Journal of Virology, October 2001, p. 9885-9895, Vol. 75, No. 20
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.9885-9895.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Center for Retrovirus Research and Department of Veterinary Biosciences,1 Comprehensive Cancer Center, The Arthur James Cancer Hospital and Research Institute,3 and Department of Molecular Virology, Immunology, and Medical Genetics,4 The Ohio State University, Columbus, Ohio 43210, and Department of Biochemistry and Molecular Biology, George Washington School of Medicine and Health Sciences, Washington, D.C. 200522
Received 17 January 2001/Accepted 18 July 2001
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ABSTRACT |
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The highly conserved coadapters CREB binding protein (CBP) and p300 form complexes with CREB as well as other DNA binding transcription factors to modulate chromatin remodeling and thus transcription. Human T-lymphotropic virus type 1 (HTLV-1) transcription is controlled, in part, by the CREB/ATF family of transcription factors which bind promoter sequences and function as complexes with the viral oncogenic protein Tax. We have reported that the nuclear localizing protein p30II of HTLV-1 functions as a transcription factor, differentially modulates CREB-responsive promoters, and is critical for maintenance of proviral loads in rabbits. In this study, we tested whether p30II directly interacts with CBP/p300 to modulate gene transcription. Gal4(BD)-p30II-mediated transactivation was enhanced following exogenous expression of p300 and was competitively repressed by the p300 binding protein, adenovirus E1A, and E1ACR2 (mutated for retinoblastoma binding but retaining p300 binding). In contrast, E1ACR1 (mutated for p300 binding) failed to alter Gal4(BD)-p30II-mediated transactivation. In addition, Gal4(BD)-p30II-mediated transactivation was competitively inhibited by the cotransfection of CMV-p30II-HA and CMV-Tax but could be rescued by exogenous p300. Importantly, we demonstrate that p30II colocalizes with p300 in cell nuclei and directly binds to CBP/p300 in cells. Deletion mutants of CBP/p300 were used to localize the site critical for binding p30II to a highly conserved KIX region. DNA binding assays confirmed the interference of p30II with the assembly of CREB-Tax-p300/CBP multiprotein complexes on 21-bp repeat oligonucleotides in vitro. Collectively, our results demonstrate that CBP/p300 is a cellular protein target for HTLV-1 p30II and mediates its transcriptional effects in vivo.
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INTRODUCTION |
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The coactivators CREB binding
protein (CBP) and p300 mediate transcriptional control of various
cellular and viral DNA binding transcription factors. These
coactivators are highly similar in nucleotide sequence, are
evolutionarily conserved, and are often referred to together as
CBP/p300, despite evidence of divergent functions (10,
25). These proteins bridge transcription factors to relevant
promoters, have intrinsic histone acetyltransferase (HAT) activity,
and form complexes with proteins such as CBP/p300 binding
protein-associated factor, which also exhibits HAT activity (26). Recent reviews provide a growing list of cellular
and viral proteins that interact with either CBP or p300, including steroid and retinoid hormone receptors, CREB, c-Jun, c-Myb,
Sap-1a, c-Fos, MyoD, p53, Stat-1/2, NF-
B,
pp90rsk, TATA-binding protein, and TFIIB
(4, 25, 29, 30). Among viral regulatory proteins, human
T-lymphotropic virus type 1 (HTLV-1) Tax, adenovirus E1A, Kaposi's
sarcoma-associated herpesvirus viral interferon regulatory
factor protein, and simian virus 40 large T antigen also target
and affect CBP and p300 functions (1-3, 17, 32, 38, 41,
52).
Complex retroviruses, like HTLV-1, must regulate their gene expression in cooperation with host cell transcription factors including CBP/p300. HTLV-1 encodes typical gag, pol, and env gene products as well as unique regulatory and accessory genes encoded in four open reading frames (ORFs) (pX ORFs I to IV) between the env gene and the 3' long terminal repeat (54). ORFs IV and III of HTLV-1 encode the well-characterized Tax and Rex proteins, respectively. Tax, a 40-kDa nuclear localizing phosphoprotein, mediates multiple virus-cell interactions by increasing viral transcription and by influencing cell proliferation, apoptosis, DNA repair, and cell cycle control (9, 33, 43, 44, 49). Rex is a 27-kDa nucleolar localizing phosphoprotein that increases the cytoplasmic accumulation of nonspliced and singly spliced viral RNA (18, 37, 51).
A number of alternatively spliced mRNAs are expressed from the pX region of HTLV-1 and have been identified from infected cell lines and freshly isolated cells from HTLV-1-infected subjects (11, 15, 36). Recently, serum antibodies and cytotoxic CD8+ T cells from HTLV-1-infected individuals have been demonstrated to recognize pX ORF I- and II-derived proteins, indicating that these viral proteins are expressed in vivo (19, 48). The HTLV-1 pX ORF II mRNA is spliced from the first tax exon and encodes two proteins, p30II and p13II. The smaller protein, p13II, is derived from initiation at the first internal methionine codon in ORF II and represents the 87 carboxyl-terminal residues of p30II. The p30II and p13II proteins are localized to the nucleus and mitochondria, respectively (16, 35, 57). p30II contains serine- and threonine-rich regions with distant homology to transcription factors Oct-1 and -2, Pit-1, and POU-1 (15). It has recently been reported that mutations in a viral clone, which destroys the initiator methionine of the mRNA encoding p13II and inserts an artificial termination codon in the mRNA encoding p30II, reduce proviral copy numbers up to 100-fold in rabbits (8). We have subsequently demonstrated that HTLV-1 p30II functions as a transcription factor, has opposing effects compared to Tax, and differentially modulates CREB-responsive promoters (57).
In this study, we sought to identify protein-protein interactions that mediate the transcription effects of p30II. Our previous work demonstrated that minimal promoter units (independent of enhancer elements) are equally influenced by p30II, and thus, we hypothesized that CBP/p300 may interact with p30II. Our data using a Gal4(BD)-p30II-mediated transactivation assay indicated that exogenous p300 enhanced the effects of p30II in transcription, whereas the p300 binding protein, wild-type adenovirus 12SE1A, and its CR2 mutant (retinoblastoma [Rb] binding mutant), but not the CR1 mutant (p300 binding mutant), reduced the effects of p30II. Gal4(BD)-p30II-mediated transactivation was also competitively inhibited by the cotransfection of CMV-p30II-HA and CMV-Tax and was rescued by exogenously expressed p300. Using immunoprecipitation and glutathione S-transferase (GST) pull-down assays, direct physical interaction between p30II and CBP/p300 was demonstrated in cells. The p30II and CBP/p300 association was confirmed by immunohistochemistry. Deletion mutants of CBP/p300 in GST pull-down assays and in functional Gal4-mediated transcription assays were used to localize the binding site of CBP/p300 for p30II to a highly conserved KIX region. Furthermore, we demonstrated that p30II inhibited the ability of CREB to bind its DNA target using biotinylated DNA precipitation assays. Taken together, our data are the first to demonstrate that CBP/p300 is a cellular protein target for HTLV-1 p30II and that these important cellular coadapters mediate the viral protein transcriptional effects in cells.
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MATERIALS AND METHODS |
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Cell lines. All cultured cells (HEK 293 cells were obtained from the American Type Culture Collection [no. CRL-1573]; HEK 293T cells were obtained from G. Franchini [National Cancer Institute, Bethesda, Md.]) were grown in 10-cm-diameter tissue culture dishes in Dulbecco's minimal essential medium containing 10% fetal bovine serum and 1% streptomycin and penicillin at 37°C. Cells were split and cultured in six-well plates to 50% confluence 16 h before transfection according to the manufacturer's protocol (Lipofectamine Plus; Gibco BRL).
Plasmids.
The luciferase reporter plasmid p5XG-E1b-Luc was a
kind gift of Y. Shi (Harvard Medical School, Cambridge, Mass.) and
contains five tandem Gal4 DNA binding sequences upstream of a TATA box derived from plasmid pE1b-CAT (50). The plasmids pCRE-Luc,
pTRE-Luc, and pRSV-
-Gal-Luc have been described previously
(57). The Gal4 effector plasmid pCMV-Gal4 (DNA binding
domain, amino acids [aa] 1 to 147)-p30II
[Gal4(BD)-p30II] was constructed by subcloning
the p30II-encoding sequence into a pCMV-Gal4(BD)
vector (Stratagene). The p30II-encoding sequence
was synthesized by PCR amplification with 5' primer
ATATGAATTCATGGCACTATGCTGTTTCGCC(5'-A) and 3'
primer TATACTGCAGTAGAGGTTCTCGGGTG (3'-A) from
the HTLV-1 molecular clone ACH (34), including the 5' EcoRI and 3' PstI restriction sites
(underlined). ACH was also used as a template to synthesize
p30II-encoding sequences for all subsequent
p30II expression constructs using appropriate
restriction sites as indicated. pBC-p30II, a
GST-p30II fusion protein expression vector, was
constructed by subcloning the same p30II-encoding
sequence into the vector pBC (12) using NdeI
and NheI restriction sites.
pBluescript-p30II
(pBS-p30II), a T7 and T3 promoter-driven
p30II expression vector, was constructed by
subcloning the p30II-encoding sequence into the
pBS vector (Stratagene) using EcoRI and PstI
restriction sites. All of the p30II expression
vectors were sequenced to confirm correct reading frames.
pCMV-p30II-HA, a p30II-HA
expression vector, was a kind gift of G. Franchini (National Cancer
Institute). Adenovirus E1A expression vectors including wild-type
12SE1A, pRSV-12SE1A, and two deletion mutants, pRSV-12SE1A
CR1 and
pRSV-12SE1A
CR2, were kindly provided by T. Kouzarides (University of
Cambridge, Cambridge, United Kingdom). pCMV-Tax expresses the HTLV-1
Tax protein and has been described previously (45).
pCMV-p300 expresses the full-length p300 protein and was kindly
provided by A. Leiter (Tufts University School of Medicine,
Boston, Mass.). All bacterial GST-p300 and GST-CBP expression
vectors have been described previously (20). pRSV-KIX (aa
379 to 654)/p300 expresses the KIX domain of p300 and was a gift of G. Louis (CNRS-University Claude Bernard, Lyon, France).
Cell transfection and reporter gene assay.
For each
transfection, 0.3 to 0.9 µg of reporter vectors was cotransfected
with various amounts of effector plasmids using Lipofectamine Plus
(Gibco BRL) (57). As an internal control for transfection
efficiency, 0.1 µg of pRSV-
-Gal (Gibco BRL) was also used in each
transfection. pBluescript (Stratagene) was used as carrier DNA to
equalize DNA concentrations for each transfection. Transfected cells
were lysed with 1× lysis buffer (Promega) using 0.4 ml of buffer per
well at room temperature for 25 min. Twenty microliters of each lysate
was used to test luciferase reporter gene activity using an Enhanced
Luciferase Assay kit (Promega). To normalize transfection experiments,
5 µl of each lysate was assayed for
-galactosidase activity
according to the manufacturer's protocol (Lumigen). Results were
expressed as mean fold increases or percentages of change ± standard deviations (SD) in arbitrary light units (ALU) of luciferase
activity in four independent trials set up as duplicates in each
experimental trial.
Western immunoblot assay. Transiently transfected cells were lysed in RIPA buffer containing 1× phosphate-buffered saline (PBS), 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Cell lysates were prepared by centrifugation at 14,000 rpm (Beckman) for 20 min at 4°C. Equal amounts of proteins were mixed with Laemmli buffer (62.5 mM Tris [pH 6.8], 2% SDS, 10% glycerol, 0.2% bromophenol blue, 100 mM dithiothreitol [DTT]). After boiling for 5 min, samples were electrophoresed through 6 or 10% polyacrylamide gels. The fractionated proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotechnology) at 100 V for 1 h at 4°C. Membranes were then blocked in Tris-buffered saline containing 5% nonfat milk and 0.1% Tween 20. Proteins were detected with the appropriate primary antibody followed by an anti-rabbit or anti-mouse (NEN Life Science) immunoglobulin G (IgG)-horseradish peroxidase-conjugated goat antibody. Blots were developed using an enhanced chemiluminescence detection system (NEN Life Science).
Biotin-labeled DNA pull-down assay.
To examine the effects
of p30II on the recruitment of CREB and p300 by
HTLV-1 Tax into a multiprotein complex bound to the HTLV-1 21-bp repeat
DNA, we labeled 1.5 µg of the annealed oligonucleotides 5'-GATCTGGGCGTTGACGACAACCCCTCACCTCAAAAAACTTTC-3'
and
5'-TTTGAAAGTTTTTTGAGGTGAGGGGTTGTCGTCAACGCCCAGATC-3' (the HTLV-1 21-bp repeat sequence is shown in bold) with
biotin-14-dATP (Life Technologies, Inc.) using 10 units of Klenow (New
England Biolabs) at 37°C for 30 min. Labeled oligonucleotides were
electrophoresed and purified from a 7.5% Tris-borate-EDTA acrylamide
gel, eluted in 250 µl of deionized distilled water, and
quantified. About 40 ng of labeled DNA (20 µl) was added to 100 µl of nuclear lysate from 293T cells transfected with pCMV-Tax,
pCMV-p300, and pCMV-p30II-HA, as indicated in the
figure legends, in a total volume of 200 µl of binding buffer (25 mM
HEPES [pH 7.9], 5 mM KCl, 0.5 mM MgCl2,
0.5 mM EDTA, 1 mg of bovine serum albumin per ml, 10% [vol/vol]
glycerol, and 0.25 mM DTT). The nuclear mixtures with biotin-labeled
oligonucleotide probes were preincubated on ice for 2 h with
gentle agitation. Sixty microliters of a 50% slurry of washed
streptavidin-agarose (Life Technologies) was added to the nuclear
lysates and kept on ice for 1 h with gentle agitation. The bound
matrices were collected, washed twice with 500 µl of binding buffer,
pelleted by centrifugation, and resuspended in 40 µl of
SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer. Each
sample was heated at 95°C for 5 min and loaded onto a precast 4 to
20% Tris-glycine-acrylamide gel (Bio-Rad) for resolution of bound
products. After electrophoresis, the gels were transferred to
nitrocellulose membranes for immunoblotting to detect CREB, Tax,
p30II-HA, and CBP/p300. CREB was detected with a
polyclonal antibody against CREB (diluted 1:1,000; New England
Biolabs), Tax was detected with a polyclonal anti-Tax antibody (diluted
1:500, Tax
-serum 6505; National Institutes of Health AIDS Research
and Reference Reagents Program), and p30II-HA was
detected by polyclonal anti-HA antibody (diluted 1:1,000; Babco). p300
was detected using a polyclonal anti-p300 antibody (diluted 1:1,000;
Santa Cruz Biotechnology, Inc.). A horseradish peroxidase-conjugated
anti-goat serum was used as secondary antibody (diluted 1:1,000; Santa
Cruz Biotechnology, Inc.).
Coimmunoprecipitation of p30II with p300. Sixty percent confluent 293T cells were cotransfected by pCMV-Gal4(BD)-p30II and pCMV-p300 or pCMV-p30II-HA and pCMV-p300 by calcium phosphate coprecipitation. After 48 h, the transfected cells were washed with PBS and resuspended in 400 µl of lysis buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 µg of leupeptin per ml, and 1 µg of aprotinin per ml. Cell suspensions were incubated on ice for 20 min and then lysed by homogenization. The lysates were centrifuged at 14,000 rpm (Beckman) for 20 min at 4°C. Supernatants were precleared by incubation with 10 µl of control rabbit IgG antibody and 30 µl of 50% protein A-agarose slurry (Amersham Pharmacia Biotechnology) for 1 h at 4°C. After centrifugation at 3,000 rpm (Beckman) for 1 min, the cleared supernatants were incubated overnight at 4°C with 100 ng of a polyclonal antibody against Gal4(BD) (Santa Cruz Biotechnology, Inc.) or 100 ng of a polyclonal antibody against hemagglutinin (HA) (Babco). After adding 40 µl of 50% of protein A-agarose slurry, the mixture was incubated at 4°C for 1 h. The immunoprecipitated complexes were washed twice with 10 volumes of lysis buffer and three times with PBS buffer. The components of the complexes were resolved on SDS-polyacrylamide gels [either 10% for Gal4(BD)-p30II and p30II-HA or 6% for p300] and detected by Western immunoblot assay.
Colocalization of p30II and CBP/p300 by immunofluorescence assay. To detect cellular colocalization of p30II and p300 by immunofluorescence, 293T cells were seeded in chamber slides (Fisher Scientific) at approximately 40% confluence 18 h prior to transfection. Transfection with 4 µg of pCMV-p30II-HA and 2 µg of pCMV-p300 was performed using Lipofectamine Plus (Gibco BRL). At 48 h posttransfection, media were removed and cells were fixed for 15 min using 4% paraformaldehyde at room temperature. Cells were then incubated with monoclonal anti-HA antiserum (diluted 1:500; Babco) and polyclonal p300 antiserum (diluted 1:1,000; Santa Cruz Biotechnology, Inc.) overnight at 4°C followed by incubation with indocarbocyanine-labeled anti-mouse immunoglobulin (diluted 1:1,000; Jackson Immunogen) and Alexa 488 goat anti-rabbit IgG (diluted 1:1,000; Molecular Probes) for 1 h at room temperature. The expression of p30II-HA and p300 was evaluated by immunofluorescence microscopy (Axioplan2; Zeiss). A digital camera (Diagnostic Instruments, Inc.) was used to produce standard light microscopic and immunofluorescent photomicrographs.
GST-p30II fusion protein pull-down assay. Eighty percent confluent 293T cells were transfected with 15 µg each of pBC-p30II and pCMV-p300 by calcium-phosphate coprecipitation. At 48 h posttransfection, cells were washed and resuspended in 400 µl of GST binding buffer containing 50 mM Tris-HCl, 150 mM NaCl, 0.5% NP-40, 5% glycerol, 1 mM PMSF, 0.5 µg of leupeptin per ml, and 1 µg of aprotinin per ml. After being kept on ice for 30 min, the cells were lysed by homogenization. Cell lysates were cleared by centrifugation at 14,000 rpm (Beckman) for 20 min at 4°C. Fifty microliters of a 50% glutathione-Sepharose slurry (Amersham Pharmacia) was added to the cell lysates, and the mixture was incubated for 1.5 h at 4°C. The GST beads were washed four times using GST washing buffer (50 mM Tris-HCl, 250 mM NaCl, 1.0% NP-40, 1 mM PMSF, 1 mM DTT, and 5% glycerol) and eluted in Laemmli buffer by boiling for 4 min. Proteins were electrophoresed in 4 to 20% gradient polyacrylamide gels and analyzed by Western immunoblot assay with anti-p300 antibody as the primary antibody (Santa Cruz Biotechnology, Inc.)
GST-CBP/p300 fusion protein pull-down assay.
To determine
the binding region of CBP/p300 with p30II, GST
fusion proteins containing defined regions of each protein were expressed in Escherichia coli BL21 cells using plasmids
expressing either GST, GST-p300 (aa 1 to 159), GST-p300 (aa 744 to
1540), GST-p300 (aa 1540 to 2368), GST-CBP (aa 1 to 250), GST-CBP (aa 251 to 450), GST-CBP (aa 451 to 682), or GST-CBP (aa 1 to 717) (20). Protein expression was induced with 0.5 mM
isopropyl-
-D-thiogalactopyranoside (IPTG) for
4 h during the exponential growth phase of the bacterial culture.
The bacteria were harvested, pelleted, and resuspended in 25 ml of PBS,
pH 7.4, containing 1 mg of lysozyme per ml and 1 mM PMSF. After an
incubation for 20 min at 4°C with gentle shaking, cells were ruptured
by mild sonication. After centrifugation to remove cell debris, 2 ml of
GST fusion protein-containing supernatant was mixed with 100 µl of
50% glutathione-Sepharose bead slurry (Amersham Pharmacia
Biotechnology) for 2 h at 4°C. After extensive washing five
times in lysis buffer, the glutathione-agarose beads were resuspended
in 200 µl of GST binding buffer and mixed with 35 µl of reaction
mixture containing 35S-labeled
p30II synthesized by the T3-driven
pBS-p30II vector using an in vitro transcription
and translation kit (Promega). After incubation at 4°C for 2 h
with gentle agitation, glutathione-agarose beads were collected by
centrifugation at 3,000 rpm (Beckman) for 1 min. After washing
three times with GST binding buffer, the GST-agarose beads were
resuspended in Laemmli buffer and subjected to SDS-10% PAGE.
Gels were dried at 80°C for 1 h, and 35S
labeled p30II was detected by autoradiography.
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RESULTS |
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Exogenous p300 enhances Gal4(BD)-p30II-mediated
transactivation.
It has been reported that
p30II functions as a transcription factor by
using both Gal4-driven and CREB-responsive element (CRE)-driven reporter gene assays (57). When fused with Gal4(BD),
p30II enhances Gal4-driven reporter gene
expression by promoting transcription at core TATA box sites. One
potential mechanism by which Gal4(BD)-p30II
may modulate transcription is through interaction with common coadapters of transcription, such as CBP/p300. To test this
possibility, we used a mammalian two-hybrid assay to determine
potential synergistic activation between
Gal4(BD)-p30II and p300. This functional
assay was designed to show novel physical interactions between proteins
regulating adenovirus virus transcription in vivo (21). We
reasoned that, if p30II and p300 physically
interact with each other,
Gal4(BD)-p30II-mediated Gal4 reporter gene
activities using the adenovirus E1B promoter system would also be
stimulated in the presence of the chimeric protein p300-Vp16 (Fig.
1A) because the interaction of p30II and p300 would recruit Vp16 to the promoter
region of the reporter gene. Vp16 as a chimeric protein with p300 is a
potent stimulator of transcription and has been useful for identifying
proteins that stabilize the transcription complex. Our data using this assay indicated that the Gal4 reporter gene activities were clearly increased by cotransfection of the p300-Vp16 vector and
Gal4(BD)-p30II (Fig. 1B).
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12SE1A protein competitively inhibits
Gal4(BD)-p30II-dependent transactivation.
Adenovirus E1A protein is a multifunctional viral protein that
modulates gene transcription by interacting with several key cellular
proteins. It is well documented that adenovirus 12SE1A protein can
inactivate p300-mediated gene transactivation by directly binding p300
(55). Since our results suggested that p300 stimulated Gal4(BD)-p30II-mediated transactivation, we
tested the possibility that 12SE1A could competitively inhibit
Gal4(BD)-p30II-dependent transactivation. We
cotransfected vectors that expressed either wild-type E1A (12SE1A) or
two deletion mutants which lack the ability to bind p300 (12SE1A
CR1)
or pRb, a cell cycle regulatory protein (12SE1A
CR2), with
CMV-Gal4(BD)-p30II in our Gal4-Luc
reporter gene system (Fig. 2A). Our data
indicated that Gal4(BD)-p30II-mediated
transactivation of our Gal4-Luc reporter gene activities was inhibited
in the presence of increasing concentrations of a pRSV-12SE1A
expression vector (Fig. 2B), whereas the control RSV vector had no
effect on Gal4(BD)-p30II transactivation
(data not shown). In contrast, RSV-12SE1A
CR1, which lacks N-terminal
amino acid residues (aa 40 to 80) necessary for its interaction with
p300, did not inhibit Gal4(BD)-p30II-mediated
transactivation when transfected at levels comparable to that of the
wild-type 12SE1A expression vector (Fig. 2C). Cotransfection with
12SE1A
CR2, which lacks C-terminal amino acid residues (aa 120 to
140) corresponding to the region that interacts with pRb, resulted in
repression of Gal4(BD)-p30II-mediated
transactivation similar to that of wild-type 12SE1A (Fig. 2D). These
data are consistent with previous reports of 12SE1A inhibition of
CBP/p300-dependent transcription (1), and the data further
implicated p300 as an essential member of a functional complex with
p30II in transcriptional regulation.
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Transactivation by Gal4(BD)-p30II through p300. Our data suggested that Gal4(BD)-p30II-mediated transactivation is dependent upon p300. CBP/p300 has been shown to interact with a variety of transcriptional activators, including Tax of HTLV-1 (23, 38). In this context, CBP/p300 serves as a coadapter between these activators and the RNA polymerase II transcriptional machinery. We reasoned, therefore, that if CBP/p300 does participate in Gal4(BD)-p30II-dependent transactivation, overexpression of those transcription factors should competitively repress the transactivation by Gal4(BD)-p30II in our Gal4-Luc reporter gene assay by reducing the available CBP/p300 pool. In contrast, by adding exogenous p300 back to the cell using transient transfection, the repression should be reversed.
We tested the necessity of p300 in Gal4(BD)-p30II-mediated transactivation using in vivo competition assays as previously described (13, 22). In this assay, saturating concentrations of p30II-HA, Tax, and E1A expression vectors were introduced by cotransfection with the Gal4-p30II expression vector in our Gal4-Luc reporter gene assay. As predicted, transactivation by Gal4(BD)-p30II was repressed by cotransfection with p30II-HA, Tax, and E1A (Fig. 3A, lanes 2, 4, and 6). However, the repression by each of the expression vectors was restored by transfection with pCMV-p300 (Fig. 3A, lanes 3, 5, and 7). Consistent with our previous data, the cotransfection of pCMV-p300 with pGal4(BD)-p30II markedly enhanced Gal4-mediated transcription (Fig. 3A, lane 8). Similar results were obtained in parallel assays performed using 293 cells (Fig. 3B), which indicated that T-antigen expression in 293T cells did not influence the interaction of p30II with p300. Importantly, our results were also confirmed using Jurkat T cells (Fig. 3C). Collectively, these data indicated that p300 actively participates in Gal4(BD)-p30II-mediated transactivation.
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p300 expression reverses p30II inhibition of CRE- and
TRE-driven gene transcription.
We have reported that
p30II, in contrast to Tax, repressed cellular
CRE-driven reporter gene activity and only at lower concentrations enhanced the HTLV-1 long terminal repeat Tax-responsive element (TRE)-driven reporter gene activity (57). Our results
suggested that p30II and Tax serve divergent
roles in the regulation of transcription. The coadapters CBP and
p300 have been demonstrated to be integral components of HTLV-1
Tax transactivation. Since our data indicated that
p30II interacts with p300, we postulated that
p30II might repress basal CRE- and TRE-driven
reporter gene activities as a consequence of competition for limited
basal quantities of CBP/p300. In this scenario, overexpression of p300
should prevent p30II-mediated repression of CRE-
and TRE-driven reporter gene expression. To test this possibility, we
performed cotransfection studies using luciferase reporter plasmids
driven by either the CRE (CRE-Luc) or the TRE (TRE-Luc) promoter in the
presence of increasing concentrations of p30II.
As has been reported (57), both reporter gene activities
were repressed by p30II in a dose-dependent
manner. Then we attempted to reverse the p30II
repressive effects by cotransfection with increasing concentrations of
the pCMV-p300 expression vector. A fixed concentration of
pCMV-p30II-HA expression vector (0.9 µg) was
cotransfected with increasing amounts of pCMV-p300 in the presence of
pCMV-CRE-Luc (Fig. 4A) or pTRE-Luc vector
(Fig. 4B). As has been reported (57), our CMV empty vector
at identical concentrations did not influence either CRE- or
TRE-mediated transcription. As predicted,
p30II-dependent repression of both CRE- and
TRE-responsive promoters was completely restored by increasing amounts
of exogenous p300. These data further support our hypothesis that p300
mediates p30II-dependent transcriptional
activity.
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p30II disrupts the assembly of CREB-Tax-p300 complexes
on TRE probes.
As a coadapter CBP/p300 functions to stabilize the
multiple protein complex that includes CREB, Tax, TATA binding protein, and other transcription machinery molecules on CRE and TRE promoters, thereby promoting transcription (23, 38, 40). To test the direct effect of p30II on the assembly of these
complexes, we expressed Tax and p300 in the presence of increasing
amounts of pCMV-p30II-HA. Nuclear lysates from
transfected cells were tested for the presence of multiprotein
complexes formed on biotin-labeled HTLV-1 21-bp repeat oligonucleotides
(Fig. 5). Increasing amounts of expressed
p30II had no effect on the expression of CREB,
Tax, or p300 (Fig. 5, lanes 1 to 4). However, increasing concentrations
of p30II dramatically reduced the binding of
CREB, Tax, and p300 to the biotin-labeled 21-bp repeat oligonucleotide
(Fig. 5, lanes 5, 6, 7, and 8). These data are consistent with
the sequestration of p300 by p30II and suggest
this tenet as a mechanism for the repression of basal TRE reporter
gene activities by p30II.
p30II did not bind directly to CRE and TRE
cis elements in electrophoretic mobility shift assays
using probes containing tandem repeats of consensus CRE or
HTLV-1 21-bp repeat TRE sequences (data not shown). Taken
together, our data indicated that p30II
negatively influences the assembly of multiprotein complexes at the
HTLV-1 21-bp repeat TRE site.
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p30II directly binds p300/CBP in vivo.
Our data
strongly suggested that p30II-mediated
transcription is dependent, at least in part, upon CBP/p300. To test
the cellular localization of each protein, we performed
immunofluorescence microscopy following the cotransfection of
pCMV-p30II-HA and pCMV-p300 in 293T cells.
As expected, p30II-HA was expressed in
the nucleus of cells and overlapped with p300 expression in cells that
expressed both proteins (Fig. 6A). To
test if p30II physically interacted with CBP/p300
in vivo, we cotransfected 293T cells with
pBC-p30II, pCMV-p30II-HA,
or pCMV-Gal4(BD)-p30II together with
pCMV-p300. Whole-cell extracts were prepared and used in GST pull-down
or coimmunoprecipitation assays. Following binding to
glutathione-Sepharose beads, GST-p30II
effectively pulled down p300 from cellular lysates, unlike the GST-vector control (Fig. 6B, compare lanes 2 and 3). p300, when coexpressed with pCMV-p30II-HA or
pCMV-Gal4(BD)-p30II, was immunoprecipitated
as a complex by using both anti-HA (Fig. 6B, lane 6) and
anti-Gal4(BD) antibodies (Fig. 6B, lane 9). In contrast, p300 could
not be detected in the complexes precipitated by using preimmune serum
(Fig. 6B, lanes 5 and 8). All three p30II fusion
proteins could be detected from their respective cellular lysates (Fig.
6B, lower panel, lanes 1 and 3 to 9). In addition, p30II failed to bind CREB directly when tested in
immunoprecipitation and Western blot assays (data not shown).
|
p30II interacts with CBP/p300 through the KIX
domain.
We next tested which domains of CBP/p300 bind with
p30II by expressing eight GST-CBP/p300 fusion
proteins containing defined portions of p300 or CBP (Fig.
7A and B). To improve the sensitivity for
detection of p30II, we synthesized and labeled
p30II with
[35S]methionine using an in vitro transcription
and translation system (46). After incubation of
glutathione-Sepharose beads bound with GST fusion proteins with
comparable amounts of in vitro-synthesized 35S-labeled p30II, bound
proteins were separated by SDS-PAGE and detected by autoradiography. Among the proteins pulled down by GST-Sepharose beads,
35S-labeled p30II could
only be detected in GST-p300, aa 1 to 595; GST-CBP, aa 451 to 682; and
GST-CBP, aa 1 to 717 (Fig. 7C). These data suggested that the region
located between aa 461 and 662, corresponding to the KIX domain, of
CBP/p300 contributed to the interaction between
p30II and CBP/p300.
|
|
| |
DISCUSSION |
|---|
|
|
|---|
It is well documented that CBP and p300, as coactivators, participate in the activation of gene expression by bridging upstream transcription factor complexes with the general transcription machinery. Furthermore, CBP/p300 has intrinsic histone acetylase activity (7) to potentially increase the state of nucleosome acetylation, which is essential for the initiation of gene transcription. In addition, CBP and p300 are general integrators of signal-dependent transcription, and a diverse array of enhancer binding factors (e.g., CREB) utilizes these coactivators for transcriptional activation in response to extracellular signals (24, 28, 39).
Previous studies have demonstrated that p30II is a transcription factor and differentially modulates CREB-responsive gene activity (57). As a Gal4 fusion protein, p30II transactivates Gal4 reporter gene activity. However, unlike Tax, p30II could also repress CRE and TRE reporter gene activity in a dose-dependent manner. We hypothesized that p30II might modify target gene activities by interacting with common coactivators like CBP and p300. Our data, herein, demonstrate that p300 enhances p30II-dependent transcription in three different cell culture models, including Jurkat T cells. Like Tax, p30II physically interacted in cells with CBP and p300 through the KIX domain, as shown by our protein binding and transcription assay data. These data are consistent with our hypothesis that p30II modulates gene expression by interacting with CBP/p300, and the data provide a mechanism to explain the p30II-dependent modulation of gene transcription.
There is precedence for a similar transcriptional repression by other viral regulatory proteins. CBP/p300 interactions are important for the biological effects of adenovirus oncoprotein E1A, including the regulation of transcription, suppression of differentiation, and immortalization of cells in culture (1, 3, 42). Simian virus T antigen regulates the expression of a group of cellular genes by modifying the HAT activity of CBP/p300 or by bridging the gap between DNA binding transcription factors and components of the general transcription machinery (4, 25). In the context of HTLV replication, Tax-mediated recruitment of CBP/p300 to the promoter has been shown to be necessary for efficient Tax-mediated transcription of HTLV-1 (23, 38, 53, 56). Our data indicate that p30II also modulates transcription in a unique manner compared to Tax but uses similar interactions with coadapter proteins important for RNA Pol II-mediated transcription.
Our results indicated that p30II acted as a repressor to down-regulate CRE- and TRE-driven reporter gene activities, which is in contrast to the role of HTLV-1 Tax. Tax is critical for high-level HTLV-1 transcription and propagation. To activate viral gene expression, Tax participates in a series of protein-protein and protein-DNA interactions, forming a stable nucleoprotein complex on the HTLV-1 promoter (56). Within this complex, Tax serves as a high-affinity binding site for the recruitment of CBP and p300 (23, 38, 56). Once associated with the viral promoter, CBP/p300 is believed to remodel chromatin and/or facilitate communication with the basal transcription machinery. To recruit CBP/p300, Tax has been shown to specifically bind to a region of CBP/p300 called the KIX domain (27, 32). This region of CBP/p300 also interacts with several other transcription factors, including Ser-133-phosphorylated CREB (14, 39). Tethering of CBP/p300 to the HTLV-1 transcriptional control region promotes the strong transcriptional activation associated with Tax (53). Interestingly, p30II shares this physical interaction with CBP/p300 through the same KIX domain. In addition, we also provide data demonstrating that p30II could destabilize Tax, p300, and CREB multiprotein complexes formed on 21-bp probes in vitro. We believe that the competitive CBP/p300 binding between p30II and Tax might explain how p30II could attenuate the formation of these multiprotein complexes and thereby repress transcription on CREB-responsive promoters. Similarly, Colgin and Nyborg (17) demonstrated that Tax expression interferes with the transcriptional activity of c-Myb and that the binding of Tax and c-Myb to the KIX domain of CBP is mutually exclusive. KIX expression, by itself, enhances the binding of Tax and CREB to CRE-containing oligonucleotides (17) and, as we have demonstrated, would be expected to enhance CRE- and TRE-mediated transcription. Additionally, Tax has been proposed to interfere with CBP-mediated transcription by binding the coactivator (53). Thus, p30II at higher concentrations may serve to promote viral persistence by reducing viral expression, thereby reducing immune elimination of virus-infected cells.
Alternatively, it has been shown that at low concentrations p30II acts to enhance TRE (viral)- over CRE (cellular)-mediated transcription (57). Thus, while promoting viral transcription, this viral protein may competitively repress CBP/p300-dependent cellular gene transcription (e.g., p53-dependent p21WAF1/CIP1 gene activity), promoting cell proliferation (5) and allowing efficient viral spread in vivo. This tenet would explain the data from the rabbit model of HTLV-1 infection, which demonstrated that proviral clones with selective mutations involving pX ORF II (p30II and p13II) fail to maintain normal viral loads in vivo (8). Like Tax and E1A, p30II appears to behave in a sequence-independent manner through sequestration of CBP/p300 (1, 25, 43, 52). Our present studies are focused on the functional significance of the interactions between p30II and CBP/p300, such as the requirement for acetyltransferase activity of CBP/p300 for the transcriptional effects of p30II.
It is noteworthy that the CBP/p300 protein is generally present at limiting concentrations within the cell nucleus, creating an environment for competition between coactivators and transcription factors, thus providing an additional layer of regulated gene expression (47, 52). Several recent studies suggest that a functional antagonism between transcription factors occurs as a consequence of direct competition for binding to common regions of CBP/p300 (6, 17, 31). HTLV-1 p30II, another accessory protein of HTLV-1, is critical for optimal viral loads and differentially modulates CREB-responsive promoters perhaps by the sequestration of cellular CBP/p300. By tightly controlling the level of viral expression in vivo, it is likely that HTLV-1 uses these accessory proteins to avoid the overexpression of viral proteins or to repress cellular genes necessary to maintain viral persistence. It will be important for future studies to define relevant target genes of p30II in addition to CREB-responsive genes to provide a more complete understanding of the role of p30II in the pathogenesis and replication of this important human pathogen.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by National Institutes of Health grant no. RR-14324 from the National Center for Research Resources and grant no. CA-70259 from the National Cancer Institute awarded through the Comprehensive Cancer Center of The Ohio State University. W. Zhang was supported by a David White Fellowship award from The Ohio State University. M. Lairmore was supported by an Independent Scientist Career Award from the National Institutes of Health (K02 AI01474).
We thank Tim Vojt for preparation of figures. We also thank S. Beebe, S. Yang, T. Kouzarides, G. Louis, P. Quinn, G. Franchini, and S. McKnight for valuable reagents. We thank Q. Xi for technical assistance. We also thank P. Green and K. Boris-Lawrie for critical reviews of the manuscript.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210-1093. Phone: (614) 292-4819. Fax: (614) 292-6473. E-mail: lairmore.1{at}osu.edu.
Present address: Center for Molecular Biology and Oral Diseases,
University of Illinois
Chicago, Chicago, IL 60612.
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