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Mol Cell Biol, April 1998, p. 2077-2088, Vol. 18, No. 4
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

NF-kappa B2 Is a Putative Target Gene of Activated Notch-1 via RBP-Jkappa

Franz Oswald,1 Susanne Liptay,2 Guido Adler,1 and Roland M. Schmid1,*

Departments of Internal Medicine1 and Pediatrics,2 University of Ulm, D-89081 Ulm, Germany

Received 18 September 1997/Returned for modification 11 November 1997/Accepted 16 January 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

NF-kappa B2 (p100/p52), a member of the NF-kappa B/Rel family of transcription factors, is involved in the regulation of a variety of genes important for immune function. Previously, we have shown that the NF-kappa B2 gene is regulated in a positive and a negative manner. Two kappa B elements within the NF-kappa B2 promoter mediate tumor necrosis factor alpha-inducible transactivation. In addition, we have shown that there exists a transcriptional repression in the absence of NF-kappa B. To identify a DNA binding activity responsible for this transcriptional repression, we have partially purified a nuclear complex, named Rep-kappa B. Here we further analyze this putative repressive binding activity. Detailed examination of Rep-kappa B-DNA interaction revealed the sequence requirements for binding to be almost identical to those of recombination signal binding protein Jkappa (RBP-Jkappa ), the mammalian homolog of the protein encoded by Drosophila suppressor of hairless [Su(H)]. In addition, in electromobility shift assays, Rep-kappa B binding activity is recognized by an antibody directed against RBP-Jkappa . By performing transient-transfection assays, we show that human RBP-Jkappa represses basal as well as RelA (p65)-stimulated NF-kappa B2 promoter activity. Studies in Drosophila melanogaster have shown that Su(H) is implicated in the Notch signaling pathway regulating cell fate decisions. In transient-transfection assays we show that truncated Notch-1 strongly induces NF-kappa B2 promoter activity. In summary, our data clearly demonstrate that Rep-kappa B is closely related or identical to RBP-Jkappa . RBP-Jkappa is a strong transcriptional repressor of NF-kappa B2. Moreover, this repression can be overcome by activated Notch-1, suggesting that NF-kappa B2 is a novel putative Notch target gene.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

NF-kappa B/Rel proteins comprise a family of inducible transcription factors which control the expression of numerous genes involved in the immune, inflammatory, and acute-phase responses (for reviews, see references 3, 5, and 18). There is increasing evidence that members of this family are also involved in the regulation of normal and malignant cellular growth, especially in hematopoietic cells (16). More recently, it was demonstrated that inhibition of NF-kappa B/Rel induces apoptosis in various cell types, suggesting an antiapoptotic potential of NF-kappa B/Rel proteins (6, 36, 56, 60, 62).

In higher vertebrates, the NF-kappa B/Rel family encompasses five different genes: those encoding NF-kappa B1 (p105/p50), NF-kappa B2 (p100/p52), RelA (p65), and RelB, and the proto-oncogene encoding c-Rel. To a limited extent, these proteins can form homo- and heterodimers with distinct DNA-binding specificity (39, 43, 49). In most cell types, NF-kappa B/Rel dimers are sequestered in the cytoplasm by a member of the Ikappa B family of inhibitory proteins. Ikappa B proteins mask the nuclear localization signal of NF-kappa B/Rel, thereby preventing the nuclear translocation of NF-kappa B/Rel. Upon stimulation, Ikappa B proteins are phosphorylated on specific serine residues, ubiquitinated, and degraded through proteasome-dependent proteolysis, thereby allowing NF-kappa B/Rel dimers to translocate into the nucleus and bind to their cognant DNA sequences (for reviews, see references 4, 5, 54, and 57).

This initial activation of NF-kappa B can occur without de novo protein synthesis. However, it was shown that maintenance of NF-kappa B activity requires ongoing protein synthesis and continuous stimulation, indicating that NF-kappa B/Rel is also regulated at a transcriptional or translational level (23).

In a previous study, we demonstrated that NF-kappa B2 is positively autoregulated via two kappa B-responsive elements (34), as shown for other family members, including NF-kappa B1 (53) and Ikappa Balpha (28). In addition, mutation of the kappa B elements resulted in a dramatic increase in the basal NF-kappa B2 promoter activity in various cell lines. Therefore, we postulated that there is a negative regulation of NF-kappa B2 transcription mediated by the kappa B elements. A putative repressive DNA binding activity, Rep-kappa B, which interacts with a kappa B motif in the NF-kappa B2 promoter was identified. Rep-kappa B binding activity was partially purified from different cell sources, indicating that its expression is ubiquitous (34).

Recombination signal binding protein Jkappa (RBP-Jkappa ), also designated KBF2 or CBF1, was originally purified based on its binding to the recombination signal of the Jkappa immunoglobulin gene (38). Subsequently it was demonstrated that RBP-Jkappa acts as a transcriptional regulator, via binding to specific DNA motifs, rather than as a recombinase in V(D)J rearrangement (55). RBP-Jkappa proteins have been highly conserved during evolution not only in vertebrates but also in invertebrates. Genetic analysis revealed that the gene encoding RBP-Jkappa is conserved as the Drosophila melanogaster suppressor of hairless [SuH)], a member of the neurogenic gene family including Notch, Delta, Enhancer of split [E(spl)], and Hairless (15, 50). These genes participate in a lateral inhibition mechanism whereby singled-out sensory mother cells prevent their neighbors from adopting the neuronal fate (2). The first evidence that RBP-Jkappa acts as a transcription factor came from studies on viral gene expression. A cellular protein with 97% identity to a RBP-Jkappa splice variant (RBP-2N), which binds specifically to a 10-bp motif in the promoter region of the adenovirus capsid protein pIX, was purified. Transcriptional repression of the pIX promoter by RBP-2N was shown in in vitro as well as in vivo experiments (11). A transcriptional repression domain within RBP-Jkappa which might directly interact with the basal transcription machinery has been identified (24). Alternatively, this domain might function as a binding interface for an as-yet-unidentified corepressor protein (58).

RBP-Jkappa -mediated repression is overcome by interaction with the Epstein-Barr virus activator protein EBNA2. EBNA2 by itself is not able to bind DNA but is targeted to its DNA-responsive elements by direct physical interaction with RBP-Jkappa , thereby masking the RBP-Jkappa repression domain and acting as a strong transcriptional activator (19, 22, 24, 59, 64).

The gene encoding RBP-Jkappa /Su(H) is genetically mapped downstream of the Notch receptor in a signaling cascade controlling cell fate decisions during Drosophila development (14). One current model of Notch signaling postulates that after ligand binding the Notch receptor is activated by a proteolytic cleavage step. This activated form of Notch, Notch-IC, exerts its function by targeting DNA-bound RBP-Jkappa /Su(H), thereby masking the repression domain of RBP-Jkappa and activating transcription (25). In mammals, Notch homologs are implicated in neurogenesis and myogenesis (41), granulocyte differentiation (40), and CD4 versus CD8 as well as alpha beta versus gamma delta lineage decisions during T-lymphocyte development (45, 61). Direct physical interaction between activated forms of the murine Notch-1 receptor and RBP-Jkappa have been demonstrated (26, 29, 52), making this signal transduction cascade an exceptional short and simple communication strategy between the cell surface and the nucleus. So far, the only known Notch-1 target gene in mammals is the hairy enhancer of split (HES-1) gene, which encodes a basic helix-loop-helix protein that is able to block MyoD-induced myogenesis (32).

Here we demonstrate, on the basis of its DNA binding requirements, its gel mobility shift pattern, and its recognition by a specific antibody, that Rep-kappa B is closely related or identical to RBP-Jkappa . Rep-kappa B binds specifically to an 8-bp DNA motif in the NF-kappa B2 promoter with a high degree of similarity to the RBP-Jkappa consensus sequence (55). This RBP-Jkappa binding site overlaps the 5'-half site of a functional kappa B element. In transient-transfection assays, we show that RBP-Jkappa strongly inhibits basal as well as RelA (p65)-induced NF-kappa B2 promoter activity. These data show that RBP-Jkappa behaves biochemically and functionally identically to the previous proposed Rep-kappa B as a repressor of NF-kappa B2 transcription. In addition, we show in transient-transfection assays that truncated Notch-1 overcomes RBP-Jkappa -mediated repression and strongly activates NF-kappa B2 transcription through the RBP-Jkappa binding site.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasmids. A 1.9-kb BamHI-XhoI fragment of the mouse RBP-Jkappa gene, kindly provided by Tasuku Honjo (Kyoto, Japan), was isolated and used to screen a cDNA library derived from HeLa cells (Stratagene). Screening was performed as described previously (35). Two independent cDNA clones with 1.8- and 1.5-kb insert sizes were isolated, subcloned into the EcoRI and XhoI sites of Bluescript SK(+) (Stratagene), and sequenced. Both clones represent 3' fragments of the RBP-Jkappa cDNA truncated in exon 2. To generate a full-length cDNA of RBP-2N, the missing 5' sequence was introduced by PCR, using BamHI and NdeI restriction sites. Using BamHI and XhoI, the full-length cDNA of RBP-2N was cloned into the pcDNA3 vector (Invitrogen), resulting in pCMV-RBP-2N. To generate an RBP-1 expression plasmid, a PCR fragment representing the 5' end of the RBP-1 gene, designated aPCR1 (1), was digested with ClaI, treated with the Klenow fragment of DNA polymerase, and digested with SphI to generate a fragment which was ligated to SpeI-digested RBP-2N DNA, blunted, and digested with SphI. The full-length RBP-1 gene was subcloned into the NotI and XhoI sites of pcDNA3. In addition, we searched a database of sequencing tags and identified a sequence identical to that of the full-length RBP-3 gene (GenBank accession no. Z36843). The plasmid was kindly provided by T. Gress (Ulm, Germany). The insert was cut out and cloned into the BamHI and NotI sites of Bluescript SK(+) and pcDNA3, resulting in pCMV-RBP-3. The identities of PCR fragments and cloning junctions were confirmed by sequencing.

The reporter plasmid pASwt was constructed by subcloning a 363-bp AvaI-to-SalI fragment of the NF-kappa B2 promoter (positions -198 to +165) (34) into the MluI and XhoI sites of the luciferase vector pGL2-basic (Promega). pAS-SL49 was generated by site-directed mutagenesis using the oligodeoxynucleotide SL49.

The expression plasmid pRSVRelA and plasmids for in vitro transcription of RelA/p65 and NF-kappa B1/p50 were described previously (12, 49). The expression plasmid for truncated murine Notch-1, pSV-mNotch-1-IC, corresponds to pSG5mNotch1IC (25) and was kindly provided by Thomas Henkel (Munich, Germany).

Cell lines. The cell lines HEK-293 (ATCC CRL 1573) and COS-7 (DSM ACC 60) were grown at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal calf serum (FCS). Jurkat T cells (ATCC TIB 152) were cultivated at 37°C under 5% CO2 in RPMI 1640 medium (Gibco) supplemented with 5% FCS.

Preparation of cell extracts. Nuclear extracts were prepared as described elsewhere (34). Whole-cell lysates were prepared as follows. Cells were washed three times in phosphate-buffered saline (PBS) and pelleted by centrifugation at 300 × g. The pellet was resuspended in 5 volumes of ice-cold CHAPS lysis buffer, consisting of 10 mM 3-[(cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM Tris-HCl (pH 7.9), 150 mM NaCl, 2 mM EDTA, 5 mM NaF, 10 µg of aprotinin per ml, 10 µg of leupeptin per ml, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride; this was followed by incubation on ice for 40 min. The lysate was cleared by centrifugation at 80,000 × g for 30 min. Protein concentrations were determined by the Bradford method (Bio-Rad), and extracts were assayed for DNA binding activity by electromobility gel shift assays (EMSAs) and Western blotting.

Translation of recombinant proteins. In vitro-translated proteins were synthesized in a reticulocyte lysate-coupled transcription-translation system in accordance with the manufacturer's instructions (Promega). The quality of translation was monitored by simultaneously labeling with [35S]methionine. After separation of the labeled translation products by sodium dodecyl sulfate (SDS) gel electrophoresis, the gels were dried and exposed to X-ray film.

Protein purification and in vitro interaction assay. Purification of Rep-kappa B was performed essentially as described previously (34). Nuclear extracts from unstimulated Jurkat-T cells were applied to DNA-cellulose columns equilibrated at 100 mM NaCl. After several washing steps, protein fractions were eluted with increasing NaCl concentrations and analyzed by EMSA. Purification of a bacterially expressed glutathione S-transferase (GST)-Notch-1-IC fusion protein and in vitro interaction assays were performed as described in references 8 and 29.

EMSAs. Approximately 5 to 10 µg of cell extract was used for EMSAs in a binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, and 4% glycerol. For binding reactions, 2 µg of poly(dI-dC) (Pharmacia) and approximately 0.5 ng of 32P-labeled oligonucleotides were added. For competition experiments, a 10- to 60-fold molar excess of unlabeled competitor was used. For antibody perturbation experiments, 0.5 µg of antibody directed against RBP-Jkappa (K0043) (21, 46), 10 µg of antibody directed against RelA (p65) (SC109X; Santa Cruz Biotechnology), or 10 µg of antibody directed against the FLAG epitope (M-5; Kodak) was added to the reaction mixture. The reaction products were separated on 5% polyacrylamide gels with 1× Tris-glycine-EDTA at room temperature. The gels were dried and exposed to X-ray film.

Oligonucleotides. The sequences of the oligodeoxynucleotides SL350 and SL366 are as follows. SL350, 5'-CTAGTAACGTCATGGGAATTCCCCCCTCCGGGGGGCCGAGAAGGGGCTTTCCCGGCCCT-3', corresponds to positions -119 to -64 of the human NF-kappa B2 gene and contains two NF-kappa B binding sites (34). SL366, 5'-GATCGTTACTGTGGGAAAGAAAGTTTGGGAAGTTTCACAC-3', spans positions -91 to -47 of the murine HES-1 promoter and contains two RBP-Jkappa binding sites. SL49, 5'-GCACGGAAACGTGAGGGGAATTCCCCC-3', was used for site-directed mutagenesis to introduce a mutation into the RBP-Jkappa consensus sequence in the reporter construct pASwt to generate pAS-SL49. The sense strand of each oligonucleotide is shown. Sequences of other oligodeoxynucleotides are shown in the respective figures.

DNA transfection and luciferase assay. A total of 106 HEK-293 cells were transfected in 90-mm-diameter culture dishes with 5 to 10 µg of plasmid DNA expressing RBP-Jkappa splice forms and murine Notch-1-IC by calcium phosphate coprecipitation, as described previously (48). Forty-eight hours after transfection, nuclear proteins were prepared and the extracts were assayed for DNA binding activity and protein expression. COS-7 cells were transfected with DEAE-dextran essentially as described previously (49). Cells (2 × 105) in 35-mm-diameter culture dishes were transfected with 2 µg of reporter plasmid DNA together with various amounts of plasmid expressing RelA (p65), RBP-2N, RBP-3, or murine Notch-1-IC. Transfection of Jurkat-T cells was performed as follows. Cells (107) were electroporated in a total volume of 300 µl of RPMI 1640 medium supplemented with 5% FCS. Electroporations were performed with a Gene Pulser II apparatus (Bio-Rad) at 260 V and 975 µF in 40-mm electroporation cuvettes. To quantitate luciferase activity, cells were harvested 48 h after transfection and lysed in 100 µl of a buffer containing 25 mM Tris-HCl (pH 7.8), 2 mM EDTA, 2 mM DTT, 10% glycerol, and 1% Triton X-100 for 10 min at room temperature. Lysates were centrifuged at 7,000 × g for 5 min. Luciferase activity of at least four independent transfections with 20 µl of cleared lysate was determined in an LB 9501 luminometer (Berthold), using a luciferase assay system (Promega). All transfections were normalized for the level of total cellular protein (Bradford assay; Bio-Rad).

Western blotting. SDS-polyacrylamide gels (7.5%) were transferred to a nitrocellulose filter (BA85; Schleicher & Schuell) for 20 min at 4°C and 150 mA, using a Tris-glycine buffer system. The membrane was blocked for 2 to 3 h in a solution of 5% dry milk powder in PBS-T (0.05% Tween 20 in PBS), incubated with the primary antibody directed against the FLAG epitope (M-5; Kodak) for 1 to 2 h in PBS-T containing 5% dry milk powder, and then washed three times in PBS-T (10 min each). The secondary antibody (1:7,000 dilution of peroxidase-conjugated sheep anti-mouse immunoglobulin G (IgG; Sigma) was incubated with the membrane for 30 min. After the membrane was washed, specific proteins were detected by using an enhanced chemiluminescence system (Amersham).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Purification of Rep-kappa B DNA binding activity. Previously, we had shown that two kappa B elements within the human NF-kappa B2 promoter are critical for responsiveness of NF-kappa B2 to tumor necrosis factor alpha (TNF-alpha ) and phorbol myristate acetate. Interestingly, mutation of the kappa B elements led to a strong induction of basal promoter activity in unstimulated cells. Therefore, we postulated that there is a kappa B element DNA binding activity which mediates transcriptional repression of the NF-kappa B2 gene in the absence of NF-kappa B. A nuclear complex, which we named Rep-kappa B, likely to be responsible for this transcriptional repression could be identified. To further characterize this putative repressive DNA binding activity, we partially purified protein complexes from nuclear extracts of unstimulated Jurkat-T cells, using DNA-cellulose columns. Proteins were eluted with increasing NaCl concentrations. DNA binding activity in the eluted fractions was monitored by EMSA using the 32P-labeled oligonucleotide SL350, which contains both kappa B sites (positions -119 to -63) of the NF-kappa B2 promoter. The specificity of DNA binding was tested by competition with increasing amounts of unlabeled oligonucleotide SL350 (Fig. 1A, lanes 3 and 4). Two major complexes, A and B, turned out to bind specifically to the NF-kappa B2 probe. On the basis of previous experiments, we identified complex A as Rep-kappa B activity. Rep-kappa B was eluted at 280 to 320 mM NaCl (Fig. 1A, fractions 10 to 13). Further characterizations of Rep-kappa B were performed with fractions 11 and 12. 


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FIG. 1.   (A) Purification of Rep-kappa B activity from Jurkat-T cells. Nuclear extracts (NE), flowthrough (Flow), and eluted fractions were tested for binding to the oligonucleotide SL350, which contains both kappa B elements in the human NF-kappa B2 promoter and flanking regions (positions -119 to -63). The specificity of binding was analyzed by adding 20- and 60-fold molar excesses of a homologous competitor (lanes 3 and 4). A and B indicate the positions of specific DNA binding complexes. Complex A was identified as Rep-kappa B activity. (B) Sequence requirements for Rep-kappa B binding. Competition experiments were performed with eluted fraction 11 (5 µl) and 20- to 60-fold molar excesses of the unlabeled competitor (Comp.) oligonucleotides shown in panel C. As a probe, the 32P-labeled oligonucleotide SL350 was used. -, no competitor added. (C) Sequences and Rep-kappa B binding abilities of different oligonucleotides derived from the first kappa B element in the NF-kappa B2 promoter. Only the positive strand of each oligonucleotide is shown. Lowercase letters represent added linker sequences; dashes indicate identity with the consensus (Consens.) sequence. (D) Rep-kappa B activity is recognized by an antibody directed against RBP-Jkappa . Treatment of eluted fractions 11 and 12 (5 µl) with an RBP-Jkappa -specific antibody (alpha -RBP) specifically abolished band A. Instead, a more slowly migrating complex (band C, lanes 2 and 4) appeared. Another DNA binding activity (complex B) was not affected by the antibody. The probe was a 32P-labeled oligonucleotide, SL332, containing the first kappa B element (positions -119 to -90) within the NF-kappa B2 promoter. -, control (antibody not added). (E) Rep-kappa B binds specifically to the RBP-Jkappa site in the murine HES-1 promoter. Nuclear extracts and eluted fractions from Jurkat-T cells were analyzed for specific DNA binding activity, using the 32P labeled oligonucleotide HES-1 as a probe. This oligonucleotide contains the known RBP-Jkappa consensus sequence 5'-CTGTGGGAAAGA-3'. As a competitor, a 50-fold molar excess of unlabeled oligonucleotide SL332 (lane 2) or HES-1 (lane 3) was used. Band A represents Rep-kappa B binding activity. -, control (competitor not added).

Sequence requirements for Rep-kappa B binding. To determine the DNA binding properties of Rep-kappa B in more detail, we carried out competition experiments with eluted fraction 11 and the 32P-labeled oligonucleotide SL350 as a probe (Fig. 1B). For competition, the oligonucleotides shown in Fig. 1C were used at a 20- or 60-fold molar excess. The oligonucleotide SL332 represents the first kappa B element (positions -119 to -90) in the human NF-kappa B2 promoter (see also Fig. 3). Since the labeled probe contains both kappa B elements, the competition experiment with SL332 (Fig. 1B, lanes 2 and 3) suggests that the first kappa B element is the major target site for Rep-kappa B binding. Therefore, we introduced various nucleotide changes in the flanking region or within the kappa B element and used the double-stranded oligonucleotides shown in Fig. 1C as competitors. As shown in Fig. 1B, excesses of oligonucleotides SL336, SL338, SL340 and SL342 did not titrate out Rep-kappa B binding. However, the competitors SL346 and SL348 were still able to bind Rep-kappa B activity. Interestingly, we identified a binding consensus element (5'-CATGGGAA-3' [Fig. 1C]) with obvious similarity to that of RBP-Jkappa (5'-CGTGGGAA-3'), the mammalian homolog of Drosophila melanogaster suppressor of hairless (54). Indeed, oligonucleotide SL344, containing the ideal RBP-Jkappa consensus sequence (Fig. 1C), did compete for Rep-kappa B binding (Fig. 1B, lanes 16 and 17). These experiments clearly demonstrate that the 5'-half site of the first kappa B element in the NF-kappa B2 promoter, rather than the whole kappa B element, is necessary for Rep-kappa B binding. In addition, the sequence requirements for Rep-kappa B DNA interaction are almost identical to those of RBP-Jkappa .

Rep-kappa B is most likely identical to RBP-Jkappa . Additional evidence that Rep-kappa B is closely related to RBP-Jkappa was obtained from gel shift analyses performed in the presence of an RBP-Jkappa -specific antibody (Fig. 1D). Eluted fractions 11 (lanes 1 and 2) and 12 (lanes 3 and 4) were incubated with or without the monoclonal rat antibody K0043 (21, 46) prior to gel shift analysis. The presence of the antibody led to the disappearance of complex A in both fractions and to the appearance of a supershifted, novel band. This complex (band C) most likely contains RBP-Jkappa . Furthermore, we performed a band shift experiment with crude nuclear extracts from Jurkat-T cells and eluted fractions 9 to 15. As a probe for DNA binding, we used a 32P-labeled oligonucleotide derived from the murine HES-1 promoter. This promoter sequence contains a perfect RBP-Jkappa consensus sequence, 5'-CTGTGGGAAAGA-3' (positions -82 to -71), without an overlapping kappa B element (8). As shown in Fig. 1E, complex A from Jurkat-T cell nuclear extracts binds specifically to the RBP-Jkappa site of the HES-1 promoter (lane 1). Competition for this binding activity was provided by a 50-fold molar excess of unlabeled oligonucleotide SL332 (lane 2) as well as by a 50-fold molar excess of the homologous competitor HES-1 (lane 3). Furthermore, the DNA binding activity associated with the HES-1 site in the eluted fractions corresponds exactly to the NF-kappa B2-specific Rep-kappa B activity shown in Fig. 1A, suggesting that Rep-kappa B is identical to RBP-Jkappa .

Finally, we tested whether recombinant RBP-Jkappa proteins bind to the kappa B element in the NF-kappa B2 promoter. Therefore, cDNAs coding for the human RBP-Jkappa splice forms RBP-1, RBP-2N, and RBP-3 were inserted into appropriate vectors (see Materials and Methods) and expressed in a cell-free translation system (Promega). Lysates (4 µl) were assayed for protein-DNA interaction in band shift experiments using the 32P-labeled oligonucleotide SL332 as a probe. Binding specificity was monitored by incubating programmed lysates with an antibody directed against RBP-Jkappa . For comparison, we used Rep-kappa B activity from eluted fraction 12. The results of this experiment are shown in Fig. 2. As previously shown, Rep-kappa B binding activity (complex A) is recognized by the RBP-Jkappa -specific antibody, supershifting a novel band (C) (lanes 1 and 2). Unfortunately, reticulocyte lysate itself contains abundant NF-kappa B activity which binds to the kappa B element within the oligonucleotide SL332 (complex B). NF-kappa B binding activity was confirmed by competition experiments (data not shown) and supershift analysis with a RelA (p65)-specific antiserum (see Fig. 6B, lane 8). However, additional kappa B binding complexes could be detected in lysates programmed with the human RBP-Jkappa proteins RBP-2N (Fig. 2, lane 5), RBP-1 (lane 7), and RBP-3 (lane 9). Incubation with an RBP-Jkappa -specific antibody completely abolished complex A (lanes 6, 8, and 10), suggesting that these bands correspond to RBP-Jkappa . This complex (A) was absent from a lysate programmed with a control plasmid, Bluescript SK(+) (Fig. 2, lanes 3 and 4).


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FIG. 2.   The RBP-Jkappa splice variants RBP-2N, RBP-1, and RBP-3 bind specifically to the first kappa B element in the NF-kappa B2 promoter. Eluted fraction 12 (5 µl) and RBP-Jkappa splice variants synthesized in a cell-free system were incubated with (+) or without (-) an antibody directed against RBP-Jkappa (alpha -RBP) prior to gel electrophoresis. Treatment of lysates with the antibody specifically abolished complex A (lanes 2, 6, 8, and 10). This complex was absent from the lysate in which a control plasmid [Bluescript SK(+) (BS)] was used in the translation assay (lane 4). The added reticulocyte lysate (5 µl) contained NF-kappa B binding activity (complex B) which covered the supershifted antibody-RBP complex (band C) detected in lane 2. The 32P-labeled oligonucleotide SL332, containing the first kappa B element of the NF-kappa B2 promoter, was used as a probe.

These experiments clearly demonstrate that (i) Rep-kappa B is closely related, if not identical, to RBP-Jkappa and that (ii) recombinant RBP-Jkappa proteins bind specifically to the first kappa B element within the NF-kappa B2 promoter.

The human NF-kappa B2 promoter is repressed by RBP-2N. Previously, the human RBP-Jkappa splice form RBP-2N was shown to act as a transcriptional repressor of adenovirus pIX gene expression via sequence-specific DNA binding (11). To test the regulation of the human NF-kappa B2 gene in response to RBP-Jkappa , NF-kappa B2-specific luciferase constructs were transiently cotransfected with plasmids expressing RBP-2N, under the control of the cytomegalovirus (CMV) promoter, into COS-7 or Jurkat-T cells. As shown in Fig. 3, the reporter construct pASwt contains NF-kappa B2-specific promoter sequences (positions -198 to +165), including both kappa B elements. The first kappa B element overlaps the putative RBP-Jkappa consensus site, 5'-CATGGGAA-3'. In pAS-SL49, this site has been mutated to 5'-GAGGGGAA-3' without affecting the functional kappa B element. Coexpression of increasing amounts of RBP-2N into COS-7 cells resulted in a 70% repression of the basal luciferase activity of pASwt. No significant effect was observed with the reporter construct pAS-SL49, which contains a mutated RBP-Jkappa site (Fig. 4A).


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FIG. 3.   Schematic representation of the luciferase (LUC) reporter constructs used for transfection assays. The constructs pASwt and pAS-SL49 contain a DNA fragment (positions -198 to +165) derived from the human NF-kappa B2 promoter. The sequences of the kappa B elements (black boxes) and the overlapping putative RBP-Jkappa elements (open boxes) are shown. In the construct pAS-SL49, the putative RBP-Jkappa consensus element is mutated as indicated. Abbreviations: A, AvaI; E, EcoRI; N, NruI; Pv, PvuI; S, SalI.


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FIG. 4.   Repression of basal as well as RelA (p65)-stimulated NF-kappa B2 promoter activity by RBP-2N depends on a functional RBP-Jkappa consensus sequence. (A) Portions (2 µg) of reporter constructs pASwt (black bars) and pAS-SL49 (open bars) were cotransfected into COS-7 cells with increasing amounts of an RBP-2N expression plasmid. (B and C) Portions (2 µg) of reporter constructs pASwt (black bars) and pAS-SL49 (open bars) were cotransfected with 500 ng of pRSVRelA and increasing amounts of CMV-RBP-2N into COS-7 cells (B) or Jurkat-T cells (C). Luciferase activity was determined from 100 µg of total cell extract, and the basal promoter activity of each construct was set to one. Mean values and standard deviations for at least four independent experiments are shown.

Next, we asked whether RBP-2N would also repress prestimulated NF-kappa B2 transcription. Therefore, we stimulated NF-kappa B2 promoter activity by transfecting a RelA (p65)-expressing plasmid together with increasing amounts of RBP-2N. The luciferase activity of pASwt was stimulated 10-fold following cotransfection with 500 ng of pRSVRelA. This activation could be abrogated to basal levels by gradually increasing the amount of RBP-2N. Using the reporter plasmid pAS-SL49, a ninefold increase in luciferase activity was detected after cotransfection with pRSVRelA. However, the repressive effect of RBP-2N was completely lost (Fig. 4B). Similar results were obtained with Jurkat-T cells (Fig. 4C).

Taken together, these results demonstrate that RBP-2N is able to suppress basal as well as RelA (p65)-stimulated NF-kappa B2 promoter activity. This transcriptional repression depends on a functional RBP-Jkappa binding site within the NF-kappa B2 promoter.

Rep-kappa B/RBP-Jkappa and NF-kappa B/Rel (p50/p65) compete for binding to the first kappa B element within the NF-kappa B2 promoter. To investigate the binding of NF-kappa B/Rel complexes and Rep-kappa B/RBP-Jkappa to their overlapping sites in more detail, we first performed a semiquantitative binding analysis for Rep-kappa B/RBP-Jkappa and NF-kappa B/Rel (p50/p65) by the method of Scatchard (12). Fraction 12, containing partially purified Rep-kappa B protein (Fig. 1A, lane 14), was used as the source of Rep-kappa B/RBP-Jkappa DNA binding activity, and cell-free system-cotranslated p50/p65 heterodimers were used to determine NF-kappa B/Rel-specific affinity for the first kappa B site. Figure 5A shows a graph of the binding of the NF-kappa B/Rel (p50/p65) heterodimers to the labeled oligonucleotide SL332, with saturation for values exceeding 1 nM free probe. For Rep-kappa B/RBP-Jkappa , no saturation could be obtained with a probe concentration of 2 nM. Although we could not precisely determine the binding affinity of Rep-kappa B/RBP-Jkappa for the NF-kappa B2-specific oligonucleotide, this experiment shows that the affinity of NF-kappa B/Rel (p50/p65) for the kappa B element is at least sixfold higher than that of Rep-kappa B/RBP-Jkappa (Fig. 5A). To investigate whether NF-kappa B/Rel proteins and Rep-kappa B/RBP-Jkappa could bind to their overlapping sites simultaneously, we carried out band shift experiments; the results are shown in Fig. 5B. Fraction 12 was incubated with increasing amounts of cell-free system-synthesized p50/p65 heterodimers and constant amounts of 32P-labeled oligodeoxynucleotide SL332, resulting in the subsequent appearance of the NF-kappa B-specific complex B (Fig. 5B, lanes 3 to 5). The specificity of this complex was tested by treatment with antiserum directed against RelA (p65), which induced a supershifted novel band, band F (Fig. 5B, lane 6). Interestingly, augmenting NF-kappa B/Rel (p50/p65)-specific DNA binding activity (complex B) was associated with a gradual loss of Rep-kappa B/RBP-Jkappa -specific DNA binding (complex A), suggesting that NF-kappa B/Rel and Rep-kappa B/RBP-Jkappa compete for binding to overlapping sites within the NF-kappa B2 promoter.


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FIG. 5.   (A) Affinity of Rep-kappa B/RBP-Jkappa and NF-kappa B/Rel (p50/p65) for the first kappa B element and the overlapping RBP-Jkappa site within the NF-kappa B2 promoter, evaluated by Scatchard analysis. Protein-DNA binding studies were performed with a range of concentrations of labeled oligonucleotide SL332 in a 20-µl reaction volume. Bound and free probe species were quantitated with a PhosphorImager (Molecular Dynamics). Reactions were performed in triplicate. (B) Competitive binding of NF-kappa B/Rel (complex B) and Rep-kappa B/RBP-Jkappa (complex A). The band shift experiment was performed with fraction 12 of partially purified nuclear extracts from Jurkat-T cells and increasing amounts of cell-free cotranslated NF-kappa B/Rel (p50/p65) heterodimers. Band F represents a RelA (p65)-containing complex which was supershifted after treatment with antiserum (alpha -p65). Radiolabeled oligonucleotide SL332 was used as a probe. -, control (no NF-kappa B/Rel added).

Activated mammalian Notch-1 interacts with Rep-kappa B/RBP-Jkappa . Truncated Notch-1 was shown to activate transcription mediated by physical interaction with RBP-Jkappa , thereby masking the repressive effect of RBP-Jkappa (25). In mammals, this was demonstrated with the murine HES-1 promoter (29). To investigate whether activated Notch-1 is also involved in the transcriptional regulation of NF-kappa B2, we first performed band shift experiments (Fig. 6). Nuclear extracts from Jurkat-T cells were incubated with and without increasing amounts of cell-free system-synthesized truncated Notch-1 protein (Notch-1-IC). As a probe, we used a 32P-labeled oligonucleotide derived from the murine HES-1 promoter (positions -91 to -47), which contains two RBP-Jkappa consensus sequences (-80 to -74 [5'-GTGGGAA-3'] and -66 to -60 [5'-TTGGGAA-3']) (Fig. 6A), or the NF-kappa B2-specific oligonucleotide SL332 (Fig. 6B). Specific protein-DNA complexes were identified by using antibodies directed against RBP-Jkappa and RelA (p65). Complex A turned out to contain RBP-Jkappa (Fig. 6A, lane 5). This complex binds to the NF-kappa B2 probe (Fig. 6B) as well as to the HES-1 probe (Fig. 6A). In contrast, an additional, more slowly migrating complex (Fig. 6B, band B) could be identified with the NF-kappa B2-specific oligonucleotide but not the HES-1 probe, which contains no kappa B site. A RelA (p65)-specific antiserum supershifted this additional complex (Fig. 6B, band F, lanes 3 and 8), suggesting that this complex contains RelA. The strong NF-kappa B binding activity is in part due to intrinsic NF-kappa B proteins in the reticulocyte lysate added to the binding reactions shown in lanes 4 to 8 (compare with Fig. 2, lanes 3 to 10). Increasing amounts of cell-free system-synthesized truncated Notch-1 protein led to the disappearance of the RBP-Jkappa -specific complex A, which bound to the HES-1 probe (Fig. 6A; compare lanes 2 to 4 with lane 1) and to the NF-kappa B2 probe (Fig. 6B; compare lanes 4 to 7 with lane 1), suggesting that activated Notch-1 interacts with Rep-kappa B/RBP-Jkappa in both cases.


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FIG. 6.   Cell-free system-synthesized Notch-1-IC interacts with Rep-kappa B/RBP-Jkappa from Jurkat T cell nuclear extracts (Jurkat-NE) at the HES-1 and NF-kappa B2 promoters. (A) Extracts (2 µg) were incubated without (lane 1) or with increasing amounts of (lanes 2 to 5) reticulocyte lysate programmed with Notch-1-IC (mNotch-1IC). Volumes were equalized by addition of pure reticulocyte lysate. A 32P-labeled HES-1-specific double-stranded oligonucleotide (SL366) was used as a probe. Rep-kappa B/RBP-Jkappa binding activity (complex A) is evidenced by an RBP-Jkappa -specific antibody (alpha -RBP) supershifting a novel band (complex C). Note that this more slowly migrating band (lane 5) does not represent a Notch-1-IC-containing complex. (B) Extracts (2 µg) were incubated without (lane 1) and with increasing amounts of (lanes 4 to 8) reticulocyte lysate programmed with Notch-1-IC. 32P-labeled NF-kappa B2-specific oligonucleotide SL332 was used as a probe. In addition to the Rep-kappa B/RBP-Jkappa -specific DNA binding activity (complex A) that was recognized by a specific antibody (alpha -RBP), a second complex (complex B) was detected in nuclear extracts and was more abundant after addition of reticulocyte lysate. Treatment with an antiserum directed against RelA (p65) (alpha -p65) interfered with this binding activity and supershifted band F (lanes 3 and 8).

Since we could not detect an additional, higher-order complex which contains Notch-1 together with RBP-Jkappa bound to DNA, we used a bacterially expressed GST-Notch-1-IC fusion protein (Fig. 7A). Fraction 12, containing partly purified Rep-kappa B/RBP-Jkappa DNA binding activity, was incubated with increasing amounts of purified GST-Notch-1-IC prior to the gel shift experiment. Radiolabeled oligonucleotide SL332 was used as a probe. As expected, increasing amounts of GST-Notch-1-IC led to a clear reduction of Rep-kappa B/RBP-Jkappa DNA binding activity (complex A). In addition, a higher-order complex (band D) could be detected (Fig. 7A, lanes 5 to 9). This complex was absent when GST-Notch-1-IC was incubated with fraction 25, which does not contain Rep-kappa B binding activity (lanes 1 to 3). GST protein incubated with fraction 12 (lanes 12 and 13) or fraction 25 (lanes 10 and 11) served as controls.


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FIG. 7.   (A) GST-Notch-1-IC forms a higher-order complex with Rep-kappa B/RBP-Jkappa bound to the RBP-Jkappa site within the NF-kappa B2 promoter. Fractions 12 and 25 were incubated with the GST proteins as indicated. Only the addition of GST-Notch-1-IC to a Rep-kappa B/RBP-Jkappa -containing fraction resulted in a decrease in RBP-Jkappa -specific DNA binding activity (complex A), and a novel, more slowly migrating complex (band D) appeared. (B) GST-Notch-1-IC interacts with recombinant RBP-Jkappa in vitro. GST proteins were immobilized with Sepharose beads and incubated with cell-free system-synthesized RBP-3 (TNT-RBP-3). After extensive washing steps, the reaction mixtures were boiled and proteins were separated by SDS-polyacrylamide gel electrophoresis. The positions of molecular size markers (in kilodaltons) are shown on the left.

Furthermore, the interaction of GST-Notch-1-IC with recombinant RBP-Jkappa protein was verified in vitro. GST-Notch-1-IC was immobilized with glutathione-Sepharose (Pharmacia) and incubated with cell-free system-synthesized, 35S-labeled RBP-3. After extensive washing, the reaction mixture was boiled and separated by SDS-polyacrylamide gel electrophoresis (Fig. 7B). Radiolabeled RBP-3 protein could be detected only when GST-Notch-1-IC was added to the reaction (Fig. 7B, lane 3). RBP-Jkappa did not bind to GST protein (Fig. 7B, lane 2) or to Sepharose beads (Fig. 7B, lane 4).

To investigate this interaction in a cellular context, we transiently transfected HEK-293 cells with plasmids expressing the RBP-Jkappa splice form RBP-3 (pCMV-RBP-3) and a truncated Notch-1 protein with an N-terminal FLAG epitope (pSV-mNotch-1-IC). Protein extracts were prepared 48 h after transfection. Expression of the activated Notch-1 protein was verified by Western blot analysis with an antibody directed against the FLAG epitope (Fig. 8B). In addition, extracts (5 µg) were assayed for protein-DNA interactions in band shift experiments using the 32P-labeled oligonucleotides SL366 (Fig. 8A) and SL332 (Fig. 8C) as probes. Using the HES-1-specific oligonucleotide SL366, an RBP-Jkappa -specific complex (band A) could already be identified in extracts of untransfected HEK-293 cells (Fig. 8A, lane 1). Transfection of 5 µg of pCMV-RBP-3 led to an increased RBP-Jkappa -specific DNA binding activity (Fig. 8A, lane 3). The endogenous RBP-Jkappa -specific DNA binding activity was almost abolished in extracts from HEK-293 cells transfected with 5 µg of pSV-mNotch-1-IC. In these extracts, a novel, more slowly migrating complex (band D) appeared (Fig. 8A, lane 5). Incubation of cell extracts with an antibody directed against the FLAG epitope supershifted complex D, resulting in complex E, which most likely contains Notch-1-IC interacting with RBP-Jkappa (Fig. 8A, lane 6). The anti-FLAG antibody did not interfere with RBP-Jkappa binding to DNA (Fig. 8A, lanes 2 and 4). The higher-order complex D and the supershifted complex E could also be identified in nuclear extracts from HEK-293 cells cotransfected with Notch-1-IC and an RBP-3 expression plasmid (Fig. 8A, lanes 7 and 8).


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FIG. 8.   Truncated mammalian Notch-1 interacts with Rep-kappa B/RBP-Jkappa DNA binding activity in transfected HEK-293 cells. (A) In cell extracts from HEK-293 cells transfected with a Notch-1-IC expression plasmid, Rep-kappa B/RBP-Jkappa -specific DNA binding activity bound to the HES-1-specific oligonucleotide (complex A) was decreased and a more slowly migrating complex appeared (complex D). Treatment with an anti-FLAG antibody (alpha -FLAG) recognized complex D and supershifted a novel complex, labeled E. (B) Cell extracts were prepared 48 h after transfection of 5 µg of pCMV-RBP-3 and/or 5 µg pSV-mNotch-1-IC and assayed for Notch-1-IC expression by Western blotting. (C) In cell lysates transfected with a Notch-1-IC expression plasmid, Rep-kappa B/RBP-Jkappa -specific DNA binding activity bound to the NF-kappa B2-specific oligonucleotide (complex A) was abolished, but no higher-order complex, could be identified; it was probably masked by additional NF-kappa B-specific DNA binding activity (complex B).

To investigate the interaction of RBP-Jkappa with Notch-1-IC in the context of the NF-kappa B2 promoter, similar experiments were performed. The oligonucleotide SL332 was used as a probe. The results are shown in Fig. 8C. Again, the RBP-Jkappa -specific complex A could be detected in lysates of untransfected HEK-293 cells (Fig. 8C, lane 1). The specificity of complex A was determined by competition experiments and supershift experiments with an RBP-Jkappa -specific antibody (data not shown). In lysates transfected with pSV-mNotch-1-IC, this complex was completely abolished, suggesting the occurrence of an interaction of truncated Notch-1 with Rep-kappa B/RBP-Jkappa at the NF-kappa B2 promoter (Fig. 8C, lane 3). A similar decrease in RBP-Jkappa binding activity was observed in extracts from HEK-293 cells transfected with a plasmid expressing the truncated form of Notch-1 together with CMV-RBP-3 (Fig. 8C, lane 5). However, we did not detect an additional, higher-order DNA binding complex (Fig. 8C, lanes 3 and 5). After lysates were incubated with the anti-FLAG antibody, a supershifted complex could not be identified (lanes 4 and 6).

Taken together, the results shown in Fig. 6 to 8 suggest the occurrence of a physical interaction between Rep-kappa B/RBP-Jkappa and truncated Notch-1, thereby changing RBP-Jkappa DNA binding complexes, both at the murine HES-1 promoter and at the human NF-kappa B2 promoter.

Activated mammalian Notch-1 stimulates NF-kappa B2 promoter activity. Since the interaction of Rep-kappa B/RBP-Jkappa with truncated Notch-1 results in alterations of the RBP-Jkappa -specific DNA binding, we were interested in the functional consequences. Therefore, we carried out transient-transfection experiments with NF-kappa B2-specific reporter constructs (Fig. 3) and expression plasmids for Notch-1-IC, RBP-2N, and RelA (p65). Luciferase activity of pASwt was stimulated 21-fold after transfecting 2 µg of pSV-mNotch-1-IC into COS-7 cells. This strong stimulation mediated by truncated Notch-1 was clearly diminished when pAS-SL49, which has a mutated RBP-Jkappa binding site, was used as a reporter (Fig. 9A). Therefore, transactivation of NF-kappa B2 by truncated mammalian Notch-1 depends on the functional RBP-Jkappa site within the NF-kappa B2 promoter.


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FIG. 9.   (A) Stimulation of the NF-kappa B2 promoter by activated Notch-1. Portions (2 µg) of reporter construct pASwt (black bars) and pAS-SL49 (open bars) were cotransfected into COS-7 cells with the indicated amounts of plasmid DNA expressing truncated Notch-1. (B) The RelA (p65)-mediated promoter activity is not further increased by the addition of Notch-1-IC, but Notch-1-IC-stimulated NF-kappa B2 promoter activity is abrogated by RBP-2N. Portions (2 µg) of reporter construct pASwt were cotransfected into COS-7 cells with the indicated amounts of plasmid DNA expressing RelA (p65), Notch-1-IC, and RBP-2N. Luciferase activity in 100 µg of total cellular extract was determined. The basal promoter activity of each construct was set to one. Mean values and standard deviations for at least four independent experiments are shown.

Cotransfection of pASwt with 1 µg of pRSVRelA stimulated the NF-kappa B2 promoter activity 16-fold, and this stimulation was only marginally increased by the addition of pSV-mNotch-1-IC. These data indicate that activated Notch-1 and RelA do not have an additive effect on the NF-kappa B2 promoter (Fig. 9B). However, the Notch-1-IC-mediated stimulation of the NF-kappa B2 promoter was greatly reduced with increasing amounts of RBP-2N, suggesting that activation versus repression is mediated by the ratio of Notch-1-IC/RBP-2N complexes to RBP-2N at the binding site (14-fold versus 1.5-fold) (Fig. 9B).

Rep-kappa B/RBP-Jkappa binds to a subset of NF-kappa B-responsive elements. In a previous study, we investigated whether Rep-kappa B binding was specific for the NF-kappa B2 kappa B elements or could also bind to other known kappa B elements. At that time, we did not know that Rep-kappa B was identical to RBP-Jkappa . Furthermore, we believed that the observed repression was mediated by kappa B elements in the NF-kappa B2 promoter. The oligonucleotides used in the former competition experiments were not designed in view of an RBP-Jkappa site overlapping the kappa B element. For these reasons, new oligonucleotides representing kappa B elements and flanking sequences from various genes were designed (Fig. 10B). Partially purified Rep-kappa B/RBP-Jkappa binding activity (fraction 12 [Fig. 1A]) was used in competition experiments with the oligonucleotide SL332 as a probe. The various competitors were added in 10- and 60-fold molar excesses, and the HES-1-specific oligonucleotide SL366 served as a control. The results are shown in Fig. 10A and summarized in Fig. 10B. The tested sequences could be divided into three subclasses with regard to Rep-kappa B/RBP-Jkappa DNA binding affinity: sequences with high, intermediate, and low levels of binding activity. Oligonucleotides with high affinity for Rep-kappa B/RBP-Jkappa DNA were derived from the promoters of the following genes, as listed in Fig. 10B: HES-1, used as a control (lanes 6 and 7); mRBP-Jkappa (lanes 10 and 11); Bcl-3 (lanes 8 and 9); interleukin-6 (IL-6) (lanes 30 and 31); Ikappa Balpha (lanes 28 and 29); beta 2-microglobulin (lanes 20 and 21); beta interferon (IFN-beta ) (lanes 24 and 25); and NF-kappa B2 (first kappa B site) (lanes 2 and 3). Moderate-level binding of Rep-kappa B/RBP-Jkappa was observed with oligonucleotides derived from the promoters of the following genes: angiotensinogen (one kappa B site) (lanes 18 and 19), invariant chain (lanes 32 and 33), major histocompatibility complex (MHC) class I (lanes 34 and 35), serum amyloid A1 (SAA-1) (lanes 14 and 15), and TNF-alpha (lanes 38 and 39). Weak binding or no affinity for Rep-kappa B/RBP-Jkappa was detected with oligonucleotides derived from the following promoters: A20 (lanes 16 and 17), immunoglobulin heavy chain (lanes 22 and 23), Igkappa enhancer (lanes 26 and 27), NF-kappa B1 (lanes 36 and 37), and NF-kappa B2 (second kappa B site) (lanes 12 and 13).


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FIG. 10.   Rep-kappa B/RBP-Jkappa binds to a subset of kappa B elements. (A) Competition experiments were performed with fraction 12 of partially purified nuclear extract from Jurkat-T cells; a radiolabeled oligonucleotide (SL332) encompassing the first kappa B element of the human NF-kappa B2 promoter (positions -119 to -90) was used as a probe. As competitors, the unlabeled double-stranded oligonucleotides (shown in panel B) were added at 20- and 60-fold molar excesses. (B) Oligonucleotides used for competition analysis. The sequences were derived from the promoters-enhancers of the genes indicated. In each case, the putative kappa B element (underlined) and the overlapping RBP-Jkappa site are marked. Their GenBank accession numbers are as follows: A20, M96756; angiotensinogen (Angioten.), M31673; Bcl-3, M31731; beta 2-microglobulin (beta 2-Micro), M12485; immunoglobulin heavy chain, U17387; HES-1, D16464; IFN-beta , V00534; Igkappa , X00268; Ikappa Balpha , U08468; IL-6, M22111; invariant chain, M35872; MHC-I, M11847; NF-kappa B1, X63942; RBP-Jkappa , X58337; SAA-1, M64088; and TNF-alpha , X59352. Symbols: +, strong binding; +/-, intermediate-level binding; -, weak or no binding.

Summarizing these data, we can reveal a Rep-kappa B/RBP-Jkappa consensus sequence, 5'-TGGGAA-3', which overlaps with a kappa B binding site. The concept of overlapping RBP-Jkappa binding sites with kappa B elements could allow the cell to tightly control kappa B-mediated gene expression in an unstimulated status through transcriptional repression by Rep-kappa B/RBP-Jkappa . However, the functional consequences of RBP-Jkappa binding to the promoters of these various genes have to be elucidated.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In a previous report, we demonstrated a negative transcriptional regulation of NF-kappa B2 mediated by kappa B elements in its promoter region. A ubiquitously expressed DNA binding activity which binds specifically to this region was identified. Because of its putative repressive effect, this binding activity was named Rep-kappa B. Rep-kappa B activity did not bind solely to the NF-kappa B2 kappa B element; it also bound to kappa B sites present in the IFN-beta and beta 2-microglobulin enhancers. In contrast, no binding to the canonical kappa B site was observed. Therefore, we postulated the existence of a novel mode of kappa B-dependent regulation mediated by Rep-kappa B (34).

Here we have provided biochemical and functional evidence that Rep-kappa B binding activity is identical or closely related to RBP-Jkappa . This assumption is based on the following findings. (i) By using partially purified Rep-kappa B binding activity in competition experiments, we identified an 8-bp Rep-kappa B binding motif (CATGGGAA) with strong sequence homology to the RBP-Jkappa consensus site (CGTGGGAA). This Rep-kappa B/RBP-Jkappa binding site partially overlaps a functional kappa B element (GGGAATTCCC). (ii) Partially purified Rep-kappa B activity binds with high affinity to a known RBP-Jkappa -responsive site in the murine HES-1 promoter. (iii) Different in vitro-translated RBP-Jkappa splice variants bind to the Rep-kappa B binding site in the NF-kappa B2 promoter. (iv) In supershift assays, Rep-kappa B activity is recognized by a specific antibody raised against RBP-Jkappa . (v) In transient-transfection assays, RBP-2N represses NF-kappa B2 promoter activity.

Several observations suggesting a role for RBP-Jkappa in the regulation of NF-kappa B-inducible genes have been published. (i) KBF2 was purified with a beta 2-microglobulin kappa B affinity column and found to be identical to RBP-Jkappa (27). (ii) A DNA binding activity with affinity for the MHC class I probe was purified from thymic nuclear extracts and identified as RBP-Jkappa (51). Binding of RBP-Jkappa to the MHC class I oligonucleotide was not evident in splenic extracts, suggesting the occurrence of tissue-specific binding. (iii) Recently, RBP-Jkappa was shown to be involved in transcriptional repression of the IL-6 gene. An RBP-Jkappa binding site overlapping with a functional kappa B element was identified as being responsible for this repression (30, 44).

In this report, we have shown that human RBP-Jkappa (RBP-2N) strongly represses basal as well as RelA (p65)-induced NF-kappa B2 promoter activity, suggesting that RBP-Jkappa acts as a repressor of NF-kappa B2 transcription. Previously, we did no