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INTRODUCTION |
Nuclear factor
B
(NF-
B)1 is an inducible
cellular transcription factor that regulates a wide variety of cellular
and viral genes including cytokines, cell adhesion molecules and human
immunodeficiency virus (1-5). The members of the NF-
B family in
mammalian cells include the proto-oncogene c-Rel, RelA (p65), RelB,
NFkB1 (p50/105), and NFkB2 (p52/p100). These proteins share a conserved
300-amino acid region known as the Rel homology domain, which is
responsible for DNA binding, dimerization, and nuclear translocation of
NF-
B (1, 2, 4, 5). In most cells, Rel family members form hetero-
and homodimers with distinct specificities in various combinations.
p65, RelB, and c-Rel are transcriptionally active members of the
NF-
B family, whereas p50 and p52 primarily serve as DNA binding
subunits (1, 2, 4, 5). These proteins play fundamental roles in immune
and inflammatory responses and in the control of cell proliferation (4,
6-9). A common feature of the regulation of NF-
B is the
sequestration in the cytoplasm as an inactive complex with a
class of inhibitory molecules known as I
Bs (2, 10). Treatment of
cells with a variety of inducers such as phorbol esters, interleukin-1
(IL-1), and tumor necrosis factor
(TNF-
) results in
phosphorylation, ubiquitination, and degradation of the I
B proteins
(5, 11, 12). The degradation of I
B proteins exposes the nuclear
localization sequence in the remaining NF-
B dimers, followed by the
rapid translocation of NF-
B to the nucleus where it activates the
target genes by binding to the DNA regulatory element (1, 2, 4, 5).
The protein regions responsible for the transcriptional activation
(called "transactivation domain") of p65, RelB, and c-Rel have been
mapped in their unique C-terminal regions. p65 contains at least two
independent transactivation domains within its C-terminal 120 amino
acids (Fig. 1A) (13-16). One of these transactivation domains, TA1, is confined to the C-terminal 30 amino acids of p65. The
second transactivation domain, TA2, is located within the N-terminally
adjacent 90 amino acids and contains TA1-like domain and leucine-rich
regions. Since the nuclear translocation and DNA binding of NF-
B
were not sufficient for gene induction (17, 18), it was suggested that
interactions with other protein molecules through the transactivation
domain (15, 19, 20) as well as its modification by phosphorylation (16)
might play a critical role.
It has been previously reported that transcriptional activation of
NF-
B requires multiple coactivator proteins including CREB-binding
protein (CBP)/p300 (19, 20), CBP-associated factor, and steroid
receptor coactivator 1 (21). These coactivators have histone
acetyltransferase activity to modify the chromatin structure and also
provide molecular bridges to the basal transcriptional machinery.
Recently, p65 was also found to interact specifically with a newly
identified coactivator complex, activator-recruited cofactor/vitamin D
receptor-interacting protein, which potentiated chromatin-dependent transcriptional activation by NF-
B
in vitro (22). In addition to general coactivators, the
transcriptional activation of gene-specific activators can be mediated
by basal transcription factors through direct interaction with the
activation domain. In the case of NF-
B, the association of p65 with
basal transcription factors such as TFIIB, TAFII105, and
TBP has been demonstrated (15, 23-27).
It is thus postulated that specific protein-protein interactions with
NF-
B determine its transcriptional competence: up-regulation of the
NF-
B transcriptional activity is mediated by interaction with basal
factors and coactivators, and its down-regulation is mediated by
interaction with inhibitors and corepressors at multiple levels. In our
previous studies, yeast two-hybrid screen yielded two novel regulators
of NF-
B. RelA-associated inhibitor was found to interact with the
central region of p65 (RelA) and block DNA binding in the nucleus,
similar to the action of cytoplasmic inhibitors I
Bs (8). The other
proteins found to interact with p65 belong to the Grg
(Groucho-related genes) family,
including amino-terminal enhancer of split (AES) and transducin-like
enhancer of split (TLE1) (7), previously known as nuclear corepressors
(28, 29).
Translocated in liposarcoma (TLS), also known as FUS, was originally
identified through its fusion to CHOP, a member of the CCAAT/enhancer-binding protein family of transcription factors, in
human myxoid liposarcoma with the t(12;16) chromosomal translocation (30, 31). TLS has high homology to hTAFII68/RBP56, EWS, and Drosophila protein SARFH (collectively called the "TET"
family (32)). These genes were found to be involved in carcinogenesis through chromosomal translocation with other genes of transcription factors; normally the N-terminal region of these proteins provides a
transcriptional activator domain, and the moiety of counterpart proteins provides a DNA-binding domain (DBD), thus making these fusion
proteins constitutively active for their transcriptional activities
(33-35). The C-terminal half of TLS spanning the
ribonucleoprotein consensus sequence domain is usually excluded by
translocation, and tumorigenic transformation is associated with the
fusion of the N-terminal portion to the DNA-binding domain of a given
transcription factor (30, 31, 36). Interestingly, TLS was shown to
associate with a subpopulation of the TFIID complex in cells (32, 37). Moreover, SARFH, a Drosophila homologue of TLS, was
colocalized with RNA polymerase II at the active chromatin (38). In
fact, TLS was shown to be associated with RNA polymerase II through its
N-terminal domain (39).
In this study, we demonstrate that TLS interacts with NF-
B p65
through the C-terminal transactivation domain and activates NF-
B-mediated transcription. The yeast two-hybrid interaction assay
revealed that the region between two core transactivation domains of
p65, TA1-like and TA1, is required for the interaction with TLS. We
confirmed the interaction between p65 and TLS in vitro using
the bacterially expressed fusion proteins and in vivo coimmunoprecipitation/Western blot assay. In transient transfection assays, TLS showed transactivation potential and activated
NF-
B-dependent gene expression. These data indicate that
TLS mediates the transcriptional activity of NF-
B.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Mammalian expression vector plasmids pCMV-TBP,
Gal4-Sp1, pCMV-NIK, ICAM-1-luc (positions
339 to
30), and
IFN-
-luc were generous gifts from Drs. T. Tamura (Chiba University),
S. T. Smale (UCLA School of Medicine), D. Wallach (Weitzmann
Institute of Science), L. A. Madge and J. S. Pober (Yale
University School of Medicine), and T. Taniguchi (Tokyo University),
respectively. pCMV-p65, pGBT-p65-(1-286), pGBT-p65-(286-551),
pGBT-p65-(286-521), pGBT-p65-(286-470), pGBT-p65-(286-442),
pGBT-p65-(286-442/477-521), and pGBT-p65-(473-522) have been
described previously (7). To generate the mammalian expression plasmid
for TLS, the full-length TLS cDNA fragment was excised from
pACT2-TLS with the BamHI and XhoI site, and
ligated in frame into pcDNA3.1/HisA vector at the BamHI-XhoI site to form pCMV-TLS. To create a
dominant negative form of TLS-(274-525), the TLS cDNA was
amplified by polymerase chain reaction using pACT2-TLS as a template
with oligoncleotides containing the BamHI-XhoI
site. These products were digested with BamHI-XhoI and subcloned in frame into
pcDNA3.1/HisA vector at the BamHI-XhoI site
to form pCMV-TLS-(274-525). To construct pM-p65-(1-551), which
expresses the fusion protein of Gal4-DBD and p65, the cDNA of human
p65 (amino acids 1-551) was amplified by polymerase chain reaction
using pCMV-p65 as a template with oligonucleotides containing BamHI sites (forward,
5'-CCCCCGGATCCCCGGCCATGGACGAACTGTTC-3'; reverse,
5'-ACCAGGGATCCGGGGAGGGCAGGCGTCACCC-3'). This fragment was digested with
BamHI and ligated in frame into the BamHI site of
pM (CLONTECH). To generate pM-p65-(1-286) and
pM-p65-(286-551), p65 cDNA fragments excised from
pGBT-p65-(1-286) with EcoRI and pGBT-p65-(286-551) with
BamHI/EcoRI were ligated in frame into the
corresponding sites of pM. Construction of a luciferase reporter plasmid, 4
Bw-luc or 4
Bm-luc, containing four tandem copies of the
human immunodeficiency virus-
B sequence upstream of minimal SV40
promoter has been described previously (40). The other luciferase
reporter plasmid, 5×Gal4-luc (pFR-luc) was purchased from Stratagene.
This plasmid contains five tandem copies of the Gal4 binding site
upstream of the TATA box.
Yeast Two-hybrid Screening and Protein-Protein Interaction
Assay--
The yeast two-hybrid screening was performed as previously
described (7-9). The various portions of the p65 C-terminal regions corresponding to amino acids 286-551, 286-521, 286-470, 286-442, 286-442/477-521, and 473-522 were fused in-frame to Gal4 DNA binding domain (positions 1-147) using the pGBT9 vector
(CLONTECH). They were tested for activation of
Gal4-dependent lacZ expression
(
-galactosidase activity). Among them, pGBT-p65 (286-442/477-521)
was chosen as a bait for library screening, since it had undetectable
background in the
-galactosidase assay. Yeast strain Y190 was
transformed with pGBT-p65-(286-442/477-521), and the human placenta
cDNA expression library was fused to the Gal4
transactivation domain in the pACT2 vector
(CLONTECH). Approximately one million transformants
were screened for the ability to grow on the plates with medium lacking tryptophan/leucine/histidine and containing 25 mM
3-aminotriazole. Plasmids were rescued from clones that were positive
for
-galactosidase activity and identified by nucleotide sequencing.
cDNA sequences and their amino acid sequences were compared with
GenBankTM and SwissProt data bases for identification of
the interacting proteins.
Cell Culture and Transfection--
293 cells were maintained in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum and
antibiotics. Cells were transfected using Fugene-6 transfection reagent
(Roche Molecular Biochemicals) according to the manufacturer's
instructions. At 48 h post-transfection, the cells were harvested,
and the extracts were prepared for luciferase assay. Luciferase
activity was measured by the Luciferase Assay System (Promega, Madison,
WI) as previously described (8). Transfection efficiency was monitored
by Renilla luciferase activity using the pRL-TK plasmid
(Promega) as an internal control. The data are presented as the -fold
increase in luciferase activities (means ± S.E.) relative to
control of three independent transfections. Human recombinant TNF-
and IL-1
were purchased from Roche Molecular Biochemicals.
In Vitro Binding Assay--
Bacterial expression of Glutathione
S-transferase (GST) fusion proteins utilize pGEX expression
vectors. To generate pGEX-TLS-(1-273) and pGEX-TLS-(274-525), which
express GST-TLS-(1-273) and GST-TLS-(274-525), the TLS cDNA was
amplified by polymerase chain reaction using pACT2-TLS as a template
with oligonucleotides containing a
BamHI-XhoI site. These products were digested
with BamHI-XhoI and subcloned in frame into
pGEX-5X-2 vector (Amersham Pharmacia Biotech) at the
BamHI-XhoI site. GST fusion proteins were
expressed in Escherichia coli strain DH5
and purified as
described (8). In vitro protein-protein interaction assays
were carried out as described previously (7, 8). p65 and proteins were
labeled with [35S]methionine by the in vitro
transcription/translation procedure using the TNT wheat germ
extract-coupled system (Promega) according to the manufacturer's
protocol. Approximately 20 µg of GST fusion proteins were immobilized
on 20 µl of glutathione-Sepharose beads and washed two times with 1 ml of modified HEMNK buffer (20 mM HEPES-KOH (pH 7.5), 100 mM KCl, 12.5 mM MgCl2, 0.2 mM EDTA, 0.3% Nonidet P-40, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). The
beads were left in 0.6 ml of HEMNK after the final wash and were
incubated with the radiolabeled proteins for 2 h at 4 °C with
gentle mixing. The beads were then washed three times with 1 ml of
HEMNK buffer and two times with 1 ml of HEMNK buffer containing 150 mM KCl. Bound radiolabeled proteins were eluted with 30 µl of Laemmli sample buffer, boiled for 3 min, and resolved by 10%
SDS-PAGE.
Coimmunoprecipitation and Western Blot Assays--
After
transfection of relevant plasmids, 293 cells were cultured for 48 h and then harvested with lysis buffer (25 mM HEPES-NaOH (pH 7.9), 150 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.3% Nonidet P-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride). The lysate was incubated with 1 µg of anti-p65 (NLS) mouse
monoclonal antibody (Roche Molecular Biochemicals) or control mouse
monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
and anti-p65 (C-terminal) rabbit polyclonal antibody (Santa Cruz
Biotechnology) or control rabbit polyclonal antibody (Santa Cruz
Biotechnology) overnight at 4 °C. 10 µl of protein G-agarose beads
were added, and the reaction was further incubated for 1 h. The
beads were washed five times with 1 ml of lysis buffer.
Antibody-bound complexes were eluted by boiling in 2×
Laemmli sample buffer. Supernatants were resolved by 10%
SDS-PAGE and transferred on nitrocellulose membrane (Hybond-C;
Amersham Pharmacia Biotech). The membrane was incubated with
anti-TLS antibody, and immunoreactive proteins were visualized by
enhanced chemiluminescence (SuperSignal; Pierce) as described
previously (41, 42). Polyclonal antibody to human TLS was a generous
gift from K. Shimizu and M. Ohki (National Cancer Center Research
Institute, Tokyo, Japan). This antibody was raised by immunizing
rabbits with purified recombinant GST-TLS (amino acids 8-134). To
evaluate the level of exogenous p65 expressed by pCMV-p65 containing
the His epitope tag, rabbit polyclonal anti-His6 antibody
(Santa Cruz Biotechnology) was used.
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RESULTS |
Identification of TLS as a p65-binding Protein in the Yeast
Two-hybrid Screen--
To identify proteins interacting with the p65
subunit of NF-
B, we performed the yeast two-hybrid screen using the
unique C-terminal region of NF-
B p65 as a bait (Table
I and Fig.
1). As depicted in Fig. 1A,
various portions of p65 (i.e. amino acids 286-551,
286-521, 286-470, 286-442, 473-522, 286-442/477-521, and
473-522) were fused to Gal4 DNA-binding domain (Gal4-DBD) in the pGBT9
vector. Among these clones, pGBT-p65 (286-442/477-521) was chosen as
a bait for the screening, since it had no detectable background in
-galactosidase assay (Table I). Yeast strain Y190 was used to screen
human placenta cDNA library fused to the Gal4 transcriptional
activation domain in the pACT2 vector (CLONTECH). From ~1.0 × 106 Y190 yeast transformants, 90 colonies grew on selective medium and turned blue when tested in a
filter lift
-galactosidase assay. Each plasmid, purified from the
positive colonies, was cotransfected with bait plasmid into the yeast
to confirm the specific interaction. DNA sequencing and comparison with
GenBankTM and SwissProt data bases revealed the gene for
TLS (one clone) in addition to Bcl-3 (one clone) and the I
B family
including I

/MAD3 (five clones), which were previously shown to
interact with p65.
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Table I
Yeast two-hybrid interaction assays between p65 and TLS
Yeast Y190 cells were cotransformed with expression vectors encoding
various proteins fused to Gal4-DBD and Gal4-AD. pACT2-TLS is a rescued
clone that encodes TLS fused to Gal4-AD. pACT2-I B encodes
full-length I B (amino acids 1-317) fused to Gal4-AD.
Leu+ Trp+ transformants were streaked on selective
medium lacking leucine/tryptophan and allowed to grow for 2 days at
30 °C. At least three colonies of each transformant were tested for
-galactosidase activity using a
5-bromo-4-chloro-3-indolyl- -D-galactopyranoside colony
filter assay (Clontech). +, positive for -galactosidase activity
(blue colony) after 2-3 h; , no -galactosidase activity (white
colony) after 24 h; ND, activity not determined.
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Fig. 1.
Interaction between p65 and TLS in the
yeast. A, schematic illustrations of various functional
domains of p65 and TLS and nomenclature of plasmid constructs.
Gal4-DBD, Gal4 DNA-binding domain; RHD,
Rel homology domain; TA1, transactivation domain 1;
TA1-like, TA1-like domain; TA2, transactivation
domain 2; LLL, leucine-rich region; QSY rich,
Gln-Ser-Tyr-rich region; G-rich; Gly-rich region;
RNP, ribonucleoprotein; Zn finger, zinc finger
motif; RGG rich, Arg-Gly-Gly-rich region. B,
growth of yeast transformants coexpressing p65 and TLS on the selective
medium. The yeast Y190 was transformed with pACT2-TLS and pGBT plasmids
expressing various portions of the p65 in fusion with Gal4-DBD. The
yeast transformants grown on plates lacking leucine and tryptophan were
streaked on plates lacking leucine, tryptophan, and histidine and
containing 25 mM 3-aminotriazole.
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To determine the region of p65 involved in the binding to TLS, various
regions of the protein were fused to Gal4-DBD in the pGBT9 vector and
cotransfected with pACT2-TLS, encoding TLS fused to Gal4
transcriptional activation domain. Interactions were tested by
-galactosidase activity (Table I) and by growth of yeast cells on
plates with medium lacking histidine, leucine, and tryptophan and
containing 25 mM 3-aminotriazole (Fig. 1B).
pGBT-p65-(1-286), pGBT-p65-(286-442), pGBT-p65-(286-442/477-521),
and pGBT-p65-(473-522) alone did not show any background in the
prototrophic selection or in the
-galactosidase assay. Among these,
pGBT-p65-(286-442/477-521) and pGBT-p65-(473-522) were shown to be
positive for the interaction with pACT2-TLS (Table I and Fig.
1B). These results indicate that the minimal region of p65
responsible for the interaction with TLS resides within the amino acid
sequence 477-521.
TLS Supports Transcriptional Activation When Tethered to the
Promoter--
It was previously implicated that the N-terminal portion
of TLS might act as a transcriptional activator in fusion with other transcription factors such as CHOP and ERG (33, 43), although transcriptional activity of the full-length TLS or its C terminus has
not been documented. It is suggested that TLS may mediate transcriptional activation by associating with transcription factors such as CHOP, ERG, and NF-
B. Thus, we asked whether TLS acts as a
transcriptional coactivator when tethered to the proximity of a given
promoter. To examine this possibility, we created various fusion
proteins of TLS and Gal4 DNA-binding domain containing full-length TLS
or either its N-terminal or C-terminal subdomain (Fig.
2A). As demonstrated in Fig.
2B, all of these TLS-Gal4 (DNA binding) fusion proteins more
or less supported transcriptional activation from the reporter plasmid
under the control of Gal4. Therefore, it was suggested that TLS could
act as a coactivator of transcription if it was recruited to the
vicinity of the promoter.

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Fig. 2.
TLS mediates transcriptional activation when
tethered to the promoter. A, plasmids expressing
various TLS domains in fusion with Gal4-DBD (Gal4-TLS plasmids).
B, transcriptional activation by Gal4-TLS. The
transcriptional activity of various TLS proteins were examined by
tethering with the Gal4-DBD using transient luciferase assay. The level
of Gal4-dependent gene expression from 5×Gal4-luc reporter
plasmid (indicated in the figure) was assessed. Plasmids
were cotransfected into 293 cells with 50 ng of 5×Gal4-luc and the
indicated amounts of various Gal4-TLS expression plasmids. pCMV control
plasmids were included such that all transfections had equivalent
amounts of expression plasmid. Total DNA was kept at 0.5 µg with
pUC19 plasmid. Cells were harvested 48 h after transfection, and
luciferase activity was measured. Extents of -fold activation of
luciferase gene expression as compared with the transfection with
reporter plasmid alone are indicated. Values (-fold activation)
represent the means ± S.E. of three independent transfections.
Similar results were achieved repeatedly.
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Binding between TLS and p65 in Vitro and in Vivo--
To
demonstrate the direct interaction between TLS and p65, we performed an
in vitro protein-protein interaction assay using various
recombinant TLS proteins in fusion with GST. The radiolabeled p65
protein was synthesized by in vitro
transcription/translation in the presence of
[35S]methionine and was incubated with GST-TLS fusion
proteins immobilized on glutathione-Sepharose beads. As shown in Fig.
3A, p65 bound to
GST-TLS-(274-525). A slightly weaker binding between p65 and GST-TLS-(1-273) was also detected. In this assay condition,
GST-I
B
(positive control for interaction with p65) beads also
bound to p65. However, no p65 binding was detected with beads
containing GST alone (negative control). A coimmunoprecipitation
experiment was carried out to examine whether TLS interacts with p65 in
cultured cells. 293 cells were transiently transfected with plasmids
pCMV-p65 and pCMV-TLS, expressing p65 and TLS, respectively. Cell
extracts from these transfected cells were immunoprecipitated with
mouse monoclonal anti-p65 (NLS) antibody (Fig. 3B,
lane 3) or rabbit polyclonal anti-p65 antibody
(recognizing its C-terminal region) (lane 5). The
immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with
rabbit polyclonal anti-TLS antibody. Either anti-p65 (NLS) or anti-p65
(C-terminal) antibodies coimmunoprecipitated TLS (lanes
3 and 5), whereas mouse or rabbit control IgGs
did not (lanes 2 and 4). These results
demonstrated that TLS specifically interacted with the p65 subunit of
NF-
B in vivo as well as in vitro.

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Fig. 3.
TLS interacts with p65. A,
p65 binds to TLS and I B in vitro. p65 was labeled with
[35S]methionine by in vitro
transcription/translation. Radiolabeled p65 was incubated with GST,
GST-TLS-(1-273), GST-TLS-(274-525), or GST-I B immobilized on
glutathione-Sepharose beads. After incubation and further washing, the
complexes were resolved by 10% SDS-PAGE and subjected to
autoradiography. B, p65 binds to TLS in cells. 293 cells
were transfected with pCMV-TLS and pCMV-p65 expression vectors. Whole
cell extract was harvested 48 h after transfection, and TLS was
immunoprecipitated with anti-p65 (NLS) mouse monoclonal IgG or anti-p65
(C-terminal) rabbit polyclonal IgG. The immunoprecipitated proteins
were resolved by 10% SDS-PAGE and immunoblotted with anti-TLS
antibody. The positions of TLS and rabbit IgG heavy chain are
indicated. WB, Western blot.
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TLS Augments NF-
B-dependent Gene Expression--
We
then investigated the effect of TLS on NF-
B-dependent
gene expression. In Fig. 4A,
the effect of TLS was examined on gene expression from the reporter
plasmid 4
Bw-luc by transfection of pCMV-TLS with or without
cotransfection of pCMV-p65 in 293 cells. TLS augmented the
NF-
B-mediated transactivation in a dose-dependent manner
when the p65-expression plasmid was cotransfected. pCMV-p65 alone
activated gene expression from 4
Bw-luc by 57-fold, but upon
cotransfection with pCMV-TLS (100 ng), the extent of gene activation
was elevated to 131-fold (2.3-fold augmentation by the effect of TLS).
However, there was no detectable effect on the basal transcription
level. Moreover, when a control luciferase reporter construct,
4
Bm-luc, in which all four
B sites were mutated, was used,
neither the activation by pCMV-p65 nor the effect of cotransfection of
pCMV-TLS was observed. These effects of TLS were not the result of an
increased p65 level, since Western blot analysis of the
transfected cell lysate revealed no increase in the protein level of
exogenously expressed p65 (Fig. 4A, lower panels). Similarly, TLS augmented p65-mediated gene
expression from human interferon
promoter containing one binding
site for NF-
B (data not shown). To further examine whether the
effect of TLS depends on the transactivation domain of p65 and its
specificity, we created expression plasmids for Gal4-p65 fusion
proteins in which the DNA-binding domain of Gal4 was fused with various
portions of p65 or Sp1. The extents of augmentation of transactivation of these Gal4-p65 and Gal4-Sp1 by TLS were compared in Fig.
4B. TLS augmented the transactivation mediated by
Gal4-p65-(1-551) and Gal4-p65-(286-551) by 5.2- and 5.5-fold,
respectively. In sharp contrast, there was no significant effect of TLS
on the actions of Gal4-p65-(1-286) and Gal4-Sp1. These observations
indicated that the effects of TLS on transactivation appeared to depend on the C-terminal domain of p65 and were relatively specific for NF-
B.

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Fig. 4.
TLS augments
NF- B-dependent gene
expression. A, 293 cells were transfected with 20 ng of
4 Bw-luc (containing wild type NF- B binding sites) or 4 Bm-luc
(containing mutated NF- B binding sites) together with the indicated
amounts of pCMV-p65 (containing His6 epitope) and pCMV-TLS
expression plasmids (upper panel). Cells were
harvested 48 h after transfection, and luciferase activity was
measured. Western blot (WB) analysis of p65 levels in
transfected cell extracts was done to confirm that an equal amount of
the exogenous p65 is expressed irrespective of TLS overexpression
(lower panel). A portion of each cell extract was
separated by 10% SDS-PAGE and immunoblotted with anti-His antibody.
B, specificity of the effects of TLS on transcriptional
activation. 293 cells were transfected with 50 ng of 5×Gal4-luc
reporter plasmid together with 5 ng of Gal4-p65-(1-551),
Gal4-p65-(286-551), Gal4-p65-(1-286), or 100 ng of Gal4-Sp1 and the
indicated amounts (ng) of pCMV-TLS. pCMV and pGal4-DBD control plasmids
were included such that all transfections had equivalent amounts of
expression plasmid. Cells were harvested 48 h after transfection,
and luciferase activity was measured. Values represent the luciferase
activity means ± S.E. of three independent transfections.
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TLS Augments NF-
B-dependent Gene Expression Induced
by Physiological Stimuli--
To examine the physiological relevance
of the interaction between TLS and p65, we have examined the effect of
TLS on the human intercellular adhesion molecule-1 (ICAM-1)
promoter containing an NF-
B binding site (44, 45). In addition to
cotransfection with p65 expression plasmid, effects of physiological
stimuli such as TNF-
or IL-1
were also examined. Various amounts
of the TLS-expressing plasmid (pCMV-TLS) were transfected into 293 cells along with ICAM-1-luc reporter plasmid, and the effects of
TNF-
and IL-1
were investigated. As demonstrated in Fig. 5A, TLS greatly augmented the
p65-mediated ICAM-1 gene expression in a dose-dependent
manner (Fig. 5A, lanes 5-8). TLS
enhanced the ICAM-1 gene expression induced by TNF-
(lanes 9-12) and IL-1
(lanes
13-16). Although it seems likely that the effect of TLS on
ICAM-1 gene expression is mediated through NF-
B, we have confirmed this by using an artificial reporter construct, 4
Bw-luc, containing only the NF-
B sites and the minimal SV40 promoter. As shown in Fig.
5B, TLS augmented NF-
B-dependent gene
expression from 4
Bw-luc induced by TNF-
and by NF-
B-inducing
kinase (NIK), an effector kinase involved in the NF-
B activation
pathway elicited by TNF-
. When a luciferase reporter construct
containing the mutated
B sites (4
Bm-luc) was used, no activation
by TNF-
or NIK was observed, and no significant effect of
cotransfection of TLS was demonstrated (data not shown). These data
demonstrated that the effects of TLS were also evident on the
signal-induced NF-
B-dependent gene expression.

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Fig. 5.
TLS augments the
NF- B-dependent gene expression
induced by physiological stimuli. A, effect of TLS on
human ICAM-1 promoter activity. 293 cells were transfected with 20 ng
of ICAM-1-luc, 10 ng of pCMV-p65 (lanes 5-8),
and the indicated amounts (ng) of pCMV-TLS. After 24 h of
transfection, cells were stimulated with 1 ng/ml TNF-
(lanes 9-12) or 10 ng/ml IL-1
(lanes 13-16) and harvested after additional
incubation for 24 h. Values represent the luciferase activity
means ± S.E. of three independent transfections. B,
effects of TLS on the NF- B-dependent gene expression
induced by TNF- or NIK. Left panel, 293 cells
were transfected with 4 Bw-luc (50 ng) and the indicated amounts (ng)
of pCMV-TLS. After 24 h of transfection, cells were stimulated
with 1 ng/ml TNF- and harvested after additional incubation for
24 h. Right panel, 293 cells were
transfected with 4 Bw-luc (50 ng), pCMV-NIK (10 ng), and the
indicated amounts (ng) of pCMV-TLS. Values represent the luciferase
activity means ± S.E. of three independent transfections.
C, induction of the interaction between TLS and p65 by
stimulation with TNF- . 293 cells were transfected with pCMV-TLS
plasmid. After 24 h of transfection, cells were stimulated with 1 ng/ml of TNF- (lanes 2 and 3).
Cells were harvested after an additional incubation of 1 h, and
TLS was immunoprecipitated with anti-p65 (C-terminal) rabbit polyclonal
IgG (lanes 1 and 2) or control IgG
(lane 3). The immunoprecipitated (IP)
proteins were resolved by 10% SDS-PAGE and immunoblotted with anti-TLS
antibody. Western blot (WB) analysis of TLS and p65 protein
levels in transfected cell extracts was performed. A portion of each
whole-cell extract was separated by 10% SDS-PAGE and immunoblotted
with anti-TLS and anti-p65 antibody (Fig. 5C,
lower panels).
|
|
To further confirm the interaction of TLS with p65 upon TNF-
signaling, the cell extract was prepared from 293 cells that were
transfected with TLS expression plasmid or pCMV control plasmid in the
presence or absence of TNF-
stimulation. The cell extract was
immunoprecipitated with anti-p65 antibody, and the immunoprecipitated proteins were separated on SDS-PAGE, transferred to a membrane, and
probed by anti-TLS antibody. As demonstrated in Fig. 5C, TLS was coimmunoprecipitated with p65 in cells stimulated by TNF-
(lane 2). A barely detectable level of TLS was
coprecipitated with p65 when cells were not stimulated by TNF-
(lane 1). No coimmunoprecipitation was detected
when control IgG was used (lane 3). In addition,
TNF-
stimulation did not alter the protein levels of p65 and TLS as
demonstrated by immunoblotting using relevant antibodies (Fig.
5C, lower panels). These results
indicate that TLS interacts with p65 upon physiological signaling for
the activation of NF-
B.
 |
DISCUSSION |
NF-
B subunit p65 contains at least two independent
transactivation domains, TA1 and TA2, located adjacently in the
C-terminal region. Although TA2 contains TA1-like domain, additional
regions are required for its full activity. For example, a previous
study demonstrated that the region between the TA1-like domain of TA2 and the TA1 domain (amino acids 477-521 of p65) was necessary for the
activity of TA2 although not sufficient for the transcriptional activity (16). Using the C-terminal portion of p65 (286-442/477-521) as a bait in the yeast two-hybrid screen, we have identified TLS as an
interacting protein. We have further narrowed down the minimal region
of p65 (amino acids 477-521) necessary for the interaction with TLS
(Fig. 1). Further molecular genetic studies have revealed that TLS acts
as a mediator of the NF-
B transactivation (Figs. 4 and 5).
TLS shares a common feature with a subgroup of TAFII
proteins including hTAFII68/RBP56 and EWS. These proteins
have been found associated with TFIID complexes (32, 37) and are
implicated in transcriptional activation (33, 34, 43, 46). TFIID is a
heterologous multiprotein complex consisting of TBP and a large number
of TAFIIs (47-51). Some of the gene-specific
transactivators are known to interact with distinct TAFIIs
through direct interaction with the activation domain (52, 53). For
example, Sp1, estrogen receptor, vitamin D receptor, p53, and p65 were
shown to interact with dTAFII110 (54), hTAFII30
(55), TAFII135 (56),
TAFII40/TAFII60 (57), and TAFII105
(26, 27), respectively. Other TAFII proteins such as
hTAFII250, hTAFII80, and hTAFII28
were also shown to bind to the C-terminal transactivation domain of p65
at least in vitro (58). In this study, we also observed that
TLS was coimmunoprecipitated with TFIID as well as with p65 in cultured
cells (data not shown). These findings suggest that TLS may facilitate
gene expression through bridging between p65 and basal transcriptional machinery.
TLS was originally identified as a fusion protein with CHOP in human
liposarcoma and later found in other malignancies such as acute myeloid
leukemia, in which the N terminus of TLS was fused to the C-terminal
region of ERG (30, 31, 36). The fusion with the N-terminal half of TLS
was suggested to be a prerequisite for these transcriptional activators
to have the oncogenic potential by augmenting their activities and/or
by changing the target gene specificity (43, 46). In fact, our present
study demonstrated that the N-terminal half of TLS exhibited a strong
transcriptional activity when fused to Gal4-DBD (Fig. 2). The
C-terminal region of TLS, which is often replaced by the DNA-binding
domain of transcription factors through chromosomal translocation, also
showed a strong transcriptional activity by fusion with Gal4-DBD.
Therefore, it was suggested that the counterpart of chromosomal
translocation involving TLS might also acquire the transcriptional
competence. Although our results indicated that TLS acts as a
transcriptional activator, additional functions were suggested by other
studies. For example, TLS binds to RNA in a sequence-independent way
in vitro and in cells (30, 33, 59) and engages in rapid
nucleocytoplasmic shuttling (43, 59). These features, together with the
fact that TLS associates with a subpopulation of the TFIID complex in
cells (32, 37), suggest that TLS may participate in both transcriptional regulation and mRNA export by participating in heterogeneous nuclear ribonucleoprotein formation (60).
Interestingly, we previously demonstrated that the same region (amino
acids 477-521) within the p65 transactivation domain interacted with
AES (7). We found that AES-mediated repression of p65-mediated
transactivation was down-regulated by TLS, and vice versa
(data not shown). Although additional experiments are needed to compare
the binding affinity of TLS and AES with p65, it is likely that the
transcriptional activity of NF-
B is regulated through the selective
binding of interacting proteins with opposing actions such as TLS and
AES.
In conclusion, these findings suggest a multiplicative mode of TLS
actions in regulation of gene expression. The capability of NF-
B to
associate with TLS, in addition to other basal transcription factors
including TFIIB (15), TBP (23, 25), TAFII105 (26, 27), and
CBP/p300 coactivators (19, 20), may be attributable to its strong
transcriptional activity as well as its susceptibility to various
transcriptional repressors such as silencing mediator of retinoic acid
and thyroid hormone receptors (61) and AES/TLE1 (7).