Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejeon 305-701, Korea1
Author for correspondence: Joonho Choe. Fax +82 42 869 5630. e-mail jchoe{at}mail.kaist.ac.kr
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Abstract |
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Introduction |
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We performed a yeast two-hybrid assay with full-length LANA as bait to identify cellular proteins that interact with LANA. Among several positive clones was a partial cDNA of activating transcription factor (ATF) 4 (Hai et al., 1989 ), also called cAMP response element (CRE)-binding protein (CREB) 2 (Karpinski et al., 1992
) or TAXREB67 (Tsujimoto et al., 1991
), which is a member of the ATF/CREB family of transcription factors. ATF4/CREB2 was initially characterized as a negative regulatory transcription factor of CRE (Karpinski et al., 1992
). It was later shown to interact with CREB-binding protein (CBP)/p300 co-activators as well as several general transcription factors, including TATA-binding protein, TFIIB and the RAP30 subunit of TFIIF, and it functions as a transcriptional activator (Liang & Hai, 1997
). Members of the ATF/CREB family of transcription factors share a relatively conserved basic region/leucine zipper (bZIP) domain required for direct contact with DNA and homo- and heterodimerization (Sassone-Corsi, 1995
). They also dimerize selectively with the Fos/Jun family of transcription factors (Hai & Curran, 1991
). Through such cross-family dimerization, in addition to homo-/heterodimerization, they extend their DNA-binding specificity to allow differential regulation of transcription.
Here, we report that LANA interacts with ATF4/CREB2 in vivo and in vitro and that the bZIP domain of ATF4/CREB2 is required for the interaction. LANA represses the transcriptional activation activity of ATF4/CREB2 and the ATF4/CREB2-binding domain of LANA is required for repression. Since the bZIP domains of members of the ATF/CREB family have been shown to be targetted by many viral proteins, including adenovirus E1a (Liu & Green, 1994 ), human T-cell leukaemia virus Tax (Gachon et al., 1998
; Bex & Gaynor, 1998
) and hepatitis B virus pX (Maguire et al., 1991
), LANA may represent a conserved strategy for transcriptional regulation during virus infection.
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Methods |
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Yeast two-hybrid assay.
Yeast two-hybrid screening was performed as described previously (Lee et al., 1999 ), except that pLexA LANA was used as the bait plasmid and 10 µg of a B cell cDNA library in the yeast vector pCgatrp was used for screening. Library plasmids containing positive clones were isolated (Lundblad, 1992
), transformed into MC1061/P3 (Invitrogen) and sequenced by using the T7 Sequenase version 2.0 DNA sequencing kit (Amersham Pharmacia Biotech). Positive clones were confirmed by co-transformation with pLexA LANA into the yeast strain EGY048, harbouring the LacZ reporter plasmid p8op-lacZ (Clontech). More than two independent
-galactosidase assays were performed from a liquid culture using ONPG (Sigma) as a substrate (Lundblad, 1992
).
Cell culture, transfection and reporter assays.
HeLa and 293T cells were maintained in Dulbeccos modified Eagles medium supplemented with 10% foetal bovine serum and transfected by the calcium phosphate method (Kingston, 1992 ). Cells of 6070% confluence were plated in 60 mm dishes at 12 h before transfection. The total amount of transfected DNA was adjusted with the appropriate blank vector. Cells were harvested at 36 h post-transfection. Luciferase and
-galactosidase assays were performed according to manufacturers instructions (Promega) and the luciferase activity was normalized with co-transfected
-galactosidase activity. The results are the means of four independent experiments.
In vivo binding assay.
HA-tagged ATF4/CREB2 expression vector (pSR ATF4/CREB2) was co-transfected with either pEBG blank vector or pEBG LANA expression vector into 293T cells. At 36 h post-transfection, the cells were harvested and lysed in ice-cold PBS containing 0·5% Nonidet P-40 and a protease inhibitor cocktail (Roche Molecular Biochemicals) with brief sonication. After precipitating cell debris, the supernatant was incubated with glutathioneSepharose 4B (Amersham Pharmacia Biotech) at 4 °C for 3 h. The beads were washed three times in PBS containing 0·1% Nonidet P-40 and a protease cocktail and then resuspended in a SDS-gel loading buffer. The eluted proteins were separated by 10% SDSPAGE, transferred to nitrocellulose membrane and then immunoblotted and detected by ECL (Amersham Pharmacia Biotech).
GST pull-down assay.
GST fusion proteins were incubated with [35S]methionine-labelled proteins synthesized by the TNT T7 coupled transcription-translation reticulocyte lysate system, according to the manufacturers instructions (Promega). After 3 h incubation at 4 °C in a binding buffer [20 mM TrisHCl (pH 7·5), 50 mM NaCl, 2 mM MgCl2, 1 mM DTT and 0·1% Nonidet P-40], glutathioneSepharose 4B beads were added and incubated further for 1 h at 4 °C. The beads were washed four times in binding buffer and resuspended in SDS-gel loading buffer. The eluted proteins were analysed by SDSPAGE and subjected to autoradiography.
Localization of GFP-fused LANA deletion mutants.
HeLa cells grown on coverslips were transfected with GFP or GFP-fused LANA deletion mutant expression plasmids by the calcium phosphate method, as described above. At 36 h post-transfection, the cells were washed three times and then fixed with formaldehyde (3·7%, v/v) in PBS. After two more washes, the coverslips were mounted and examined with a confocal laser scanning microscope (LSM510, Carl Zeiss).
Production of rabbit anti-LANA polyclonal antiserum.
The C-terminal 212 amino acids of LANA fused to MBP were expressed in bacteria, purified and used in intradermal immunization. One month after the priming immunization with complete Freunds adjuvant (Sigma), four additional boosting immunizations were administered with incomplete Freunds adjuvant at intervals of 1 week. Rabbit anti-LANA polyclonal antiserum diluted 1:500 was used in Western blots of total cell lysates.
Electrophoretic mobility shift assay (EMSA).
Synthetic oligonucleotides containing CRE (5' AAGATTGCCTGACGTCAGAGAGCTAG 3') were labelled with [-32P]ATP by T4 polynucleotide kinase and annealed with the complementary strand. Labelled probe (
100fmol) was incubated with 12·5 ng bacterially expressed GST or GSTATF4/CREB2 in the absence or presence of in vitro-translated LANA in binding buffer [25 mM TrisHCl (pH 7·5), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol and 1 µg poly(dI±dC)] at room temperature for 30 min. The total amount of reticulocyte lysate added in the reaction was adjusted with control reticulocyte lysate. The reaction was terminated by loading on a 5% native polyacrylamide gel containing 0·5x TBE [45 mM Tris base, 45 mM boric acid, 1 mM EDTA (pH 8·0)]. After electrophoresis in 0·5x TBE, the gel was dried and subjected to autoradiography. To confirm the specificity of the DNAprotein complex, a 10-fold excess of unlabelled competitor oligonucleotides containing CREB- or Oct-1-binding sites (Promega) was used.
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Results |
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Assay of binding in vivo between LANA and ATF4/CREB2
In order to confirm that LANA interacts with ATF4/CREB2 in mammalian cells, an in vivo binding assay was performed. Plasmids expressing GST or GSTLANA were co-transfected with HA-tagged ATF4/CREB2 expression plasmid into 293T cells. After cells were harvested, proteins were precipitated with glutathioneSepharose beads and immunoblotted by using a monoclonal antibody against GST (Fig. 1a). In Fig. 1(b)
, total cell lysates (lane T) or glutathione-bound proteins (lane P) were immunoblotted by using a monoclonal antibody against HA. Although HA-tagged ATF4/CREB2 protein was expressed at comparable levels (lanes 3 and 5), it was only co-precipitated with GSTLANA protein (lane 6). When the GSTLANA expression plasmid alone (lane 2) or GST- and HAATF4/CREB2-expressing plasmids (lane 4) were transfected, no such band was observed, confirming that the pulled-down protein was not a non-specific protein of similar size. This result shows that LANA associates with ATF4/CREB2 in vivo.
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Discussion |
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The PSORT II program (http://psort.nibb.ac.jp/) predicted three putative NLSs in the published amino acid sequence of LANA (GenBank accession no. AAC57158). These were PRRKH (amino acids 4347), RKRR (amino acids 200203) and PGVRMRR (amino acids 9951001). The two N-terminal putative NLSs seem to be essential, since all C-terminal deletion mutants of LANA fused to GFP localized exclusively in the nucleus, but an N-terminal deletion mutant lacking these motifs (LANA N) did not. Also, we found that the N-terminal 213 amino acids of LANA were necessary and sufficient for exclusively nuclear localization and that the C-terminal 212 amino acids of LANA were also necessary for nuclear localization in an N-terminal deletion mutant of LANA, which showed a similar localization pattern to that of a GFP control (data not shown).
Three models of active repression have been defined (Galvin & Shi, 1997 ). From our results, we deduced that LANA belongs by definition to the type II quenching repressors; that is, a protein that represses transcription by interacting with the activator itself. In order to elucidate the repression mechanism of LANA, we examined whether LANA acted as a direct repressor tethered to a promoter by using the GAL4LANA fusion system. GAL4LANA repressed a thymidine kinase promoter containing tandem GAL4-binding sites weakly in a dose- and GAL4 DBD-dependent manner (data not shown). Because the expression level of GAL4LANA in our study was relatively low, we could not determine the reason for the observed weak repression. We also tested whether LANA recruited a co-repressor complex containing histone deacetylase activity (Knoepfler & Eisenman, 1999
) to the promoter via ATF4/CREB2. LANA did not interact directly with GST-fused histone deacetylases 1, 2 and 3 in an in vitro GST pull-down assay. The repression of LANA upon transcriptional activation by GAL4ATF4/CREB2 was not relieved by treatment with trichostatin A, a histone deacetylase inhibitor (Johnson & Turner, 1999
), in a transient co-transfection experiment (data not shown). The above result indicated that the repression of LANA is not related to histone deacetylase activity.
Moderate repression of ATF4/CREB2-dependent transcription by LANA can be explained by multiple interactions of ATF4/CREB2 with general transcription factors and CBP (Liang & Hai, 1997 ). The C-terminal bZIP domain of ATF4/CREB2, in addition to DNA binding/dimerization, has the ability to interact with general transcription factors/CBP and activates transcription weakly. However, LANA may be unable to inhibit the transactivation activity of ATF4/CREB2 completely because the N terminus of ATF4/CREB2 can also interact with general transcription factors and still activates transcription more efficiently than the C-terminal bZIP domain.
The physiological functions of ATF4/CREB2 have not been defined. It can repress protein kinase A-dependent transcriptional activation (Karpinski et al., 1992 ) or activate transcription when tethered to a promoter by a GAL DBD fusion or via ATF4/CREB2-binding sites (Liang & Hai, 1997
). A previous study reported that LANA shows preferential binding to different regions of KSHV DNA in vitro (Cotter & Robertson, 1999
). However, it is uncertain whether LANA binds directly to DNA with sequence preference, because in vitro-translated LANA used in a DNA-binding assay may be contaminated with cellular proteins in a rabbit reticulocyte lysate. LANA may use sequence-specific DNA-binding adaptor proteins, such as ATF4/CREB2, for sequence-preferential DNA binding or may target a viral or cellular promoter to regulate a transcriptional network during virus infection. It would be interesting to investigate a viral or cellular promoter targetted by LANA via ATF4/CREB2, in addition to the repression mechanism of LANA.
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Acknowledgments |
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References |
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Received 19 April 2000;
accepted 14 July 2000.