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

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 k B (NF- k B) using a yeast two-hy-brid 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. How-ever, the role of TLS in transcriptional regulation has not yet been clearly elucidated. We found that TLS enhanced the NF- k B-mediated transactivation induced by physiological stimuli such as tumor necrosis factor a , interleukin-1 b , and overexpression of NF- k B-inducing kinase. TLS augmented NF- k B-dependent promoter activity of the intercellular adhesion molecule-1 gene and interferon- b gene. These results suggest that TLS acts as a coactivator of NF- k the Bam HI and Xho I site, and ligated in frame into pcDNA3.1/HisA vector at the HI– Xho I 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 Bam HI– Xho I site. These products were digested with Bam HI– Xho I and subcloned in frame into pcDNA3.1/HisA vector at the Bam HI– Xho I 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 Bam HI sites (forward, 5 9 -CCCCCGGATCCCCGGCCATG-GACGAACTGTTC-3 9 ; reverse, 5 9 -ACCAGGGATCCGGGGAGGGCAG- GCGTCACCC-3 9 ). This fragment was digested with Bam HI and ligated in frame into the Bam HI 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 Eco RI and pGBT-p65-(286–551) with Bam HI/ Eco RI were ligated in frame into the corresponding sites of pM. Con-struction of a luciferase reporter plasmid, 4 k Bw-luc or 4 k Bm-luc, con- taining four tandem copies of the human immunodeficiency virus- k B sequence upstream of minimal SV40 promoter has been described pre- viously (40). The other luciferase reporter plasmid, 5 3 Gal4-luc (pFR-luc) was purchased from Stratagene. This plasmid contains five tandem copies of the Gal4 binding site upstream of the TATA box. indicate that TLS interacts with p65 upon physiological signaling for the activation of NF- k B.

From the ‡Department of Molecular Genetics and §First Department of Internal Medicine, Nagoya City University Medical School, 1 Kawasumi,Nagoya,Japan 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 B (NF-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-B-mediated transactivation induced by physiological stimuli such as tumor necrosis factor ␣, interleukin-1␤, and overexpression of NF-B-inducing kinase. TLS augmented NF-B-dependent promoter activity of the intercellular adhesion molecule-1 gene and interferon-␤ gene. These results suggest that TLS acts as a coactivator of NF-B and plays a pivotal role in the NF-B-mediated transactivation.
Nuclear factor B (NF-B) 1 is an inducible cellular transcription factor that regulates a wide variety of cellular and viral genes including cytokines, cell adhesion molecules and human immunodeficiency virus (1)(2)(3)(4)(5). The members of the NF-B family in mammalian cells include the proto-oncogene c-Rel, RelA (p65), RelB, NFkB1 (p50/105), and NFkB2 (p52/p100). These proteins share a conserved 300-amino acid region known as the Rel homology domain, which is responsible for DNA binding, dimerization, and nuclear translocation of NF-B (1,2,4,5). In most cells, Rel family members form hetero-and homodimers with distinct specificities in various combinations. p65, RelB, and c-Rel are transcriptionally active members of the NF-B family, whereas p50 and p52 primarily serve as DNA binding subunits (1,2,4,5). These proteins play fundamental roles in immune and inflammatory responses and in the control of cell proliferation (4, 6 -9). A common feature of the regulation of NF-B is the sequestration in the cytoplasm as an inactive complex with a class of inhibitory molecules known as IBs (2,10). Treatment of cells with a variety of inducers such as phorbol esters, interleukin-1 (IL-1), and tumor necrosis factor ␣ (TNF-␣) results in phosphorylation, ubiquitination, and degradation of the IB proteins (5,11,12). The degradation of IB proteins exposes the nuclear localization sequence in the remaining NF-B dimers, followed by the rapid translocation of NF-B to the nucleus where it activates the target genes by binding to the DNA regulatory element (1,2,4,5).
The protein regions responsible for the transcriptional activation (called "transactivation domain") of p65, RelB, and c-Rel have been mapped in their unique C-terminal regions. p65 contains at least two independent transactivation domains within its C-terminal 120 amino acids (Fig. 1A) (13)(14)(15)(16). One of these transactivation domains, TA1, is confined to the C-terminal 30 amino acids of p65. The second transactivation domain, TA2, is located within the N-terminally adjacent 90 amino acids and contains TA1-like domain and leucine-rich regions. Since the nuclear translocation and DNA binding of NF-B were not sufficient for gene induction (17,18), it was suggested that interactions with other protein molecules through the transactivation domain (15,19,20) as well as its modification by phosphorylation (16) might play a critical role.
It has been previously reported that transcriptional activation of NF-B requires multiple coactivator proteins including CREB-binding protein (CBP)/p300 (19,20), CBP-associated factor, and steroid receptor coactivator 1 (21). These coactivators have histone acetyltransferase activity to modify the chromatin structure and also provide molecular bridges to the basal transcriptional machinery. Recently, p65 was also found to interact specifically with a newly identified coactivator complex, activator-recruited cofactor/vitamin D receptor-interacting protein, which potentiated chromatin-dependent transcriptional activation by NF-B in vitro (22). In addition to general coactivators, the transcriptional activation of gene-specific activators can be mediated by basal transcription factors through direct interaction with the activation domain. In the case of NF-B, the association of p65 with basal transcription factors such as TFIIB, TAF II 105, and TBP has been demonstrated (15,(23)(24)(25)(26)(27).
It is thus postulated that specific protein-protein interactions with NF-B determine its transcriptional competence: up-regulation of the NF-B transcriptional activity is mediated by interaction with basal factors and coactivators, and its down-regulation is mediated by interaction with inhibitors and corepressors at multiple levels. In our previous studies, yeast two-hybrid screen yielded two novel regulators of NF-B. RelAassociated 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 IBs (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 hTAF II 68/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)(34)(35). The C-terminal half of TLS spanning the ribonucleoprotein consensus sequence domain is usually excluded by translocation, and tumorigenic transformation is associated with the fusion of the N-terminal portion to the DNA-binding domain of a given transcription factor (30,31,36). Interestingly, TLS was shown to associate with a subpopulation of the TFIID complex in cells (32,37). Moreover, SARFH, a Drosophila homologue of TLS, was colocalized with RNA polymerase II at the active chromatin (38). In fact, TLS was shown to be associated with RNA polymerase II through its N-terminal domain (39).
In this study, we demonstrate that TLS interacts with NF-B p65 through the C-terminal transactivation domain and activates NF-B-mediated transcription. The yeast two-hybrid interaction assay revealed that the region between two core transactivation domains of p65, TA1-like and TA1, is required for the interaction with TLS. We confirmed the interaction between p65 and TLS in vitro using the bacterially expressed fusion proteins and in vivo coimmunoprecipitation/Western blot assay. In transient transfection assays, TLS showed transactivation potential and activated NF-B-dependent gene expression. These data indicate that TLS mediates the transcriptional activity of NF-B. , 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, 5Ј-CCCCCGGATCCCCGGCCATG-GACGAACTGTTC-3Ј; reverse, 5Ј-ACCAGGGATCCGGGGAGGGCAG-GCGTCACCC-3Ј). 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, 4Bw-luc or 4Bm-luc, containing four tandem copies of the human immunodeficiency virus-B sequence upstream of minimal SV40 promoter has been described previously (40). The other luciferase reporter plasmid, 5ϫGal4-luc (pFRluc) 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)(8)(9). The various portions of the p65 C-terminal regions corresponding to amino acids 286 -551, 286 -521, 286 -470, 286 -442, 286 -442/477-521, and 473-522 were fused in-frame to Gal4 DNA binding domain (positions 1-147) using the pGBT9 vector (CLONTECH). They were tested for activation of Gal4-dependent lacZ expression (␤-galactosidase activity). Among them, pGBT-p65 (286 -442/477-521) was chosen as a bait for library screening, since it had undetectable background in the ␤-galactosidase assay. Yeast strain Y190 was transformed with pGBT-p65-(286 -442/477-521), and the human placenta cDNA expression library was fused to the Gal4 transactivation domain in the pACT2 vector (CLONTECH). Approximately one million transformants were screened for the ability to grow on the plates with medium lacking tryptophan/leucine/histidine and containing 25 mM 3-aminotriazole. Plasmids were rescued from clones that were positive for ␤-galactosidase activity and identified by nucleotide sequencing. cDNA sequences and their amino acid sequences were compared with GenBank TM 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 Ϯ S.E.) relative to control of three independent transfections. Human recombinant TNF-␣ and IL-1␤ were purchased from Roche Molecular Biochemicals.
Ϫ ϩ Ϫ purified as described (8). In vitro protein-protein interaction assays were carried out as described previously (7,8). p65 and proteins were labeled with [ 35 S]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 g of GST fusion proteins were immobilized on 20 l 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 MgCl 2 , 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 l 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 MgCl 2 , 0.2 mM EDTA, 0.3% Nonidet P-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). The lysate was incubated with 1 g 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 l 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 2ϫ 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 Insti-tute, 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-His 6 antibody (Santa Cruz Biotechnology) was used.

Identification of TLS as a p65-binding Protein in the Yeast
Two-hybrid Screen-To identify proteins interacting with the p65 subunit of NF-B, we performed the yeast two-hybrid screen using the unique C-terminal region of NF-B p65 as a bait (Table I and Fig. 1). As depicted in Fig. 1A, various portions of p65 (i.e. amino acids 286 -551, 286 -521, 286 -470, 286 -442, 473-522, 286 -442/477-521, and 473-522) were fused to Gal4 DNA-binding domain (Gal4-DBD) in the pGBT9 vector. Among these clones, pGBT-p65 (286 -442/477-521) was chosen as a bait for the screening, since it had no detectable background in ␤-galactosidase assay (Table I). Yeast strain Y190 was used to screen human placenta cDNA library fused to the Gal4 transcriptional activation domain in the pACT2 vector (CLONTECH). From ϳ1.0 ϫ 10 6 Y190 yeast transformants, 90 colonies grew on selective medium and turned blue when tested in a filter lift ␤-galactosidase assay. Each plasmid, purified from the positive colonies, was cotransfected with bait plasmid into the yeast to confirm the specific interaction. DNA sequencing and comparison with GenBank TM and SwissProt data bases revealed the gene for TLS (one clone) in addition to Bcl-3 (one clone) and the IB family including I␤␣/MAD3 (five clones), which were previously shown to interact with p65.
TLS Supports Transcriptional Activation When Tethered to the Promoter-It was previously implicated that the N-terminal portion of TLS might act as a transcriptional activator in fusion with other transcription factors such as CHOP and ERG (33,43), although transcriptional activity of the full-length TLS or its C terminus has not been documented. It is suggested that TLS may mediate transcriptional activation by associating with transcription factors such as CHOP, ERG, and NF-B. Thus, we asked whether TLS acts as a transcriptional coactivator when tethered to the proximity of a given promoter. To examine this possibility, we created various fusion proteins of TLS and Gal4 DNA-binding domain containing full-length TLS or either its N-terminal or C-terminal subdomain ( Fig. 2A). As demonstrated in Fig. 2B, all of these TLS-Gal4 (DNA binding) fusion proteins more or less supported transcriptional activation from the reporter plasmid under the control of Gal4. Therefore, it was suggested that TLS could act as a coactivator of transcription if it was recruited to the vicinity of the promoter.
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 [ 35 S]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-IB␣ (positive control for interaction with p65) beads also bound to p65. However, no p65 binding was detected with beads containing GST alone (negative control). A coimmunoprecipitation experiment was carried out to examine whether TLS interacts with p65 in cultured cells. 293 cells were transiently transfected with plasmids pCMV-p65 and pCMV-TLS, expressing p65 and TLS, respectively. Cell extracts from these transfected cells were immunoprecipitated with mouse monoclonal anti-p65 (NLS) antibody (Fig. 3B, lane 3) or rabbit polyclonal anti-p65 antibody (recognizing its C-terminal region) (lane 5). The immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with rabbit polyclonal anti-TLS antibody. Either anti-p65 (NLS) or anti-p65 (C-terminal) antibodies coimmunoprecipitated TLS (lanes 3 and 5), whereas mouse or rabbit control IgGs did not (lanes 2 and 4). These results demonstrated that TLS specifically interacted with the p65 subunit of NF-B in vivo as well as in vitro.
TLS Augments NF-B-dependent Gene Expression-We then investigated the effect of TLS on NF-B-dependent gene expression. In Fig. 4A, the effect of TLS was examined on gene expression from the reporter plasmid 4Bw-luc by transfection of pCMV-TLS with or without cotransfection of pCMV-p65 in 293 cells. TLS augmented the NF-B-mediated transactivation in a dose-dependent manner when the p65-expression plasmid was cotransfected. pCMV-p65 alone activated gene expression from 4Bw-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, 4Bmluc, in which all four B sites were mutated, was used, neither the activation by pCMV-p65 nor the effect of cotransfection of pCMV-TLS was observed. These effects of TLS were not the result of an increased p65 level, since Western blot analysis of the transfected cell lysate revealed no increase in the protein level of exogenously expressed p65 (Fig. 4A, lower panels). Similarly, TLS augmented p65-mediated gene expression from human interferon ␤ promoter containing one binding site for NF-B (data not shown). To further examine whether the effect of TLS depends on the transactivation domain of p65 and its specificity, we created expression plasmids for Gal4-p65 fusion proteins in which the DNA-binding domain of Gal4 was fused with various portions of p65 or Sp1. The extents of augmentation of transactivation of these Gal4-p65 and Gal4-Sp1 by TLS were compared in Fig. 4B. TLS augmented the transactivation mediated by Gal4-p65-(1-551) and Gal4-p65-(286 -551) by 5.2and 5.5-fold, respectively. In sharp contrast, there was no significant effect of TLS on the actions of Gal4-p65-(1-286) and Gal4-Sp1. These observations indicated that the effects of TLS on transactivation appeared to depend on the C-terminal domain of p65 and were relatively specific for NF-B.
TLS Augments NF-B-dependent Gene Expression Induced by Physiological Stimuli-To examine the physiological relevance of the interaction between TLS and p65, we have examined the effect of TLS on the human intercellular adhesion molecule-1 (ICAM-1) promoter containing an NF-B binding site (44,45). In addition to cotransfection with p65 expression plasmid, effects of physiological stimuli such as TNF-␣ or IL-1␤ were also examined. Various amounts of the TLS-expressing plasmid (pCMV-TLS) were transfected into 293 cells along with ICAM-1-luc reporter plasmid, and the effects of TNF-␣ and IL-1␤ were investigated. As demonstrated in Fig. 5A, TLS greatly augmented the p65-mediated ICAM-1 gene expression in a dose-dependent manner (Fig. 5A, lanes 5-8). TLS enhanced the ICAM-1 gene expression induced by TNF-␣ (lanes 9 -12) and IL-1␤ (lanes 13-16). Although it seems likely that the effect of TLS on ICAM-1 gene expression is mediated through NF-B, we have confirmed this by using an artificial reporter construct, 4Bw-luc, containing only the NF-B sites and the minimal SV40 promoter. As shown in Fig. 5B, TLS augmented NF-B-dependent gene expression from 4Bw-luc induced by TNF-␣ and by NF-B-inducing kinase (NIK), an effector kinase involved in the NF-B activation pathway elicited by TNF-␣. When a luciferase reporter construct containing the mutated B sites (4Bm-luc) was used, no activation by TNF-␣ or NIK was observed, and no significant effect of cotransfection of TLS was demonstrated (data not shown). These data demonstrated that the effects of TLS were also evident on the signal-induced NF-B-dependent gene expression.
To further confirm the interaction of TLS with p65 upon TNF-␣ signaling, the cell extract was prepared from 293 cells that were transfected with TLS expression plasmid or pCMV control plasmid in the presence or absence of TNF-␣ stimulation. The cell extract was immunoprecipitated with anti-p65 antibody, and the immunoprecipitated proteins were separated on SDS-PAGE, transferred to a membrane, and probed by anti-TLS antibody. As demonstrated in Fig. 5C, TLS was coimmunoprecipitated with p65 in cells stimulated by TNF-␣ (lane 2). A barely detectable level of TLS was coprecipitated with p65 when cells were not stimulated by TNF-␣ (lane 1). No coimmunoprecipitation was detected when control IgG was used (lane 3). In addition, TNF-␣ stimulation did not alter the protein levels of p65 and TLS as demonstrated by immunoblotting using relevant antibodies (Fig. 5C, lower panels). These results indicate that TLS interacts with p65 upon physiological signaling for the activation of NF-B. DISCUSSION NF-B subunit p65 contains at least two independent transactivation domains, TA1 and TA2, located adjacently in the C-terminal region. Although TA2 contains TA1-like domain, additional regions are required for its full activity. For example, a previous study demonstrated that the region between the TA1-like domain of TA2 and the TA1 domain (amino acids 477-521 of p65) was necessary for the activity of TA2 although not sufficient for the transcriptional activity (16). Using the C-terminal portion of p65 (286 -442/477-521) as a bait in the yeast two-hybrid screen, we have identified TLS as an interacting protein. We have further narrowed down the minimal region of p65 (amino acids 477-521) necessary for the interaction with TLS (Fig. 1). Further molecular genetic studies have revealed that TLS acts as a mediator of the NF-B transactivation (Figs. 4 and 5).
TLS shares a common feature with a subgroup of TAF II proteins including hTAF II 68/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 TAF II s (47)(48)(49)(50)(51). Some of the genespecific transactivators are known to interact with distinct TAF II s through direct interaction with the activation domain (52,53). For example, Sp1, estrogen receptor, vitamin D recep-tor, p53, and p65 were shown to interact with dTAF II 110 (54), hTAF II 30 (55), TAF II 135 (56), TAF II 40/TAF II 60 (57), and TAF II 105 (26,27), respectively. Other TAF II proteins such as hTAF II 250, hTAF II 80, and hTAF II 28 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 tran-  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). scriptional activator, additional functions were suggested by other studies. For example, TLS binds to RNA in a sequenceindependent way in vitro and in cells (30,33,59) and engages in rapid nucleocytoplasmic shuttling (43,59). These features, together with the fact that TLS associates with a subpopulation of the TFIID complex in cells (32,37), suggest that TLS may participate in both transcriptional regulation and mRNA export by participating in heterogeneous nuclear ribonucleoprotein formation (60).
Interestingly, we previously demonstrated that the same region (amino acids 477-521) within the p65 transactivation domain interacted with AES (7). We found that AES-mediated repression of p65-mediated transactivation was down-regulated by TLS, and vice versa (data not shown). Although additional experiments are needed to compare the binding affinity of TLS and AES with p65, it is likely that the transcriptional activity of NF-B is regulated through the selective binding of interacting proteins with opposing actions such as TLS and AES.
In conclusion, these findings suggest a multiplicative mode of TLS actions in regulation of gene expression. The capability of NF-B to associate with TLS, in addition to other basal transcription factors including TFIIB (15), TBP (23,25), TAF II 105 (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).