Involvement of the Pro-oncoprotein TLS (Translocated in Liposarcoma) in Nuclear Factor-kappa B p65-mediated Transcription as a Coactivator*

Hiroaki UranishiDagger §, Toshifumi TetsukaDagger , Mayumi YamashitaDagger , Kaori AsamitsuDagger , Manabu Shimizu§, Makoto Itoh§, and Takashi OkamotoDagger

From the Dagger  Department of Molecular Genetics and § First Department of Internal Medicine, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan

Received for publication, December 12, 2000, and in revised form, January 11, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we have demonstrated that translocated in liposarcoma (TLS), also termed FUS, is an interacting molecule of the p65 (RelA) subunit of the transcription factor nuclear factor kappa B (NF-kappa B) using a yeast two-hybrid screen. We confirmed the interaction between TLS and p65 by the pull-down assay in vitro and by a coimmunoprecipitation experiment followed by Western blot of the cultured cell in vivo. TLS was originally identified as part of a fusion protein with CHOP arising from chromosomal translocation in human myxoid liposarcomas. TLS has been shown to be involved in TFIID complex formation and associated with RNA polymerase II. However, the role of TLS in transcriptional regulation has not yet been clearly elucidated. We found that TLS enhanced the NF-kappa B-mediated transactivation induced by physiological stimuli such as tumor necrosis factor alpha , interleukin-1beta , and overexpression of NF-kappa B-inducing kinase. TLS augmented NF-kappa B-dependent promoter activity of the intercellular adhesion molecule-1 gene and interferon-beta gene. These results suggest that TLS acts as a coactivator of NF-kappa B and plays a pivotal role in the NF-kappa B-mediated transactivation.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nuclear factor kappa B (NF-kappa 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-kappa 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-kappa 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-kappa 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-kappa B is the sequestration in the cytoplasm as an inactive complex with a class of inhibitory molecules known as Ikappa Bs (2, 10). Treatment of cells with a variety of inducers such as phorbol esters, interleukin-1 (IL-1), and tumor necrosis factor alpha  (TNF-alpha ) results in phosphorylation, ubiquitination, and degradation of the Ikappa B proteins (5, 11, 12). The degradation of Ikappa B proteins exposes the nuclear localization sequence in the remaining NF-kappa B dimers, followed by the rapid translocation of NF-kappa 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-kappa 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-kappa 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-kappa 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-kappa 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-kappa B determine its transcriptional competence: up-regulation of the NF-kappa 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-kappa 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 Ikappa 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-kappa B p65 through the C-terminal transactivation domain and activates NF-kappa 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-kappa B-dependent gene expression. These data indicate that TLS mediates the transcriptional activity of NF-kappa B.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- Mammalian expression vector plasmids pCMV-TBP, Gal4-Sp1, pCMV-NIK, ICAM-1-luc (positions -339 to -30), and IFN-beta -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, 4kappa Bw-luc or 4kappa Bm-luc, containing four tandem copies of the human immunodeficiency virus-kappa 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 (beta -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 beta -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 beta -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-alpha and IL-1beta 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 DH5alpha 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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of TLS as a p65-binding Protein in the Yeast Two-hybrid Screen-- To identify proteins interacting with the p65 subunit of NF-kappa B, we performed the yeast two-hybrid screen using the unique C-terminal region of NF-kappa 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 beta -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 beta -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 Ikappa B family including Ikappa beta alpha /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-Ikappa Balpha encodes full-length Ikappa Balpha (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 beta -galactosidase activity using a 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside colony filter assay (Clontech). +, positive for beta -galactosidase activity (blue colony) after 2-3 h; -, no beta -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.

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 beta -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 beta -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-kappa 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.

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-Ikappa Balpha (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-kappa B in vivo as well as in vitro.


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Fig. 3.   TLS interacts with p65. A, p65 binds to TLS and Ikappa Balpha 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-Ikappa Balpha 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.

TLS Augments NF-kappa B-dependent Gene Expression-- We then investigated the effect of TLS on NF-kappa B-dependent gene expression. In Fig. 4A, the effect of TLS was examined on gene expression from the reporter plasmid 4kappa Bw-luc by transfection of pCMV-TLS with or without cotransfection of pCMV-p65 in 293 cells. TLS augmented the NF-kappa B-mediated transactivation in a dose-dependent manner when the p65-expression plasmid was cotransfected. pCMV-p65 alone activated gene expression from 4kappa 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, 4kappa Bm-luc, in which all four kappa 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 beta  promoter containing one binding site for NF-kappa 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-kappa B.


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Fig. 4.   TLS augments NF-kappa B-dependent gene expression. A, 293 cells were transfected with 20 ng of 4kappa Bw-luc (containing wild type NF-kappa B binding sites) or 4kappa Bm-luc (containing mutated NF-kappa 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.

TLS Augments NF-kappa 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-kappa B binding site (44, 45). In addition to cotransfection with p65 expression plasmid, effects of physiological stimuli such as TNF-alpha or IL-1beta 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-alpha and IL-1beta 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-alpha (lanes 9-12) and IL-1beta (lanes 13-16). Although it seems likely that the effect of TLS on ICAM-1 gene expression is mediated through NF-kappa B, we have confirmed this by using an artificial reporter construct, 4kappa Bw-luc, containing only the NF-kappa B sites and the minimal SV40 promoter. As shown in Fig. 5B, TLS augmented NF-kappa B-dependent gene expression from 4kappa Bw-luc induced by TNF-alpha and by NF-kappa B-inducing kinase (NIK), an effector kinase involved in the NF-kappa B activation pathway elicited by TNF-alpha . When a luciferase reporter construct containing the mutated kappa B sites (4kappa Bm-luc) was used, no activation by TNF-alpha 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-kappa B-dependent gene expression.


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Fig. 5.   TLS augments the NF-kappa 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-alpha (lanes 9-12) or 10 ng/ml IL-1beta (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-kappa B-dependent gene expression induced by TNF-alpha or NIK. Left panel, 293 cells were transfected with 4kappa 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-alpha and harvested after additional incubation for 24 h. Right panel, 293 cells were transfected with 4kappa 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-alpha . 293 cells were transfected with pCMV-TLS plasmid. After 24 h of transfection, cells were stimulated with 1 ng/ml of TNF-alpha (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-alpha 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-alpha 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-alpha (lane 2). A barely detectable level of TLS was coprecipitated with p65 when cells were not stimulated by TNF-alpha (lane 1). No coimmunoprecipitation was detected when control IgG was used (lane 3). In addition, TNF-alpha 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-kappa B.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NF-kappa 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-kappa 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-kappa 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-kappa 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).

    ACKNOWLEDGEMENTS

We thank Drs. T. Tamura, S. T. Smale, D. Wallach, L. A. Madge, J. S. Pober, and T. Taniguchi for plasmids. We thank Drs. K. Shimizu and M. Ohki for the generous gift of anti-TLS antibody.

    FOOTNOTES

* This work was supported in part by grants-in-aid from the Ministry of Health and Welfare, the Ministry of Education, Science, and Culture of Japan, and the Japanease Health Sciences Foundation.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.: 81-52-853-8204; Fax: 81-52-859-1235; E-mail: tokamoto@med.nagoya-cu.ac.jp.

Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M011176200

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; AES, amino-terminal enhancer of split; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; CMV, cytomegalovirus; DBD, DNA-binding domain; AD, transcriptional activation domain; GST, glutathione S-transferase; ICAM-1, intercellular adhesion molecule-1; IL, interleukin; NIK, NF-kappa B-inducing kinase; NLS, nuclear localization signal; TLE, transducin-like enhancer of split; TLS, translocated in liposarcoma; TNF, tumor necrosis factor; PAGE, polyacrylamide gel electrophoresis.

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