulatory elements capable of affecting CD40L promoter
activity (data not shown). In contrast, the 809-1284-bp segment was
sufficient to substantially augment CD40L promoter activity in CD4 T
cells, indicating that it contained an important transcriptional
activation element.
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Fig. 6.
The NF- B/Rel
binding site is a key element for CD40L 3'-flanking enhancer activity
in nontransformed T cells. A, luciferase activity
directed by either the CD40L promoter alone (pCD40L) or by the CD40L
promoter in conjunction with either bp 1-1284 (pCD40L.3'FL),
1-808 (pCD40L.3'FL/1-808), or 809-1284
(pCD40L.3'FL/809-1284) of the CD40L 3'-flanking segment.
B, luciferase activity directed by pCD40L, pCD40L.3'FL, or a
pCD40L.3'FL construct containing either one (pCD40L.3'FL.mut1) or two
(pCD40L.3'FL.mut2) dinucleotide substitution mutations of the
canonical NF-B/Rel site (see "Experimental Procedures").
B/Rel-binding segment of the
1284-bp 3'-flanking region was essential for enhancement of CD40L
promoter activity. This segment AGGGAATTTTCCC includes not only a 9 of 10 match (underlined) for the consensus NF-B decamer-binding site (GGGRNTYYCC (50)) but also contains a 7 of
7 match (italic type) on the noncoding strand for an NFAT binding site
(HGGAAAA (54)). This finding could be of functional importance, since
there are examples where NF-B and NFAT transcription factors can
independently contribute to transcriptional activation by binding to
such NF-B/NFAT composite cis-elements (e.g.
the human immunodeficiency virus-1 long terminal repeat promoter (55, 56).
B/Rel proteins, based on the results of in vitro studies using
recombinant NF-B/Rel proteins to define ideal binding sites (57),
while leaving the NFAT binding site intact. In contrast, the double mutant was expected to abrogate both NF-B/Rel and NFAT binding (54,
57). We found that the single dinucleotide mutation resulted in more
than 50% loss of the enhancement of CD40L activity by the 1284-bp
3'-flanking region in CD4 T cells (Fig. 6B). On the other
hand, the double dinucleotide mutation led to only a slight additional
decrease in enhancement, suggesting that NFAT binding at this site made
a minor contribution to enhancer activity. These results suggested that
NF-B/Rel proteins, rather than NFAT, were crucial for the enhancer
activity mediated by the NF-B/Rel site.
B/Rel Site Binds CD4 T Cell p50 NF-B1 in Vitro and in
Vivo--
Using EMSAs, we found that an oligonucleotide probe
containing the NF-B/Rel site of the CD40L 3'-flanking region formed
a complex with nuclear protein from CD3 and CD28 mAb-activated CD4 T
cells (Fig. 7A,
lane 1). Formation of this complex was inhibited by either a 20- or 200-fold molar excess of unlabeled oligonucleotide (Fig. 7A, lanes 2 and 3,
Self). In contrast, a 20- or 200-fold molar excess of
oligonucleotides containing mutations of the NF-B/Rel site were
ineffective in competing complex (Fig. 7A, lanes
4-7). This indicated that complex formation at the
NF-B/Rel site with nuclear protein correlated with the ability of
this site to contribute to CD40L enhancer activity. The specific
complex was also effectively competed by an oligonucleotide containing
the canonical NF-B/Rel site of the murine immunoglobulin chain,
indicating that it might bind NF-B/Rel proteins (Fig. 7A,
lanes 8 and 9). In contrast, the
putative NF-B/Rel element located in the 5'-flanking region of the
human CD40L gene (24) was ineffective as a competitor (Fig.
7B, lanes 1-4), suggesting that it
did not specifically bind the same proteins as the 3'-flanking
site.
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Fig. 7.
The NF- B/Rel element
of the CD40L 3'-flanking segment and T cell nuclear protein form a
specific complex that contains p50. EMSAs in which nuclear protein
from CD3 and CD28 mAb-stimulated CD4 T cells was reacted with the CD40L
3'FL NF-B/Rel probe are shown. A, nuclear protein was
preincubated with either nothing (lane 1) or
unlabeled oligonucleotides (see Table I) containing either the wild
type CD40L 3'-flanking NF-B/Rel site (Self;
lanes 2 and 3), the single
dinucleotide mutant CD40L 3'-flanking NF-B/Rel site (SM;
lanes 4 and 5), the double
dinucleotide mutant CD40L 3'-flanking NF-B/Rel site (DM;
lanes 6 and 7), or the wild type
NF-B/Rel site of the murine Ig chain gene enhancer
(Ig; lanes 8 and 9).
Competing unlabeled oligonucleotides were added at either a 20-fold
(lanes 2, 4, 6, and
8) or a 200-fold molar excess (lanes
3, 5, 7, and 9) with
respect to the labeled probe. B, nuclear protein was
preincubated with either nothing (lane 1), the
wild type CD40L 3'-flanking NF-B/Rel site (self;
lane 2), or the putative NF-B/Rel site of the
CD40L 5'-flanking region (5' FL; lanes
3 and 4). Competing unlabeled oligonucleotides
were added at either a 20-fold (lanes 2 and
3) or a 200-fold molar excess (lanes
4) with respect to the probe. In lanes
5-10, nuclear protein was preincubated with either
preimmune rabbit antiserum (PI; lane
5) or postimmune antiserum reactive with either p50
(lane 6), the p52 product of NF-B2
(lane 7), p65 (lane 8),
c-Rel (lane 9), or a NFATc2 mAb (lane
10) prior to the addition of probe. The positions of free
probe, the specific protein-probe complex, and the complex supershifted
by the addition of p50-specific antiserum are indicated.
B1 gene product resulted in a virtually complete supershift of
the specific complex formed with the CD40L 3'-flanking NF-B
oligonucleotide, suggesting that p50 was a major component (Fig.
7B, lanes 5 and 6). In
contrast, antisera to p52 (NF-B2), p65 (RelA), c-Rel, or NFAT1, the
major NFAT protein contained in these extracts (28), had little or no
effect on the formation or the mobility of the complex (Fig.
7B, lanes 7-10). We also failed to
obtain supershifts using a variety of commercial antisera or mAbs
specific for p65 and c-Rel. Together, these results indicated that p50
homodimers were likely to be the predominant NF-B/Rel species in the
DNA-binding complex and that the CD40L 3'-flanking NF-B/Rel site was
an important cis-element for the enhancement of CD40L
promoter activity by binding NF-B p50.
B1 p50 to the 3'-flanking
NF-B/Rel site occurred in intact CD4 T cells, ChIP assays were performed. Primary CD4 T cells were briefly stimulated with CD3 and
CD28 mAbs, and, after formaldehyde cross-linkage and sonication, proteins were immunoprecipitated with preimmune antiserum or an antiserum specific for the N-terminal region of NF-B p50. CD40L 3'-flanking region primers encompassing the NF-B/Rel site were then
used to amplify immunoprecipitated DNA in a real time PCR assay. The Ct
value (Ct = 27.6) obtained with NF-B p50 antiserum was
consistently lower than that obtained with preimmune antiserum (Ct = 29). This indicated that the NF-B p50 antiserum specifically immunoprecipitated the CD40L 3' DNA template containing the NF-B/Rel site (Fig. 8) and that NF-B p50 binds
to this site in vivo in human CD4 T cells.
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Fig. 8.
The p50 form of
NF- B1 binds to the 3'-flanking enhancer region
of the CD40L gene in intact CD4 T cells. ChIP assays were
performed using NF-B p50 antiserum versus
preimmune antiserum and CD3 and CD28 mAb-activated CD4 T cells.
Oligonucleotide primers encompassing the enhancer site were used to
amplify immunoprecipitated DNA in real time PCRs. For each ChIP
reaction, the accumulation of PCR products from cycles 20-40 and the
Ct value are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-T cell receptor-CD3 complex by antigenic peptide bound to
class II MHC molecules or by microbe-derived superantigens.
B/Rel cis-elements of other genes (see Table I and
references therein). In addition, a DNase I hypersensitivity site that
corresponded to the NF-B/Rel element within the enhancer was
identified. Consistent with the functional importance of the NF-B/Rel site, an oligonucleotide containing the NF-B/Rel element formed a specific complex with nuclear protein contained in CD4 T
cells, of which NF-B p50 was a prominent component. ChIP assays confirmed the association of NF-B p50 with the 3'-enhancer site. Importantly, the ability of this element to form an NF-B-containing protein complex with CD4 T cells strictly correlated with the ability
of the 3'-flanking region to enhance CD40L promoter activity in this
cell type. In contrast, the presence of an additional mutation in the
3' region of the element, which would be expected to also prevent
binding of NFAT proteins (12), only led to a modest additional decrease
in enhancer activity. This suggests that the binding of NF-B/Rel
proteins, rather than NFAT proteins, is necessary for most of the
enhancer activity of this element in CD4 T cells and is consistent with
our inability to detect NFAT proteins in CD4 T cell nuclear protein
complexes formed with the CD40L 3'-flanking NF-B/Rel element (data
not shown). In support of our findings, NF-B p50 was noted to be
required for the induction of CD40L expression, based on studies using
mice with genetic disruption of the NF-B1 gene (63). However, these
results do not formally exclude the possibility that NFAT proteins may
not act independently but only cooperatively with NF-B/Rel proteins in mediating enhancer activity. While there are clear examples of
composite NF-B/Rel and NFAT cis-elements in which both
transcription factor families independently contribute to
transcriptional activation in T-lineage cells (e.g. the
human immunodeficiency virus long terminal repeat promoter (55, 56) and
the first intron interferon-gamma gene enhancer (64)), we conclude that
this does not appear to apply to the CD40L 3'-flanking enhancer.
B/Rel proteins are dimers, which
may consist of any two members of the NF-B/Rel family, including p50
(a derivative of the NF-B1 gene), p52 (a derivative of the NF-B2
gene), p65, c-Rel, and RelB (50, 57, 65). With the exception of RelB,
all of these NF-B/Rel proteins are expressed by T cells (65). We
found that the p50 product of the NF-B1 gene was the predominant
type of NF-B/Rel protein contained in the nuclear extracts of human
CD4 T cells that bound to the 3'-flanking enhancer site in
vitro and that p50 antisera immunoprecipitated the enhancer region
of the CD40L 3'-flanking segment. No detectable binding of p52,
p65, or c-Rel was found, although the nuclear protein extracts we
employed contain substantial amounts of these proteins (data not
shown). p50 homodimers have a tendency to bind with high affinity to
palindromic NF-B/Rel sites (57), as defined by using in
vitro binding site selection assays (66). It is also interesting
to note that, with the exception of one nucleotide indicated in
italics, the NF-B/Rel element consists of an 11-bp palindromic
sequence, GGGAATTTTCCC, and that a similar 11-bp palindromic
sequence, found in the enhancer of the class I MHC gene (see Table I),
has previously been shown to preferentially bind p50 homodimers (67).
These results, taken together, suggest that p50, probably in the form
of homodimers, enhances CD40L promoter activity by binding to a
3'-flanking cis-activation element.
B/Rel DNA-binding proteins in mediating transcription (65),
such as members of the CCAAT enhancer-binding protein, CREB/ATF, HMG, and SP-1 families, and with the proto-oncogene product,
bcl-3, which can act as a transcriptional co-activator (73).
Therefore, it is plausible that p50 homodimers could contribute to
increased CD40L gene transcription in T-lineage cells. This is
particularly the case, since such homodimers are a major component of
the nuclear extracts of freshly isolated T cells (74), which have a
high capacity to rapidly express the CD40L gene (6, 7).
B/Rel cis-elements (e.g. immunoglobulin chain genes
(78)) in B-lineage cells. However, to the best of our knowledge,
the enhancer of the CD40L gene described here is the first in which a
NF-B/Rel element located in the 3'-flanking region has been
implicated in activation-dependent gene expression by T
cells. This is in contrast to previously reported NF-B/Rel sites
utilized by T cell activation-dependent genes, which have
been identified either in the 5'-flanking promoter (e.g. the
IL-2 (79) and granulocyte-macrophage colony-stimulating factor genes
(80)) or the first intron, such as for the interferon- gene (64). As
discussed above, the 3'-flanking NF-B/Rel site we identified in the
CD40L gene also differs from these other sites in that it appears to
preferentially bind p50 homodimers rather than other NF-B Rel
complexes commonly found in human T cells, such as heterodimers of p50
or p52 with Rel A (p65) or with c-Rel (74).
B/Rel cis-element in
the 3' genomic region immediately flanking the last exon, and this
element may augment lipopolysaccharide-induced TNF transcription by
mononuclear phagocyte lineage cells (19). This suggests the possibility that such NF-B/Rel-dependent 3'-flanking enhancers may
be a previously unappreciated feature of other members of the TNF
ligand superfamily. Whether this 3'-flanking TNF enhancer and its
NF-B/Rel cis-element also have activity in T-lineage
cells is not known but, given our results, is of potential interest.
| |
ACKNOWLEDGEMENTS |
|---|
We thank C. Wilson (University of Washington)
for providing the J.DNAX Jurkat thymoma and U937 cell lines, E. Mellins
(Stanford University) for providing the 8.1.6 B cell line, M. Sweetser
for providing the p-actin-RL plasmid, D. Hollenbaugh
(Bristol-Myers Squibb) for providing a bacteriophage
clone of the human CD40L gene, and Dr. N. R. Rice for providing
the NF-B/Rel-specific antisera.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health (NIH) R01 Grants HD-36291 (to D. B. L.) and DK-35008 (to A. M. K.), a graduate student stipend from NIH Grant CA-09537 (to L. A. S.), NIH Child Health Research Center Grant P30-HH 2815-08 (to R. Q. C. for support of M. B.), an Arthritis Foundation Investigator Award Grant (to R. Q. C.), a postdoctoral fellowship for physicians from the Howard Hughes Medical Institute (to R. Q. C.), and a Walter V. and Idun Y. Berry Fellowship in Children's Health (to A. M. C.).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.
§ Present address: Virginia Mason Research Center, Rm. 130, 1201 9th Ave., Seattle, WA 98101.
** The Shelagh Galligan Professor of Pediatrics.
To whom correspondence should be addressed. Tel.: 650-498-4189;
Fax: 650-498-6077; E-mail: dblewis@leland.stanford.edu.
Published, JBC Papers in Press, December 19, 2001, DOI 10.1074/jbc.M110350200
2 P. Jullien and D. B. Lewis, unpublished observations.
3 L. A. Schubert, R. Q. Cron, and D. B. Lewis, unpublished results.
| |
ABBREVIATIONS |
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The abbreviations used are:
CD40L, CD40 ligand;
TNF, tumor necrosis factor;
IL, interleukin;
NFAT, nuclear
factor of activated T cells;
PMA, phorbol-12-myristate 13-acetate;
GAPDH, glyceraldehyde- 3-phosphate dehydrogenase;
Ct, cycle threshold;
mAb, monoclonal antibody;
EMSA, electrophoretic mobility shift assay;
Ig, immunoglobulin kappa;
ChIP, chromatin immunoprecipitation;
MHC, major histocompatibility complex.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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| 4. | Conlon, K., Osborne, J., Morimoto, C., Ortaldo, J. R., and Young, H. A. (1995) Eur. J. Immunol. 25, 644-648[Medline] [Order article via Infotrieve] |
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