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SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway

2002; Springer Nature; Volume: 21; Issue: 20 Linguagem: Inglês

10.1093/emboj/cdf549

1460-2075

Franz Oswald,

Wnt/β-catenin signaling in development and cancer

Article15 October 2002free access SHARP is a novel component of the Notch/RBP-Jκ signalling pathway Franz Oswald Franz Oswald Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Ulrike Kostezka Ulrike Kostezka Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Kathy Astrahantseff Kathy Astrahantseff Department of Biochemistry, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Soizic Bourteele Soizic Bourteele Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Karin Dillinger Karin Dillinger Department of Biochemistry, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Ulrich Zechner Ulrich Zechner Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Leopold Ludwig Leopold Ludwig Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Monika Wilda Monika Wilda Department of Human Genetics University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Horst Hameister Horst Hameister Department of Human Genetics University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Walter Knöchel Walter Knöchel Department of Biochemistry, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Susanne Liptay Susanne Liptay Department of Pediatrics, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Roland M. Schmid Corresponding Author Roland M. Schmid Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Franz Oswald Franz Oswald Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Ulrike Kostezka Ulrike Kostezka Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Kathy Astrahantseff Kathy Astrahantseff Department of Biochemistry, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Soizic Bourteele Soizic Bourteele Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Karin Dillinger Karin Dillinger Department of Biochemistry, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Ulrich Zechner Ulrich Zechner Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Leopold Ludwig Leopold Ludwig Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Monika Wilda Monika Wilda Department of Human Genetics University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Horst Hameister Horst Hameister Department of Human Genetics University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Walter Knöchel Walter Knöchel Department of Biochemistry, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Susanne Liptay Susanne Liptay Department of Pediatrics, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Roland M. Schmid Corresponding Author Roland M. Schmid Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany Search for more papers by this author Author Information Franz Oswald1, Ulrike Kostezka1, Kathy Astrahantseff2, Soizic Bourteele1, Karin Dillinger2, Ulrich Zechner1, Leopold Ludwig1, Monika Wilda3, Horst Hameister3, Walter Knöchel2, Susanne Liptay4 and Roland M. Schmid 1 1Department of Internal Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany 2Department of Biochemistry, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany 3Department of Human Genetics University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany 4Department of Pediatrics, University of Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany ‡F.Oswald and U.Kostezka contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:5417-5426https://doi.org/10.1093/emboj/cdf549 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Notch proteins are the receptors for an evolutionarily highly conserved signalling pathway that regulates numerous cell fate decisions during development. Signal transduction involves the presenilin-dependent intracellular processing of Notch and nuclear translocation of the intracellular domain of Notch, Notch-IC. Notch-IC associates with the DNA-binding protein RBP-Jκ/CBF-1 to activate transcription of Notch target genes. In the absence of Notch signalling, RBP-Jκ/CBF-1 acts as a transcriptional repressor through the recruitment of histone deacetylase (HDAC) corepressor complexes. We identified SHARP as an RBP-Jκ/CBF-1-interacting corepressor in a yeast two-hybrid screen. In cotransfection experiments, SHARP-mediated repression was sensitive to the HDAC inhibitor TSA and facilitated by SKIP, a highly conserved SMRT and RBP-Jκ-interacting protein. SHARP repressed Hairy/Enhancer of split (HES)-1 promoter activity, inhibited Notch-1-mediated transactivation and rescued Notch-1-induced inhibition of primary neurogenesis in Xenopus laevis embryos. Based on our data, we propose a model in which SHARP is a novel component of the HDAC corepressor complex, recruited by RBP-Jκ to repress transcription of target genes in the absence of activated Notch. Introduction The differentiation of cells during development is often regulated by cell–cell interactions. An important function for Notch signalling is to control a mechanism called lateral inhibition, which ensures that two distinct cell types are produced in correct numbers from a population of initially homogenous cells. The number of neurons that develop from neural precursor cells is controlled in this way (for a review, see Beatus and Lendahl, 1998). During this process, neural precursors arise via the activity of basic helix–loop–helix (bHLH) transcriptions factors encoded by proneural genes, such as those in the Achaete-Scute (A-Sc) gene complex in Drosophila, and MASH in mammals. A second class of bHLH repressor proteins downstream of Notch, such as those encoded by the genes within the Enhancer of Split [E(Spl)] complex in Drosophila and HES (Hairy/Enhancer of Split) genes in mammals, appear to inhibit the formation of neural precursors. This mechanism ultimately restricts the number of cells that form the neural precursors. Despite their diverse function in multiple developmental programs, Notch receptors and ligands are highly conserved. The mammalian family of Notch proteins consists of at least four transmembrane receptors (Notch-1 to Notch-4). The Notch ligands (Jagged-1, Jagged-2, Delta-1, Delta-2 and Delta-3) represent transmembrane proteins of the DSL (Delta, Serrate, and Lag-2) family that, like Notch, contain multiple epidermal growth factor (EGF)-like repeats in their extracellular domains (Egan et al., 1998). After ligand binding, an extracellular cleavage step mediated by TACE, an ADAM family protease, occurs (Brou et al., 2000) followed by a cleavage step near the transmembrane region of the C-terminal protein fragment. This final proteolytic cleavage, which has been linked to presenilins (De Strooper et al., 1999; Struhl and Greenwald, 1999), releases the intracellular domain of Notch (Notch-IC, activated Notch), which then translocates to the nucleus and associates with the ubiquitous DNA-binding protein RBP-Jκ/CBF-1, the mammalian homologue of Drosophila Suppressor of Hairless [Su(H)] (Schroeter et al., 1998; Struhl and Adachi, 1998). RBP-Jκ has been shown to act as a repressor of transcription (Dou et al., 1994; Oswald et al., 1998). RBP-Jκ-mediated repression includes destabilization of the general transcription factor IID (TFIID)/TFIIA interaction (Olave et al., 1998) and recruitment of histone deacetylase (HDAC) corepressor complexes. Indeed, CIR-1, an RBP-Jκ-interacting corepressor protein, was shown to associate with a corepressor complex including SAP-30 and HDAC-2 (Hsieh et al., 1999). RBP-Jκ has also been shown to interact with the corepressor complex proteins SMRT and HDAC-1 (Kao et al., 1998). Moreover, the LIM protein KyoT2 negatively regulates transcription by association with RBP-Jκ (Taniguchi et al., 1998). Here we present the identification and characterization of SHARP as an RBP-Jκ-interacting protein. SHARP, originally identified as an SMRT-associated protein in a yeast two-hybrid screen, was previously implicated in nuclear receptor signalling (Shi et al., 2001). We show that SHARP physically interacts with RBP-Jκ in vitro and in vivo. In cotransfection experiments, SHARP repressed transcription in an HDAC-dependent fashion and transactivation mediated by Notch-1 was inhibited by SHARP. In addition, overexpression of SHARP induced a neurogenic phenotype in Xenopus laevis embryos and rescued loss of primary neurogenesis resulting from overexpression of dominant active Notch-1. Our data suggest that SHARP is recruited by RBP-Jκ to a HDAC corepressor complex regulating the Notch signalling pathway. Results SHARP interacts with RBP-Jκ The RBP2N cDNA was fused to the Gal4 DNA-binding domain in pGBT9, and a yeast two-hybrid screen for RBP-Jκ-interacting proteins was performed using a human embryonic liver cDNA library (Stratagene). We identified an 1100 bp open reading frame having no significant homology to known sequences at the cDNA or predicted amino acid level. While this study was in progress, the corresponding full-length protein was identified as the SMRT-interacting corepressor, SHARP, implicated in nuclear receptor signalling (Shi et al., 2001). A schematic representation of the SHARP protein fragments used in this study is shown in Figure 1. Figure 1.Schematic representation of SHARP-specific expression constructs. Reported and putative functional domains are shown in the full-length construct. The protein contains four putative RRM and five putative nuclear localization signals (NLS). The receptor interaction domain (RID) and the SMRT interaction/repression domain (SID/RD) were characterized previously (Shi et al., 2001). The RBP interaction domain (RBPID) is characterized in this study. The black bars represent protein fragments used for antibody production. The first and last amino acids compared with the full-length protein are indicated in parentheses. The SHARP-Δ constructs represent in-frame deletions of the indicated amino acids. Download figure Download PowerPoint Interaction of SHARP with RBP-Jκ was verified in vitro by pull-down assays with glutathione S-tranferase (GST)–RBP2N. Glutathione–Sepharose beads were coated with bacterially expressed GST or GST–RBP2N proteins, and used as bait for cell-free synthesized and radiolabelled SHARP fragments (Figure 2A). Radiolabelled mNotch-1- IC protein was used as a positive control for RBP binding. As expected, a clear interaction of mNotch-1-IC with GST–RBP2N was observed (Figure 2A, left). No interaction could be detected with glutathione–Sepharose or with GST alone. Two fragments of SHARP (2770–3127 and 2790–3127) interacted with GST–RBP2N (Figure 2A, middle). No interaction was detected with a smaller SHARP fragment (2834–3127) (Figure 2A, right). These results show that SHARP physically interacts with GST–RBP2N, and that the region between amino acids 2790 and 2840 is required for interaction. Figure 2.SHARP interacts with RBP-Jκ. (A) Cell-free synthesized SHARP(2770–3127) and the N-terminal-deleted SHARP(2790–3127) bind specifically to GST–RBP2N (middle). A further N-terminal- truncated SHARP(2843–3127) construct fails to interact with GST–RBP2N (right). Interaction of GST–RBP2N with the intracellular form of Notch-1 (Notch-1-IC) was used as a control (left). GST–RBP2N was immobilized on Sepharose beads and incubated with in vitro translated, radiolabelled proteins. After extensive washing, proteins were eluted and separated on SDS–PAGE. (B) RBP-Jκ binding to GST–Notch-1-IC is reduced by the addition of SHARP. Cell-free synthesized proteins alone are shown in lanes 1, 2, 7 and 8. GST–Sepharose beads and GST protein alone were used as negative controls (lanes 3, 4, 9 and 10). RBP-Jκ binding in the absence (lanes 5 and 11) and presence of either SHARP (2770–3127, lane 6) or SHARP lacking the RBP-binding site (lane 12) is shown. (C) Supernatants from GST–Notch-1-IC pull-downs (B, lanes 5, 6, 11 and 12) were coimmuno precipated with anti-Flag-agarose (lanes 1–4, respectively). RBP2N was only immunoprecipitated with a SHARP protein containing the RBP-binding domain (compare lanes 2 and 4). (D) SHARP and RBP interact in the mammalian two-hybrid assay. HeLa cells were cotransfected with the indicated Gal4–SHARP constructs and increasing amounts of CMV–RBP–VP16 together with pFR-Luc. Fold-activation was determined by the relative luciferase activity after cotransfection of the Gal4–SHARP(2770–3127) construct alone. Luciferase activity was determined from 100 μg portions of total cell extract. Mean values and standard deviations from four experiments are shown. Download figure Download PowerPoint Since RBP binds SHARP and Notch, the question arose whether both proteins could bind RBP simultaneously. Therefore, we performed a pull-down using GST–Notch-1-IC as bait (Figure 2B). Cell-free synthesized RBP2N (lane 1) did not bind to beads or GST protein alone (lanes 3 and 4), but bound specifically to GST–Notch-1-IC (lane 5). Addition of cell-free synthesized SHARP (2770–3217, lane 2) reduced the amount of RBP2N protein bound to Notch-1-IC (lane 6). A cell-free synthesized SHARP fragment lacking the RBP-binding site did not reduce RBP binding to Notch-1-IC (lane 12). The supernatants from Figure 2B, lanes 5, 6, 11 and 12 were used in an anti-Flag coimmunoprecitation (Figure 2C). The RBP protein displaced from GST–Notch-1-IC was bound to the SHARP protein (lane 2). These experiments suggest that RBP binds either Notch or SHARP exclusively. To test SHARP RBP-Jκ interaction on a cellular background, we performed a mammalian two-hybrid assay. SHARP(2770–3127) and SHARP(2843–3127) were fused to the Gal4 DNA-binding domain. The SHARP–Gal4 fusion vectors were cotransfected into HeLa cells together with increasing amounts of CMV–RBP–VP16 and the reporter plasmid, pFR-Luc, containing five copies of the Gal4-binding site upstream of a luciferase gene. Luciferase activity was detected when RBP–VP16 was coexpressed with Gal4–SHARP(2770–3127), but not after coexpression of RBP–VP16 with Gal4–SHARP(2843–3127, Figure 2D). As in the pull-downs, the N-terminally deleted SHARP(2843–3127) fragment had lost the capacity to interact with the RBP fusion protein in the mammalian two-hybrid assay. To characterize the RBP-Jκ-binding domain of SHARP in more detail, we performed additional pull-down assays using GST–RBP2N and the C-terminal part of SHARP, SHARP(2002–3664), as well as the in frame deletions, SHARP-ΔA to ΔD (see Figure 1). As shown in Figure 3A, cell-free synthesized SHARP(2002–3664) interacted specifically with GST–RBP2N. A strong interaction could also be detected with SHARP-ΔA containing a seven amino acid in-frame deletion. GST–RBP2N did not bind the SHARP-ΔB, -ΔC and -ΔD deletion constructs (Figure 3A). The results from these mapping experiments suggest that amino acids 2804–2816 within SHARP are required for interaction with RBP-Jκ. Figure 3.SHARP binds RBP in vivo and requires a 14 amino acid region. (A) Cell-free synthesized SHARP(2002–3664) binds specifically to GST–RBP2N. Deletion of seven amino acids within SHARP-ΔA had no effect on binding to GST–RBP2N. The in-frame deletion mutants SHARP-ΔB, -ΔC and -ΔD failed to bind to GST–RBP2N. (B) Expression of transfected RBP2N–GFP was detected in lysate (lys, lanes 4, 7, 10 and 13) as well as supernatant (sn, lanes 5, 8, 11 and 14) using an antibody against GFP. Coimmunoprecipitated RBP2N–GFP was detected in the IP fraction after cotransfection with SHARP(2770–3127) (IP, lane 9) and SHARP(2002–3664) (IP, lane 12). RBP2N was not coimmunoprecipitated after cotransfection of SHARP(2002–3664)-ΔB (IP, lane 15). (C) Full-length SHARP binds to RBP2N–GFP in vivo. Expression of transfected RBP2N–GFP was detected in the lysate (lanes 2 and 3). RBP2N–GFP was coimmunoprecipitated after cotransfection of SHARP (lane 6). HEK-293 cells were transiently transfected with an expression plasmid for RBP2N–GFP alone or together with expression plasmids for the indicated N-terminal Flag-tagged SHARP proteins. The SHARP proteins were immunoprecipitated using an antibody directed against the Flag-tag. Coimmunoprecipitated RBP2N–GFP proteins were detected by western blotting using an anti-GFP antibody. The asterisks indicate the heavy and light chains of the anti-Flag antibody. Download figure Download PowerPoint In vivo interaction of RBP and SHARP was tested by coimmunoprecipitation assays. HEK-293 cells were transfected with an expression plasmid for RBP2N–green fluorescent protein (GFP) alone or together with expression plasmids for various N-terminally Flag-tagged SHARP proteins. The SHARP proteins were immuno precipitated using an antibody against the Flag tag. Coimmunoprecipitated RBP2N–GFP proteins were detected by western blotting using an anti-GFP antibody (Figure 3B). No cellular protein was detected by the anti-GFP antibody in either, the lysate, or supernatant after immunoprecipitation of untransfected HEK-293 cells (Figure 3B, lanes 1 and 2). After transfection of RBP2N–GFP alone, the GFP fusion protein was detected in the lysate (lane 4) and in the supernatant (lane 5), but not in the IP fraction (lane 6). The RBP2N–GFP fusion protein was coimmunoprecipitated from lysates of HEK-293 cells cotransfected with the SHARP(2770–3127) expression construct (lane 9), suggesting an interaction of SHARP and RBP2N in vivo. Interaction was also observed in lysates of cells cotransfected with SHARP (2202–3664, lane 12). In contrast, the RBP2N–GFP protein was not coimmunoprecipitated from lysates of cells cotransfected with the SHARP-ΔB expression plasmid (lane 15). Finally, the RBP protein was also coimmunoprecipitated after cotransfection of both RBP2N–GFP and the full-length SHARP construct (Figure 3C, lane 6). These results show that (i) full-length SHARP is able to interact with RBP2N–GFP in vivo, and (ii) amino acids 2804–2816 within SHARP are required for interaction. To investigate the formation of SHARP/RBP DNA-binding complexes, we performed band shift experiments using in vitro translated RBP2N (TNT–RBP2N) together with the in vitro translated SHARP fragments, TNT–SHARP(2770–3127) and TNT–SHARP(2843–3127). Translation of TNT–SHARP proteins was controlled in the anti-Flag western blot shown in Figure 4A (WB). The RBP DNA-binding complexes A and a (lane 1) were supershifted after addition of an anti-RBP antibody to generate complexes B and b (lanes 3, 6 and 9). Addition of an anti-Flag antibody had no effect on the RBP DNA-binding complexes (lane 2). Addition of cell-free synthesized SHARP(2770–3127) to the reaction mixture resulted in the formation of a novel, higher order complex (complex C, lane 4). This novel complex disappeared after addition of an anti-Flag antibody to generate the supershifted complex D containing the flag-tagged SHARP (lane 5). No higher order complex was detected after addition of cell-free synthesized SHARP(2843–3127) to the reaction mixture (lane 7). The RBP DNA-binding complexes A and a could also be detected in nuclear extracts from SUP-T1 cells (Figure 4B). Additionally, the slower migrating complex B was present, and this complex was supershifted with an antibody against the C-terminus of human Notch-1 (lane 4), indicating that complex B represents an RBP–Notch-1 complex. Addition of cell-free synthesized SHARP(2770–3127) but not SHARP(2843–3127) lacking the RBP-binding site results in the formation of complex C at the expense of complexes A, a and interestingly also B. Again, complex C was supershifted with the anti-Flag antibody (lane 7). These results show that (i) SHARP(2770–3127) can form a DNA-binding complex with both cell-free RBP2N and endogenous RBP-Jκ, whereas SHARP lacking the RBP-binding site cannot, and (ii) the DNA-bound RBP–SHARP complex in SUP-T1 lysates is formed at the expense of both the DNA-bound RBP complex and the RBP–Notch-1 complex. These data combined with those shown in Figure 2B and C suggest that the presence of either SHARP or Notch in the DNA-bound RBP complex is exclusive. Figure 4.(A) Detection of RBP/SHARP DNA-binding complexes. The insert (left) shows the TNT–SHARP translation products visualized in an anti-Flag western blot. Cell-free synthesized RBP2N formed specific DNA-binding complexes (complexes A and a, lane 1). These complexes were supershifted after addition of an anti-RBP antibody to generate complexes B and b (lanes 3, 6 and 9). Addition of cell-free synthesized SHARP(2770–3127) resulted in the formation of a novel higher order complex C (lane 4), which was supershifted by an antibody directed against the N-terminal Flag tag on the SHARP protein to generate complex D (lane 5). Cell-free synthesized SHARP(2843–3127) failed to form a higher order complex with RBP (lane 7), and no supershifted complex could be detected after addition of the anti-Flag antibody (lane 8). (B) SHARP or Notch presence in the DNA-bound RBP complex is exclusive. Sup-T1 cell lysate formed specific DNA-binding complexes A, a and B (lane 1). Complex B was not supershifted with α-Flag (lane 2) or an antibody against the N-terminus of human Notch-1 (α-N1/N, lane 3), but was supershifted to complex D with α-N1/C, an antibody against the C-terminus of human Notch-1, (lanes 4, 8 and 12). Addition of cell-free synthesized SHARP(2770–3127) created an additional higher order complex, C (lanes 5 and 6), which could be supershifted with α-Flag (lane 7). Note that this complex migrates at the same position as complex B (compare to A, lane 5). No changes in the DNA-binding complexes A, a and B were observed with the addition of cell-free synthesized SHARP missing the RBP-binding site (lanes 9–12). The 32P-labelled oligonuleotide FO233 was used as a probe. The asterisk designates a non-specific complex. Download figure Download PowerPoint Expression of SHARP mRNA in the mouse SHARP is ubiquitously expressed in the embryonic and adult mouse. Hybridization of a SHARP riboprobe to 9.5 and 14.5 d.p.c. mouse embryo sections showed little differences in SHARP expression between organs, although expression in the liver and lung were slightly higher (see Supplementary data, available at The EMBO Journal Online). All adult mouse tissues examined expressed SHARP, although at variable levels according to real-time RT–PCR analysis (see Supplementary data). The highest expression was measured in the brain and testis. SHARP expression overlapped with, but was not restricted to, the expression pattern of Notch-1 during mouse development. SHARP is a nuclear protein and colocalizes with RBP-Jκ and SKIP To test subcellular localization of SHARP proteins, we performed transient transfection experiments with plasmids expressing SHARP(2770–3127), SHARP(2002–3664) and the full-length SHARP protein in HEK-293 cells. The expressed proteins were localized using the α-SHARP.1 rabbit polyclonal antiserum and confocal microscopy. Whereas SHARP(2770–3127) was localized in the cytoplasm as well as in the nucleus (Figure 5Aa), the C-terminal part of SHARP(2002–3664) was only found in the cytoplasm (Figure 5Ab). The full-length SHARP protein was localized exclusively in the nucleus where it was expressed in a speckled pattern (Figure 5Ac). Subcellular localization of endogenous SHARP protein from various human cell lines was investigated by immunofluorescence using a second polyclonal antiserum directed against the N-terminus of SHARP (α-SHARP.2, see also Figure 1). Again, the SHARP protein was located in the nuclei of HeLa as well as pancreatic carcinoma cell lines, MiaPaCa-2 and PANC-1 (Figure 5B). HEK-293 cells were transiently transfected with expression plasmids for SHARP and RBP2N–GFP. Immunofluorescence staining showed that RBP2N–GFP and SHARP were colocalized in speckles in the nuclei (Figure 5C). Figure 5.(A) Subcellular localization of SHARP constructs. HEK-293 cells were transfected with plasmids expressing N-terminal Flag-tagged SHARP(2770–3127) (a and d), SHARP(2002–3664) (b and e), and full-length SHARP(1–3664) (c and f). At 24 h after transfection, cells were fixed, permeabilized and immunostained with an antiserum directed against human SHARP (α-SHARP-1). (B) Nuclear localization of endogenous SHARP. The human cell lines HeLa (a and d), MiaPaCa-2 (b and e) and PANC-1 (c and f) were fixed and immunostained with a rabbit polyclonal antiserum (α-RCor.2) directed against human SHARP (upper) or the pre-immune serum from the same rabbit (lower). (C) SHARP colocalizes with RBP2N–GFP. HEK-293 cells were transiently transfected with an expression plasmid for SHARP and RBP2N–GFP. Cells were fixed 24 h after transfection, permeabilized and immunostained with the SHARP antiserum. The subcellular localization of SHARP (red, a) and RBP2N-GFP (green, b) was assayed by confocal microscopy. (D) SHARP colocalizes with SKIP. HEK-293 cells were transiently transfected with an expression plasmid for Flag-tagged human SKIP. Cells were fixed 24 h after transfection, permeabilized and immunostained with an antibody directed against the Flag epitope and with the SHARP antiserum. The subcellular localization of SHARP (red, a) and SKIP proteins (green, b) was assayed by confocal microscopy. Download figure Download PowerPoint It was shown previously that RBP-Jκ acts as a transcriptional repressor by recruiting a corepressor complex involving SMRT and HDAC-1 (Kao et al., 1998). Originally identified as an RBP-Jκ-associated protein, SKIP (Zhou et al., 2000b) was shown to be a component of this corepressor complex (Zhou et al., 2001). To investigate the role of SHARP in this context, we performed transient transfection experiments in HEK-293 cells with expression plasmids for N-terminally Flag-tagged SKIP. Localization of SKIP and SHARP was assayed by immunofluorescence using the anti-Flag antibody and α-SHARP.2 antiserum. Overexpressed SKIP protein was localized in a halo around the nuclear membrane as well as in speckles in the nucleus (Figure 5Db). Interestingly, SHARP localized to these nuclear spots (Figure 5Da) resulting in a colocalized signal in the overlay (Figure 5Dc). Transcriptional repression via SHARP depends on HDAC activity and is facilitated by SKIP To characterize the function of SHARP in RBP-Jκ-mediated transcriptional regulation, we performed transient cotransfection experiments with expression plasmids for RBP–VP16 and full-length SHARP together with the RBP-Jκ responsive reporter construct, pGa981/6. This luciferase reporter plasmid carries six repeats of the EBNA-2 responsive element within the Epstein–Barr Virus (EBV) TP-1 promoter upstream of a minimal β-globin promoter. As shown previously (Oswald et al., 2001), cotransfection of a plasmid expressing RBP fused to the VP16 transactivation domain (RBP–VP16) into HeLa cells resulted in stimulation of luciferase activity (Figure 6A). Transactivation mediated by RBP–VP16 was gradually reduced after cotransfection of increasing amounts of SHARP suggesting that SHARP acts as an RBP-interacting corepressor. In addition, this repression was HDAC-dependent since incubation of the cells with increasing amounts of the HDAC inhibitor trichostatin A (TSA) for 6 h resulted in a complete loss of repression (Figure 6A). This appeared to be specific, since TSA could not further activate the reporter in the presence of Notch-1-ΔE, a dominant active form of Notch-1. Taken together, incubation of the cells with increasing amounts of TSA resulted in histone hyperacetylation and completely restored RBP–VP16-mediated activation (Figure 6A and B). Figure 6.(A) SHARP-mediated repression depends on HDAC activity. Portions (2 μg) of reporter construct pGa981/6 were transfected alone into HeLa cells or cotransfected with 100 ng of plasmids expressing RBP–VP16 together with increasing amounts (200 and 500 ng) of plasmid expressing SHARP. Cotransfected cells were incubated with increasing amounts (25, 50 and 100 nM) of TSA 6 h prior to harvesting. Cells transfected with reporter construct alone had 3-fold higher activation in the presence of 100 nM TSA. Cotransfection of Notch-1-ΔE with the reporter plasmid shows the activated condition, which could not be further increased with 100 mM TSA treatment. (B) Treatment of HeLa cells with TSA results in hyperacetylation of histones. Extracts (20 μg) from cells treated with the indicated amounts of TSA were used fo