From the Division of Human Immunology, Hanson Centre
for Cancer Research, Institute of Medical and Veterinary Science, Frome
Road, Adelaide, South Australia, 5000 and the ¶ Division of
Biochemistry and Molecular Biology, John Curtin School of Medical
Research, Australian National University,
Canberra, Australian Capital Territory 2601, Australia
Received for publication, October 27, 2000
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ABSTRACT |
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Cold shock domain (CSD) family members have been
shown to play roles in either transcriptional activation or repression
of many genes in various cell types. We have previously shown that CSD
proteins dbpAv and dbpB (also known as YB-1) act to repress granulocyte-macrophage colony-stimulating factor transcription in human
embryonic lung (HEL) fibroblasts via binding to single-stranded DNA
regions across the promoter. Here we show that the same CSD factors are
involved in granulocyte-macrophage colony-stimulating factor
transcriptional activation in Jurkat T cells. Unlike the mechanisms of
CSD repression in HEL fibroblasts, CSD-mediated activation in Jurkat T
cells is not mediated through DNA binding but presumably through
protein-protein interactions via the C terminus of the CSD protein with
transcription factors such as RelA/NF- Cold shock domain (CSD)1
proteins were originally identified in bacteria and have been shown to
be highly conserved throughout evolution from bacteria to humans
(1-3). CSD proteins have three functional domains: an N terminus, the
central CSD, and a C-terminal domain. The N terminus region of the
protein has not been well characterized but has been shown to
contribute to single-stranded DNA binding (4, 5). The highly conserved
central cold shock domain (from which this family of proteins derives
its name) contains an RNP1 motif that is essential for
sequence-specific DNA and RNA binding (5-9) (see Fig.
1C). The C terminus of the
protein has alternating basic and acidic domains and has been
implicated in both nonsequence specific RNA binding and protein-protein
interactions with transcriptional regulators like RelA, ZO-1, TATA
binding protein, NF-Y, YY-1, and AP-2 (10-15) (Fig.
1C). As a whole, the family of CSD proteins has been
reported to bind to double- and single-stranded DNA and RNA and is
involved in transcriptional repression and activation and in mRNA
packaging, transport, localization, masking, stability, and translation
(1-3, 16-20).
B p65. We demonstrate that
Jurkat T cells lack truncated CSD factor subtypes present in HEL
fibroblasts, which raises the possibility that the cellular content of
CSD proteins may determine their final role as activators or repressors
of transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The GM-CSF proximal promoter and CSD factor
dbpB truncation constructs. A, the sequence of the
human GM-CSF proximal promoter is shown. Domain 1 ( 114 to
71) and
domain 2 (
70 to
31) regions are indicated. The binding sites for
many double-stranded DNA binding transcription factors that mediate
GM-CSF expression, including NF-kB, CBF, AP1, ETS/NFAT, and CD28RC, are
underlined (20, 38). Nuclear NF-GMb/c and recombinant CSD
factor binding sites are indicated on the noncoding (
) strand in
domain 1 and the coding (+) strand in domain 2 with shaded
boxes. GM-CSF promoter, luciferase reporter constructs used,
pGMCK-1(2)-TK/Luc, pGM1-Luc, pGM1(mut1)-Luc, pGM2-Luc, and pGM4-Luc are
shown diagrammatically underneath. Numbers indicate distance
from the transcriptional start site, and boxes represent CSD
binding sites. Mutation of the CSD binding site in pGM1(mut1)-Luc is
represented by a box with an X in it, and the altered
sequence is given below it. B, the sequence of the coding
(+) and noncoding (
) wild-type domain 1 (
114 to
79)
oligonucleotides, GM and GM
, are shown (21, 22). Sequences needed for
nuclear NF-GMb/c and recombinant CSD factor binding to the noncoding
strand are indicated by a box. Base changes in CSD binding
sites of the noncoding (
) strand for each mutant oligonucleotide
GMm19, GMm22, and GMm23 are shown (22). The sequence of the
wild-type-coding strand (+) domain 2 GM93+ oligonucleotide (
70 to
31) is given with CSD, CBF, AP1, and ETS/NFAT sites shown (21, 22).
Base changes in the CSD binding sites of the coding (+) strand for each
mutant oligonucleotide GMm95+, GMm103+, and GMm105+ are shown (23).
C, the coding regions of dbpB contained in the expression
plasmids pSGdbpB, pSGdbpB
1, and pSGdbpbB
2 are shown
schematically. The N-terminal, CSD, and C-terminal regions of the
protein are represented, and the numbers represent the amino acid
number. The construct pSGdbpB encodes for full-length dbpB, pSGdbpB
2
(the first 173 amino acids), and pSGdbpbB
1 (the first 47 amino
acids).
An important role of CSD proteins is in transcriptional regulation of genes involved in growth and stress responses (13, 21-28). The CSD proteins dbpB (also known as YB-1) and dbpA have been shown to act as activators of viral genes (15, 29-32) and both as activators and repressors of many cellular genes (13, 14, 25-28, 33-37), including the gene for the hemopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) (21-23).
GM-CSF belongs to a family of growth factors that control survival,
proliferation, and differentiation of cells from the hematopoietic lineage of granulocytes and macrophages (20, 38-41). GM-CSF is expressed by a wide variety of cells including myeloid, mesenchymal, and lymphoid cells in response to stress signals such as those derived
from infection, inflammation, and blood loss (20, 28, 38, 39). GM-CSF
expression is triggered in T cells primarily via T cell receptor and
coreceptor activation and is tightly regulated at the level of
transcription (20, 38). The human GM-CSF proximal promoter can be
conveniently divided into two domains (domain 1, 114 to
71 and
domain 2,
70 to
31), which are both important for GM-CSF regulation
(21-23) (Fig. 1A). The two domains have many binding sites
for transcription factors including: NF-
B/Rel, CBF, AP-1,
ETS/NFAT, and CD28-responsive complex (CD28RC), which all act to
mediate GM-CSF expression (reviewed in Refs. 20 and 38). In addition to
the double-stranded DNA binding transcription factors mentioned above,
we have shown previously that there are four CSD binding sites along
the GM-CSF proximal promoter. There are two single-stranded DNA CSD
binding sites along the noncoding (
) strand of domain 1 and two along
the coding (+) strand of domain 2 (Fig. 1, A and
B) (21-23). We demonstrated that these CSD sites acted as
repressor sites in human embryonic lung (HEL) fibroblasts, that CSD
proteins could bind to these sites, and that overexpression lead to
repression of TNF
-mediated activation of GM-CSF (21-23).
T cells are a major source of GM-CSF in vivo, and we wished
to determine whether CSD proteins played a role in GM-CSF transcription in these cells. We now report that CSD proteins are present in Jurkat T
cells and bind to the defined CSD binding sites along the GM-CSF
promoter. The Jurkat T cell nuclear CSD complexes that form on the
GM-CSF promoter are distinct from those found in HEL fibroblasts. Using
transient transfection assays, we previously showed that the CSD
binding sites acted as repressor elements in HEL fibroblasts. However,
when Jurkat T cells are stimulated with PMA/Ca2+ ionophore
to mimic T cell receptor activation, the CSD factors behave as
coactivators. The coactivation seen does not function through the CSD
binding sites but through potential protein-protein interactions
mediated via the C-terminal domain of CSD proteins.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructs--
Luciferase reporter plasmids were
constructed by inserting oligonucleotides encoding human GM-CSF
promoter fragments into the pXP1 luciferase vector (Promega). The
number after the pGM in each luciferase construct name indicates how
many CSD binding sites are present. The plasmids pGM1-Luc, pGM2-Luc,
and pGM4-Luc were constructed by cloning oligonucleotides (with
HindIII 5' and BamHI 3' ends) spanning regions of
the GM-CSF promoter (65 to +28), (
70 to +28), and (
114 to +28)
into the HindIII/BamHI sites of pXP1 (Fig.
1A). The QuikChangeTM site-directed mutagenesis
kit (Stratagene) was used to mutate the CSD binding site in pGM1-Luc
from 5'-ACCA-3' to 5'-AGGA-3' to create the pGM1-mut1-Luc reporter
plasmid (Fig. 1A). The backbone expression vector pRcCMV was
obtained from Invitrogen. Construction of the RelA expression plasmid
has been described previously (42). Construction of the expression
plasmids pSGdbpAv and pSGdbpB has been described previously (22). The
dbpB deletion plasmid pSGdbpB
1 was created by digesting pSGdbpB with
NarI, which removes the CSD and C-terminal domain of dbpB,
followed by religation (pSGdbpB
1 contains the first 47 amino acids
of dbpB) (Fig. 1C). The dbpB deletion plasmid pSGdbpB
2
was created by digesting pSGdbpB with EcoRI and
Sau3AI, which removes most of the C-terminal domain of dbpB,
and ligating this fragment into an
EcoRI/BglII-digested pSG5, pSGdbpB
2,
containing the first 173 amino acids of dbpB (Fig. 1C).
Oligonucleotides and Probe Preparation--
All oligonucleotides
were purchased from GeneWorks (Adelaide, Australia), and full-length
product was purified from nondenaturing polyacrylamide gels (43).
Single-stranded DNA probes for gel retardation assays were prepared by
end-labeling oligonucleotides with T4 polynucleotide kinase and
[-32P]ATP followed by gel purification.
Preparation of Recombinant Proteins--
Construction of the
bacterial expression construct for CSD protein dbpB (pGEXBT) has been
previously described (23). The Escherichia coli strain
MC1061 transformed with pGEXBT was induced with
isopropyl-1-thio--D-galactopyranoside to produce
recombinant GST-dbpB fusion protein. Recombinant GST-dbpB was purified
on glutathione-Sepharose beads as described by the manufacturer
(Amersham Pharmacia Biotech). The bacterial expression construct for
recombinant RelA was a gift of Dr. Steven Gerondakis, and protein was
prepared according to the procedure described by Dunn et al.
(42).
Gel Retardation Analysis, Competitions, and Antibody Blocking Experiments-- Nuclear extracts were prepared from HUT78 T cells and Jurkat T cells as previously reported (44 45). Gel retardations were carried out using 0.25 ng of single-stranded 32P-labeled oligonucleotide probes (Fig. 1A) in a 10-µl reaction mix of 0.5× TM buffer (21, 22, 45) containing 200 mM KCl, 0.4 µg of poly(dI-dC), and either 1 µg of nuclear extract or 25 ng of recombinant CSD fusion protein (GST-dbpB) or recombinant RelA. Retardation assays using recombinant protein also contained 2 µg of bovine serum albumin. Reactions were incubated at room temperature for 20 min and analyzed on 12% (for nuclear extracts) and 6% (for recombinant proteins) nondenaturing polyacrylamide gels run in 0.5× TBE (46). Competitions with unlabeled single strand oligonucleotides were performed by mixing protein and unlabeled probe followed by the immediate addition of the 32P-labeled probe (22). Antibody blocking experiments were performed by adding protein and antibody and incubating for 5 min at room temperature before adding the 32P-labeled probe. The reaction was then incubated for an additional 20 min at room temperature before being analyzed on polyacrylamide gels.
UV Cross-linking-- For UV cross-linking, nuclear extracts were bound to 32P-labeled single-stranded DNA probes (Fig. 1B), and the complexes were separated on a 12% polyacrylamide gel as described above. The gel was exposed to UV light (340 nm) for 15 min to cross-link bound protein to the DNA. The gel was exposed to x-ray film for 12 h at 4 °C, and the retarded complexes were excised. Protein in the excised bands was analyzed on a 12% SDS-polyacrylamide gel (21, 43).
Antibodies-- The anti-CSD peptide antibody was raised by immunizing rabbits with the peptide (IKKNNPRKYLRSVGD) (dbpB amino acids 89-103) conjugated to keyhole limpet hemocyanin (Imject conjugation kit, Pierce). Immunoglobulins were partially purified via ammonium sulfate precipitation. The specificity of the anti-CSD peptide antibody was verified by dot blot immunoblots against the CSD peptide, peptides to different regions of the dbpB protein, and recombinant glutathione S-transferase (data not shown).
Cell Culture, Transfections, and Luciferase Assays-- The Jurkat T cell line was cultured in RPMI medium containing 10% fetal calf serum supplemented with L-glutamine, penicillin, and gentamycin antibiotics. Electroporation with a Bio-Rad Gene Pulser was used for transfection of Jurkat T cells at 270 V and a capacitance of 960 microfarad. 5 × 106 cells were electroporated in 500 µl of RPMI with 20% fetal calf serum per transfection with 5 µg of reporter plasmid and 10 µg of expression plasmid. Cells were stimulated 24 h post-transfection at a final concentration of 20 ng/ml PMA and 1 µM calcium ionophore (A23187) and, 8 h post-stimulation, were assayed for luciferase activity as described by Osborne et al. (47).
Western Blot and Southwestern Analysis--
Nuclear extracts
from Jurkat and HUT78 T cells were isolated as described
previously (44 45), and 10 µg of protein was separated by 12%
SDS-polyacrylamide gel electrophoresis before transfer to a
nitrocellulose membrane via a Bio-Rad protein transfer apparatus. The
filter was probed with CSD peptide antibody (1:1000 dilution) and
developed with an ECL detection kit according to the recommendations of
the manufacturer (Amersham Pharmacia Biotech). For Southwestern analysis, nuclear extracts were prepared as described above, and the
filter was probed with 32P-labeled oligonucleotides as
described by Silva et al. (48).
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RESULTS |
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Detection of CSD Complexes in Jurkat T Cells--
We have
previously shown that both HEL fibroblasts and HUT78 T cells contain
nuclear CSD proteins and that they bind to the human GM-CSF proximal
promoter (21-23). We have also shown previously that the nuclear CSD
complexes in HEL fibroblasts and HUT78 T cells are the same (21-23).
To determine whether Jurkat T cells contained CSD proteins, Jurkat T
cell nuclear extract was bound in a gel shift assay to single-stranded
oligonucleotides spanning domain 1 and domain 2 of the human proximal
GM-CSF promoter (Fig. 1B) and compared with HUT78 T cell CSD
factor binding on the same oligonucleotides. When HUT78 T cell nuclear
extract was used in a gel shift assay with the noncoding () strand of
domain 1 (GM
), which contains two CSD binding sites, we observed the
previously reported NF-GMb and NF-GMc nuclear complexes (Fig.
2A, lane 2). Specific competitions, cross-linking, and antibody analysis have previously demonstrated that these HUT78 T cell complexes contain CSD
proteins (21-23). In HEL fibroblast and HUT78 T cells, the nuclear
NF-GMb complex migrates in multiple apparent conformational forms
NF-GMb (1) and NF-GMb (2), and we have shown the different conformational forms have identical sequence specificity and protein content (21-23).2 When
Jurkat T cell nuclear extract was used in a gel shift assay we saw
complexes that comigrated with the HUT78 T cell NF-GMb complexes but
could not detect a NF-GMc-like complex (Fig. 2A, lane
1). Using HUT78 T cell nuclear extract in a gel shift with the
coding (+) strand of domain 2 (GM93+), which also contains two CSD
binding sites, we observed three bands making up the NF-GMb complex and
a single NF-GMc band (Fig. 2A, lane 4)
(21-23).2 Jurkat T cell nuclear extract gave two
bands that comigrated with the two slowest-migrating HUT78 T cell
NF-GMb bands, and again, no NF-GMc-like complex was seen (Fig.
2A, lane 3).
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To verify that the Jurkat T cell NF-GMb complexes seen on domains 1 and
2 were authentic CSD-containing NF-GMb complexes, competition assays
were performed. As shown in Fig. 2B, the unlabeled, wild-type, noncoding () oligonucleotide GM
(contains two CSD sites), when used as a competitor, inhibited formation of the Jurkat T
cell NF-GMb complex on both domain 1 (GM
) (Fig. 2B, lane 7) and domain 2 (GM93+) probes (Fig.
3A, lane 7).
Competing with GMm23, domain 1 noncoding strand oligonucleotide, which
has both CSD sites mutated, had no effect on NF-GMb complex formation (Fig. 2B, lanes 3 and 8). As a
positive control, we used an oligonucleotide from the coding (+) strand
of the human papillomavirus 18 enhancer (HPV+), which has been shown to
bind recombinant and nuclear CSD proteins (22, 49). HPV+ was able to
compete the Jurkat T cell NF-GMb complex binding almost as well as the
wild-type GM
oligonucleotide (Fig. 2B, lanes 4 and 9). The NF-GMb complex was not competed by an
oligonucleotide (non-specific) that we and others have shown is
unable to bind nuclear or recombinant CSD proteins (Fig. 2B, lanes 5 and 10) (5, 22, 50).
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As further evidence that the Jurkat T cell NF-GMb complex contained CSD
factors, we made polyclonal antibodies to the peptide (IKKNNPRKYLRSVGD,
dbpB amino acids 89-103) that represents a small region of the highly
conserved CSD domain which is required for single-stranded DNA binding
(7). This region of the CSD domain has identical sequence in both CSD
factors, dbpB and dbpAv. The antibody raised to the CSD peptide was
used in a gel shift with recombinant CSD protein dbpB (23). Increasing
amounts of antibody in a gel shift assay with recombinant dbpB
demonstrated that the CSD peptide antibody could block recombinant CSD
protein dbpB binding to the GM domain1 oligonucleotide (Fig.
2C, top panel (GST-dbpB), compare
lane 1 to lanes 2-4). In the bottom
panel of Fig. 2C we show that the CSD peptide
antibody was also able to block binding of the Jurkat NF-GMb complex,
demonstrating that the Jurkat NF-GMb complex contained CSD factors
(Fig. 2C, bottom panel (Jurkat nuclear extract),
compare lane 1 to lanes 2-4). The addition of
pre-immune antibody (P.I) to the binding reaction mixture at
the maximal concentration of CSD peptide antibody used showed no effect
on mobility or complex formation in either the recombinant dbpB protein
or the Jurkat nuclear extract gel shift (Fig. 2C, compare
lanes 1 and 5 in both panels). The CSD
peptide antibody was also able to block binding of the recombinant dbpB and Jurkat T cell nuclear extract to the coding (+) strand of domain 2 (GM93+) (data not shown).
To determine whether the Jurkat T cell NF-GMb CSD-containing complex
bound to the GM-CSF promoter CSD binding sites, nuclear extract was
used in a gel shift analysis with wild-type domain 1 (GM) and domain
2 (GM93+) oligonucleotides (each contain two CSD binding sites) and
their respective CSD binding site mutants. These CSD binding site
mutations (see Fig. 1B) have been previously described and
characterized for HUT78 T cell and HEL fibroblast CSD binding (21,
23).2 Gel shift analysis using the CSD mutant
oligonucleotides on the noncoding (
) strand of domain 1 were compared
with the binding on the wild type oligonucleotide GM
(Fig.
2D, lane 1). Mutating one CSD site in GMm19
decreased binding significantly (Fig. 2D, lane
2), whereas mutating the other CSD site in GMm21 actually increased CSD binding but also resulted in an altered mobility of the
complex (Fig. 2D, lane 3). Only when both CSD
sites were mutated, in the GMm23 oligonucleotide, was all binding
abolished (Fig. 2D, lane 4). Similar results were
seen when we compared binding of Jurkat T cell nuclear extract on the
wild-type coding (+) strand of domain 2 (GM93+) (Fig.
2D, lane 5) to the CSD mutant oligonucleotides.
Mutating one CSD binding site in GMm95 almost abolished binding (Fig.
2D, lane 6) and mutating the other CSD site in
GMm103 also significantly reduced binding (Fig. 2D,
lane 7). Again, it was not until we mutated both CSD binding
sites, in the oligonucleotide GMm105, that we lost all binding (Fig. 2D, lane 8), indicating that both CSD binding
sites were needed for full binding. These results agree with previous
observations for NF-GMb CSD complex formation in HUT78 T cells
and HEL fibroblasts (21-23).
In summary, Jurkat T cells contain CSD-like proteins that can form a NF-GMb complex on domains 1 and 2 of the GM-CSF proximal promoter. This binding is dependant on the presence of the previously defined CSD binding sites. However, unlike other cells previously examined, Jurkat T cell nuclear extracts do not form a NF-GMc complex on the GM-CSF promoter.
Jurkat T Cells Lack the 22- and 25-kDa Nuclear CSD
Proteins--
To determine the complement of CSD proteins present in
the Jurkat T cell NF-GMb-containing complexes, UV cross-linking
experiments were performed. As reported previously (21, 23), the HUT78 T cell NF-GMb complexes, which form on the noncoding () strand of
domain 1 (GM
oligonucleotide), contain both 42- and 22-kDa proteins
(Fig. 3A, lanes 3 and 4). The NF-GMc
complex, shown to be lacking from Jurkat T cells (Fig. 2A),
contained only a 22-kDa protein (Fig. 3A, lane
5). The 42-kDa protein apparently represents full-length CSD,
whereas published reports where CSD proteins in the 22-kDa protein size
range have been identified indicate that the 22-kDa protein is a
potential splice variant or proteolytic cleavage product of the
full-length protein (10, 23, 31, 51-53). On the noncoding (
) strand
of domain 1 (GM
oligonucleotide), the Jurkat T cell NF-GMb complex
contained only the 42-kDa protein (Fig. 3A, lanes
1 and 2). On the coding (+) strand oligonucleotide of
domain 2 (GM93+) in HUT78 T cells, the NF-GMb complex contained the
42-, 22-, and an extra 25-kDa protein (Fig. 3A, lanes
8-10), whereas the NF-GMc complex contained only the 22- and
25-kDa proteins (Fig. 3A, lane 11). The 22-kDa
protein has been shown to bind the most 5' CSD binding site on domain 2 and the 25-kDa protein, the 3' CSD binding site (21-23). The 25-kDa
protein may represent an additional CSD splice variant or a different
conformational binding of the 22-kDa protein (10, 23, 31, 51-53). In
Jurkat T cells, we could only detect the 42-kDa protein in the NF-GMb complexes (Fig. 3A, lanes 6 and 7),
and the 22- and 25-kDa proteins were lacking.
To determine whether the 22- and 25-kDa proteins were completely lacking from Jurkat T cells or simply unable to bind DNA, Western and Southwestern experiments were performed. Jurkat and HUT78 T cell nuclear extracts and recombinant dbpB-GST fusion protein were fractionated on a Laemmli SDS-polyacrylamide gel electrophoresis gel and transferred to nitrocellulose, resulting in three replicate filter panels. One panel was probed with the CSD peptide antibody in a Western blot. The CSD peptide antibody recognized a single protein of ~75 kDa for recombinant dbpB-GST, which is the predicted size for the dbpB-GST fusion protein (Fig. 3B, lane 3). In the Jurkat and HUT78 T cell nuclear extract lanes the CSD antibody cross-reacted with many proteins (Fig. 3B, lanes 1 and 2). Proteins of the approximate size for the CSD factors identified in UV-cross-linking experiments (42, 25, and 22 kDa) were detected. A strongly reacting band of ~42 kDa was seen in both Jurkat and HUT78 T cell extracts, whereas the smaller CSD factors 25 and 22 kDa were seen only in the HUT78 T cell extract (Fig. 3B, lanes 1 and 2).
To confirm that these were the CSD factors identified by
UV-cross-linking analysis above, Southwestern experiments were
performed. In the Southwestern experiments, one panel was probed with
the wild-type GM-CSF domain 1 GM- oligonucleotide (contains two CSD binding sites; Fig. 3C, lanes 1-3), and the
other panel was probed with the double CSD binding site mutant of the
GM oligonucleotide, GMm23- (Fig. 3C, lanes
4-6). Recombinant dbpB-GST can be seen to bind to the wild-type
GM
probe (Fig. 3C, lane 3) but not to the CSD
mutant GMm23
probe (Fig. 3C, lane 6). Proteins
of the same size, as detected by UV cross-linking the HUT78 T cell
NF-GMb/c complexes (42, 25, and 22 kDa), were detected binding to the
GM
probe in this assay (Fig. 3C, lane 2).
Several other bands were also observed that may represent CSD family
members. In Jurkat T cell extracts, a band of 42 kDa (and a smaller
protein of 39 kDa) was the strongest band observed when the GM
oligonucleotide was used as a probe; of most significance, the 22- and
25-kDa bands were not observed (Fig. 3B, lane 1).
In summary, UV cross-linking the Jurkat T cell NF-GMb complex
identified only a 42-kDa protein, which is also detected here by the
CSD peptide antibody, and as expected, the 22- and 25-kDa proteins seen
in the HUT78 T cell nuclear extract were not detected in Jurkat T cells
(Fig. 3B, lane 1). As expected, the 42-, 25-, and
22-kDa proteins either did not bind or the binding was significantly less to the CSD mutant probe GM23 (Fig. 3C, lanes
4-6). Other proteins are seen binding to the GM
probe and not
to the CSD mutant GMm23
. CSD proteins of greater than 42 kDa in size
have been detected in nuclear extracts by other groups, and these
proteins, seen binding here to the GM
probe, may represent other CSD
family members that do not form part of the NF-GMb/c complexes.
CSD Factors Activate the GM-CSF Proximal Promoter in Jurkat T
Cells--
We have previously reported that overexpression of CSD
proteins repress tumor necrosis factor -mediated activation of the human GM-CSF proximal promoter in HEL fibroblasts (22). We wished to
determine whether CSD proteins also acted as repressors of GM-CSF in
Jurkat T cells. Surprisingly, we saw that when the GM-CSF reporter
plasmid (pGM4-Luc), which contained all four CSD binding sites, was
cotransfected into Jurkat T cells with CSD overexpression constructs
for dbpAv (pSGdbpAv) and dbpB (pSGdbpB), that the CSD proteins behaved
as activators in the presence of PMA/Ca2+ ionophore (Fig.
4A). That they were only
involved in activation of the GM-CSF promoter construct when the cells
were stimulated with PMA/Ca2+ ionophore indicated the CSD
proteins either needed to be activated to function or that they
cooperate with other proteins to function.
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We wished to determine which region of the GM-CSF proximal promoter was
required for CSD-mediated activation and the role, if any, that the
four CSD sites across the promoter played in this activation. To do
this, CSD overexpression constructs were cotransfected with reporter
constructs containing variable numbers of CSD sites (Fig.
4B). Initially the pGMCK-1(2)-TK/Luc construct (114 to
70) containing only the two domain 1 CSD sites of the GM-CSF promoter
was used in experiments. Cotransfections showed that both CSD factors
(pSGdbpAv and pSGdbpB) could coactivate this construct in the presence
of PMA/Ca2+ ionophore (Fig. 4B). Since the
domain 1 region was cloned upstream of the basal thymidine kinase (TK)
promoter in pGMCK-1(2)-TK/Luc, CSD factors were tested for activator
function on the TK promoter. We observed that CSD factors could not
activate the TK promoter in the presence of PMA/Ca2+
ionophore (data not shown). We next tested a construct containing only
the domain 2 CSD sites to determine whether they too were targets for
CSD-mediated activation. When pGM2-Luc (
71 to +28, containing only
the domain 2 CSD sites) was used in cotransfection experiments,
CSD-mediated activation was still observed, but both basal (pSG5)- and
CSD (pSGdbpAv, pSGdbpB)-coactivated expression was dramatically reduced
relative to pGM4-Luc (Fig. 4B). When however, the pGM2-Luc
construct was truncated by five base pairs to create pGM1-Luc, higher
levels of activity and CSD factor-mediated activation were restored
(Fig. 4B). The five-base pair truncation removed the 5' CSD
site in domain 2, leaving only the single 3' domain 2 CSD site. These
results therefore indicated that the 5' CSD binding sites had repressor activity.
To determine whether CSD-mediated coactivation of pGM1-Luc was acting
through the single CSD site in this construct, coactivation levels were
compared between pGM1-Luc (which contains one CSD site) and
pGM1(mut1)-Luc (where the one CSD site has been mutated) (Fig.
4B). Both pGM1-Luc and pGM1(mut1)-Luc were cotransfected into Jurkat T cells with constructs encoding for CSD factors dbpAv and
dbpB. The PMA/Ca2+ ionophore stimulated levels of both
basal- and CSD-mediated coactivation in pGM1(mut1)-Luc were greater
than those seen on pGM1-Luc (up to 2-fold greater) (Fig.
4B). These data taken altogether therefore demonstrate that
both the domain 2 CSD sites have repressor activity. Our results also
show that the coactivation effect of CSD proteins on the GM-CSF
promoter did not require contact with these CSD sites. We were unable
to repeat similar experiments on domain 1 CSD sites due to the overlap
of the NF-B sites with the CSD sites. Mutation of the domain 1 NF-
B sites results in loss of GM-CSF promoter activity (54).
The C-terminal Domain of dbpB Is Required for CSD-mediated
Activation of the GM-CSF Proximal Promoter--
Because interactions
between CSD factors and other transcriptional regulators have been
implicated in mechanisms of activation of a number of viral and
cellular genes, a potential mechanism for the coactivation effect seen
here was via interaction of CSD factors with other proteins. The
C-terminal domain of dbpB has been demonstrated to be required for
protein-protein interactions (10-15). To explore the possibility that
the coactivation effect was mediated via CSD protein-protein
interactions, expression constructs were made encoding truncations of
the dbpB CSD protein. In the dbpB deletion construct pSGdbpB2, most
of the C-terminal region of dbpB was deleted, and in pSGdbpB
1, both
the C-terminal and CSD domain were deleted (Fig. 1B). The
dbpB and dbpB deletion constructs were cotransfected into Jurkat T
cells with either the pGM1-Luc or pGM1(mut1)-Luc domain 2 constructs or
the pGMCK-1(2)-TK/Luc domain 1 construct, and the cells were stimulated
with PMA/Ca2+ ionophore. Removal of most of the C-terminal
region of the dbpB in pSGdbpB
2 resulted in a significant decrease in
the level of PMA/Ca2+ ionophore coactivation on all
constructs (Fig. 5). Removal of both the
C-terminal and CSD domain of dbpB in pSGdbpb
1 resulted in a return
to near basal levels of expression on both domain 1 and 2 constructs
(Fig. 5).
|
Taken altogether the results presented in Figs. 4 and 5 indicate, first, that at least the domain 2 CSD binding sites in the GM-CSF proximal promoter act as repressor elements, second, that the PMA/Ca2+ ionophore coactivation by CSD proteins is not associated with the CSD binding sites (at least in domain 2), and third, that the coactivation seen is mediated primarily via the C-terminal protein interaction domain of dbpB.
CSD Factors and RelA Act to Synergistically Activate the GM-CSF
Proximal Promoter--
The C-terminal domain of CSD proteins has been
shown to be involved in interactions with other transcription factors
and, to date, of the transcription factors acting across the GM-CSF proximal promoter; only RelA/NF-B p65 has been reported to interact with CSD proteins (10-15, 55). To investigate the potential role of a
CSD-RelA interaction in the CSD-mediated activation of the GM-CSF
promoter domain 1 sequences (which contains RelA/NF-
B p65 sites;
Refs. 20 and 54), RelA and CSD expression constructs were
cotransfected with the GM-CSF domain 1 reporter construct pGMCK-1(2)-TK-Luc (Fig. 6). In Fig. 6, we
show as previously reported that RelA/NF-
B p65 stimulated the
pGMCK-1(2)-TK-Luc construct in unstimulated cells, whereas the CSD
factors could not (56). When the pGMCK-1(2)-TK/Luc reporter construct
was cotransfected with expression constructs for both RelA (pRcCMVRelA)
and dbpAv (pSGdbpAv) or dbpB (pSGdbpB), synergistic activation was
observed (Fig. 6). Repeating the triple cotransfection with the dbpB
C-terminal truncation (pSGdbpB
2) significantly reduced the
synergistic activation (Fig. 6), suggesting that the C terminus of the
CSD protein dbpB protein may be involved in interactions with RelA to
bring about activation. Cotransfecting with the dbpB construct
pSGdbpB
1 (missing both the C-terminal and CSD domains) further
reduced the activation to basal pSG5 levels (Fig. 6). These experiments
show that RelA/NF-
B p65 and the CSD proteins can cooperate to
activate GM-CSF promoter function and that the C terminus of dbpB is
essential for this cooperation.
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DISCUSSION |
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Cold shock domain family members have been associated with diverse functions including the ability to both activate and repress transcription and translation. Here we investigated the role of the CSD proteins dbpAv and dbpB in regulation of GM-CSF promoter in Jurkat T cells. We have previously reported that HUT78 T cell and HEL fibroblast nuclear CSD proteins bind to defined single-stranded regions along domain 1 and domain 2 of the GM-CSF promoter as NF-GMb and NF-GMc complexes (21-23). We proposed that the binding of NF-GMb/c CSD proteins along the GM-CSF promoter resulted in or stabilized a single-stranded DNA structure, thereby preventing the binding of transcriptional activators that are dependent on double-stranded DNA for binding and activity. This proposed structure would be an efficient means of silencing the GM-CSF promoter and many other promoters where NF-GMb/c CSD proteins have been shown to act as repressors of transcription (13, 23, 25-27, 33, 57). The specific arrangement of NF-GMb/c CSD binding sites observed on the GM-CSF promoter, a pair of distal sites binding on the noncoding strand and a pair of proximal sites binding the coding strand, can also be found in the promoters of the granulocyte-colony stimulating factor and interleukin-3 cytokine genes with overlapping GM-CSF expression patterns (58, 59).
Analysis of Jurkat T cell extracts identified nuclear CSD proteins binding to the CSD binding sites across the GM-CSF promoter as a NF-GMb-like complex. Competitions with single-stranded DNA CSD site sequences and CSD antibody experiments in addition to UV cross-linking indicated that the Jurkat T cell NF-GMb complex was made up of only a 42-kDa CSD protein. With the lack of the NF-GMc complex in Jurkat T cells, we also observed a lack of the 22- and 25-kDa CSD proteins previously identified in HEL fibroblasts and HUT78 T cells (21, 23). These 22- and 25-kDa CSD proteins probably represent CSD splice variants or proteolytic cleavage products, as reported by others, that lack the C-terminal protein-protein interaction domain (22, 31-33, 35, 53).
Transient transfection in Jurkat T cells showed that in contrast to the
repression observed in HEL fibroblasts, the CSD proteins, dbpAv and
dbpB, functioned as coactivators on a 140-base pair region of the human
GM-CSF proximal promoter. This activation was only observed when T
cells were activated with PMA/Ca2+ ionophore. We showed
that both the GM-CSF domain 1 region (containing NF-kB/RelA sites) and
the domain 2 region (containing the CBF, AP1, and ETS/NFAT sites) are
able to independently respond to CSD-mediated activation. We also
showed that this response to CSD-mediated activation did not require
the CSD binding sites, at least in the 60 to +28 region of domain 2. The CSD binding sites, however, still retain repressor function as
defined by deletion and mutation.
CSD factors have been reported to activate a few cellular genes
including c-Myc, 1(I) procollagen, and metalloproteinase (MMP-2) and
also regulate many viral promoters such as human immunodeficiency virus, human T-cell lymphotrophic virus, and Rous sarcoma virus long
terminal repeats and human polyomavirus JC late viral promoter (5, 31, 32, 35, 36, 60), but the precise mechanisms of CSD-mediated
activation has not been well defined. To determine the mechanism of
CSD-mediated activation in Jurkat T cells, we examined the possible
role of CSD protein interactions with other transcription factors. Our
data indicate that CSD factors dbpB and dbpAv are able to act with RelA
in transient transfections to synergistically activate the GM-CSF
promoter in the absence of PMA/Ca2+ ionophore stimulation.
Our data suggest that this coactivation of GM-CSF by CSD and RelA
proteins is via protein-protein interactions. We demonstrated this by
truncating dbpB, removing the C terminus of the protein, which has been
shown to be essential for CSD protein-protein interactions (11, 13, 30,
61-63). When the dbpB deletion construct removing the C terminus was
used, PMA/Ca2+ ionophore-mediated coactivation was
significantly reduced as was the ability of dbpB and RelA to synergize,
indicating that the coactivation was mediated primarily through the
C-terminal region of the CSD protein and, therefore, most likely via
protein-protein interactions. Raj et al. (15, 32) find that
CSD and RelA can interact in solution and that this interaction leads
to increased RelA binding to the human polyomavirus JC viral promoter.
In these experiments RelA conversely decreased CSD binding to its
single-stranded DNA binding site. This CSD-RelA interaction was
implicated in activation of the human polyomavirus JC early promoter
(32). Similarly, we have found that RelA can decrease CSD binding to its GM-CSF single-stranded DNA binding site (data not shown).
Taken together our data suggest a model whereby, first, in unstimulated
Jurkat T cells, CSD proteins bind to single-stranded DNA in a structure
as described for the repression of the GM-CSF gene by CSD proteins in
HEL fibroblasts (21-23). This model has been suggested for a number of
other genes repressed by CSD proteins (26, 27, 50). Upon T cell
stimulation (mimicked by PMA/Ca2+ ionophore) it has been
well documented that many transcriptional activators (like NF-B p50,
and RelA) are up-regulated, whereas the level of CSD proteins in the
nucleus remains constant (reviewed in Refs. 38 and 20).2 We
and others (15)2 have shown that RelA is able to dissociate
dbpB from its single-stranded target, and we propose that this results
in destabilization of the single-stranded DNA structure, facilitating a
return of the DNA to its double-stranded conformation . This would
allow the transcriptional activators (like NF-
B, AP-1, CBF,
ETS/NFAT) to bind initiating transcriptional activation.
We have presented data here that indicate a functional CSD-RelA
interaction that is involved in activation of the GM-CSF promoter, domain 1 region. We have also shown CSD mediated activation via the
domain 2 region, which contains no RelA sites, and hence, CSD proteins
may also be able to interact with other transcription factors binding
to this region. There is a growing list of proteins where CSD
protein interactions have been implicated to affect promoter function
(NF-B, mitrochondrial SSB, ZO-1, heterogeneous nuclear
ribonucleoprotein K, TBP, NF-Y, YY-1, AP-2, SP-1, Tat, PuR
)
(5, 10-13, 15, 23, 29, 30, 55, 64). Potential targets on the GM-CSF
promoter domain 2 for CSD interactions could be SP1 and YY1, both of
which have been shown to regulate GM-CSF function in T cells (20, 65,
66).
The complement of CSD protein subtypes in the cell may also affect CSD function. We have previously observed in HEL fibroblasts (with 42-, 22-, and 25-kDa CSD proteins) that CSD proteins acted to repress activation, whereas in T cells (with only the 42-kDa protein) CSD factors function to activate when the cells were PMA/Ca2+ ionophore-stimulated. The reason for this difference between the cell types is as yet unclear but may be related to the proposed lack of C-terminal-protein interaction sequences in the 22- and 25-kDa CSD subtypes observed in HEL fibroblasts (22, 31-33, 35, 53). Taking into account our previous results and the reported function associated with each of the CSD protein domains, the 42-kDa CSD factor is implicated in repression (via the CSD domain) and activation (via the C-terminal domain), whereas the 22-kDa CSD factors may be involved only in repression (21-23). In fibroblasts, where CSD factors act to repress GM-CSF promoter function, UV-cross-linking experiments indicated that the 22-kDa CSD protein sub-population makes up a greater proportion than the 42-kDa sub-population, suggesting that the CSD intracellular environment is predisposed toward truncated forms of the CSD proteins and, hence, repression (21-23). In Jurkat T cells, where CSD factors act to mediate activation of the GM-CSF promoter, only the full-length 42-kDa CSD protein is present. The 42-kDa CSD protein, although still able to bind DNA and potentially repress in the unstimulated state, is also able to interact with proteins in a stimulated cell via the C-terminal protein-protein interaction domain to bring about activation. Therefore the differences in effect when full-length CSD factors were overexpressed in Jurkat T cells and HEL fibroblasts may be due to the intrinsic difference in the ratio of CSD factor subtypes and resulting intracellular environment already present in the two cell types.
We have shown, therefore, that CSD proteins can act to repress or
activate the GM-CSF gene in different cell types. Repression is
associated with the CSD binding sites located across the promoter, whereas activation may relate to CSD cooperation with other
transcription factors. We also raise the possibility that the cellular
content of CSD proteins predetermines the functional outcome in the
transcription response.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 61-8-82223712; Fax: 61-8-82324092; E-mail: peter.diamond@imvs.sa.gov.au.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M009836200
2 P. Diamond, M. F. Shannon, M. A. Vadas, and L. S. Coles, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CSD, cold shock domain; TK, thymidine kinase; GM-CSF, granulocyte-macrophage colony-stimulating factor; HEL, human embryonic lung; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase; CBF, core binding factor; NFAT, nuclear factor of activated T cell.
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