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• W2058733797 abstract "The Notch receptor that plays an important role in cell fate determination is intracellularly cleaved by interaction with the ligand. The cleaved intracellular region (RAMIC) of Notch is translocated into the nucleus and interacts with a DNA-binding protein RBP-J to activate transcription of genes that regulate cell differentiation. Although RAMIC has been shown to facilitate the RBP-J-mediated transactivation by displacing the histone deacetylase corepressor complex from RBP-J, there is no evidence demonstrating the involvement of histone acetyltransferases (HATs) in the transactivation. Here we show that mouse Notch1 RAMIC interacts with two conserved HATs, mouse PCAF and GCN5, and recruits each of the HATs to RBP-J. The ankyrin repeats and the transactivation domain of RAMIC and the N-terminal regions of PCAF and GCN5, respectively, are required for the interaction. We also show that not only mouse Notch1 but alsoDrosophila Notch RAMIC interacts with mouse PCAF and GCN5 in mammalian cells. Furthermore, the RBP-J-mediated transactivation activity of RAMIC is repressed by two HAT inhibitor proteins, E1A and Twist. These results suggest that HATs including PCAF and GCN5 play an important role in the RBP-J-mediated transactivation by RAMIC. The Notch receptor that plays an important role in cell fate determination is intracellularly cleaved by interaction with the ligand. The cleaved intracellular region (RAMIC) of Notch is translocated into the nucleus and interacts with a DNA-binding protein RBP-J to activate transcription of genes that regulate cell differentiation. Although RAMIC has been shown to facilitate the RBP-J-mediated transactivation by displacing the histone deacetylase corepressor complex from RBP-J, there is no evidence demonstrating the involvement of histone acetyltransferases (HATs) in the transactivation. Here we show that mouse Notch1 RAMIC interacts with two conserved HATs, mouse PCAF and GCN5, and recruits each of the HATs to RBP-J. The ankyrin repeats and the transactivation domain of RAMIC and the N-terminal regions of PCAF and GCN5, respectively, are required for the interaction. We also show that not only mouse Notch1 but alsoDrosophila Notch RAMIC interacts with mouse PCAF and GCN5 in mammalian cells. Furthermore, the RBP-J-mediated transactivation activity of RAMIC is repressed by two HAT inhibitor proteins, E1A and Twist. These results suggest that HATs including PCAF and GCN5 play an important role in the RBP-J-mediated transactivation by RAMIC. Suppressor of Hairless basic helix-loop-helix RBP-J-associating molecule ankyrin nuclear localization signal transcriptional activation domain Epstein-Barr virus EBV nuclear antigen 2 EBNA2-responsive element histone deacetylase histone acetyltransferase CREB-binding protein P300/CBP-associated factor thymidine kinase cytomegalo-virus DNA-binding domain immunoprecipitation polyacrylamide gel electrophoresis trichostatin A general transcription factor monoclonal antibody electrophoretic mobility shift assay The Notch family consists of single transmembrane receptors that are involved in cell fate determination in multiple steps of development (1.Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4766) Google Scholar). Notch has been found in a variety of organisms from nematode and fruit fly to higher vertebrates, and four Notch proteins have been identified in mammals. It was shown that the Notch receptor is proteolytically processed in the trans-Golgi network into the N-terminal fragment containing most of the extracellular region and the remaining C-terminal fragment, and that the two fragments are presented on the cell surface as a functional heterodimer (2.Blaumueller C.M. Qi H. Zagouuras P. Artavanis-Tsakonas S. Cell. 1997; 90: 281-291Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 3.Logeat F. Bessia C. Brou C. LeBail O. Jarriault S. Seidah N.G. Israël A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8108-8112Crossref PubMed Scopus (562) Google Scholar). The Notch signal triggered by interaction with the ligand like Delta (4.Jarriault S. LeBail O. Hirsunger E. Pourquié O. Logeat F. Strong C.F. Brou C. Seidah N.G. Israël A. Mol. Cell. Biol. 1998; 18: 7423-7431Crossref PubMed Scopus (277) Google Scholar, 5.Kuroda K. Tani S. Tamura K. Minoguchi S. Kurooka H. Honjo T. J. Biol. Chem. 1999; 274: 7238-7244Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) or Jagged/Serrate (6.Lindsell C.E. Shawber C.J. Boulter J. Weinmaster G. Cell. 1995; 80: 909-917Abstract Full Text PDF PubMed Scopus (536) Google Scholar, 7.Luo B. Aster J.C. Hasserjian R.P. Kuo F. Sklar J. Mol. Cell. Biol. 1997; 17: 6057-6067Crossref PubMed Scopus (172) Google Scholar) blocks differentiation of stem or progenitor cells and keeps them in a multipotential state. Recent studies indicate that interaction with the ligand induces a second cleavage at a site within or near the transmembrane region of the C-terminal fragment of the heterodimer, resulting in the release of the intracellular region and its nuclear translocation (8.Struhl G. Adachi A. Cell. 1998; 93: 649-660Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 9.Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1328) Google Scholar). The translocated nuclear Notch intracellular region binds to a ubiquitous DNA-binding protein Drosophila Suppressor of Hairless (Su(H))1 or its mammalian homolog RBP-J/CBF1 (10.Honjo T. Genes to Cells. 1996; 1: 1-9Crossref PubMed Scopus (179) Google Scholar), and thereby transcription of the downstream target genes such as Enhancer of split [E(spl)]complex genes (11.Lecourtois M. Schweisguth F. Genes Dev. 1995; 9: 2598-2608Crossref PubMed Scopus (378) Google Scholar, 12.Bailey A.M. Posakony J.W. Genes Dev. 1995; 9: 2609-2622Crossref PubMed Scopus (501) Google Scholar) or mammalian homologs of Hairy andEnhancer of split, HES-1 and HES-5genes (4.Jarriault S. LeBail O. Hirsunger E. Pourquié O. Logeat F. Strong C.F. Brou C. Seidah N.G. Israël A. Mol. Cell. Biol. 1998; 18: 7423-7431Crossref PubMed Scopus (277) Google Scholar, 5.Kuroda K. Tani S. Tamura K. Minoguchi S. Kurooka H. Honjo T. J. Biol. Chem. 1999; 274: 7238-7244Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 13.Jarriault S. Brou C. Logeat F. Schroeter E.H. Kopan R. Israël A. Nature. 1995; 377: 355-358Crossref PubMed Scopus (1206) Google Scholar, 14.Otsuka T. Ishibashi M. Gradwohl G. Nakanishi S. Guillemot F. Kageyama R. EMBO J. 1999; 18: 2196-2207Crossref PubMed Google Scholar) is up-regulated. Basic helix-loop-helix (bHLH) proteins encoded by the E(spl) and HES genes antagonize other bHLH transcription factors that induce cell differentiation (15.Kageyama R. Nakanishi S. Curr. Biol. 1997; 7: 659-665Google Scholar). Thus the Notch signal can be mimicked by forced expression of the Notch intracellular region (16.Lieber T. Kidd S. Alcamo E. Corbin V. Young M.W. Genes Dev. 1993; 7: 1949-1965Crossref PubMed Scopus (359) Google Scholar, 17.Kopan R. Nye J.S. Weintraub H. Development. 1994; 120: 2385-2396Crossref PubMed Google Scholar, 18.Kato H. Taniguchi Y. Kurooka H. Minoguchi S. Sakai T. Okazaki-Nomura S. Tamura K. Honjo T. Development. 1997; 124: 4133-4141Crossref PubMed Google Scholar). The intracellular region of mouse Notch1 (hereafter designated as RAMIC) contains two evolutionarily conserved RBP-J-binding domains (10.Honjo T. Genes to Cells. 1996; 1: 1-9Crossref PubMed Scopus (179) Google Scholar,19.Tamura K. Taniguchi Y. Minoguchi S. Sakai T. Tun T. Furukawa T. Honjo T. Curr. Biol. 1995; 5: 1416-1423Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar); the transmembrane-proximal RAM domain and the CDC10/ankyrin (ANK) repeats flanked by two nuclear localization signals (NLSs) (see Fig.1). While the RAM domain binds to RBP-J strongly (19.Tamura K. Taniguchi Y. Minoguchi S. Sakai T. Tun T. Furukawa T. Honjo T. Curr. Biol. 1995; 5: 1416-1423Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 20.Roehl H. Bosenberg M. Blelloch R. Kimble J. EMBO J. 1996; 15: 7002-7012Crossref PubMed Scopus (69) Google Scholar, 21.Hsieh J.J.-D. Henkel T. Salmon P. Robey E. Peterson M.G. Hayward S.D. Mol. Cell. Biol. 1996; 16: 952-959Crossref PubMed Scopus (393) Google Scholar), the ANK repeats bind to it weakly (18.Kato H. Taniguchi Y. Kurooka H. Minoguchi S. Sakai T. Okazaki-Nomura S. Tamura K. Honjo T. Development. 1997; 124: 4133-4141Crossref PubMed Google Scholar, 22.Aster J.C. Robertson E.S. Hasserjian R.P. Turner J.R. Kieff E. Sklar J. J. Biol. Chem. 1997; 272: 11336-11343Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Hence, IC lacking the RAM domain can activate transcription of the HES genes but less strongly than RAMIC (13.Jarriault S. Brou C. Logeat F. Schroeter E.H. Kopan R. Israël A. Nature. 1995; 377: 355-358Crossref PubMed Scopus (1206) Google Scholar, 18.Kato H. Taniguchi Y. Kurooka H. Minoguchi S. Sakai T. Okazaki-Nomura S. Tamura K. Honjo T. Development. 1997; 124: 4133-4141Crossref PubMed Google Scholar). In the C-terminal region of RAMIC, there are the glutamine-rich OPA sequence and the PEST sequence, the functions of which are not clear yet. We recently identified a novel transcriptional activation domain (TAD) between the second NLS and the PEST sequence of mouse Notch1 (23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar). Although it was reported thatDrosophila Notch RAMIC also contains a potent TAD in the region corresponding to the TAD of mouse Notch1 RAMIC (24.Kidd S. Lieber T. Young M.W. Genes Dev. 1998; 12: 3728-3740Crossref PubMed Scopus (192) Google Scholar), the amino acid sequences in the corresponding regions of the two species are not homologous. RBP-J is known to associate with the viral protein Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) which is essential for the immortalization of human primary B lymphocytes (10.Honjo T. Genes to Cells. 1996; 1: 1-9Crossref PubMed Scopus (179) Google Scholar). EBNA2 has an acidic TAD similar to the TAD of the virus protein VP16 (25.Cohen J.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8030-8034Crossref PubMed Scopus (39) Google Scholar) and binds to RBP-J to activate transcription from the EBV promoter through the EBNA2-responsive element (EBNA2RE) carrying canonical RBP-J-binding sites (26.Zimber-Strobl U. Strobl L.J. Meitinger C. Hinrichs R. Sakai T. Furukawa T. Honjo T. Bornkamm G.W. EMBO J. 1994; 13: 4973-4982Crossref PubMed Scopus (174) Google Scholar, 27.Henkel T. Ling P.D. Hayward S.D. Peterson M.G. Science. 1994; 265: 92-95Crossref PubMed Scopus (364) Google Scholar). Since the EBV promoter and the HES-1 promoter are stimulated by either RAMIC or EBNA2, respectively (18.Kato H. Taniguchi Y. Kurooka H. Minoguchi S. Sakai T. Okazaki-Nomura S. Tamura K. Honjo T. Development. 1997; 124: 4133-4141Crossref PubMed Google Scholar, 28.Sakai T. Taniguchi Y. Tamura K. Minoguchi S. Fukuhara T. Strobl L.J. Zimber-Strobl U. Bornkamm G.W. Honjo T. J. Virol. 1998; 72: 6034-6039Crossref PubMed Google Scholar), it is expected that the cellular (RAMIC) and viral (EBNA2) proteins activate transcription from a similar set of promoters by a common mechanism. Evidently, provision of the TAD is at least one mechanism of the RBP-J-mediated transactivation. In addition, several lines of evidence have suggested that RBP-J is associated with a putative corepressor(s) and that RAMIC and EBNA2 displace the corepressor(s) from RBP-J to facilitate the transactivation (18.Kato H. Taniguchi Y. Kurooka H. Minoguchi S. Sakai T. Okazaki-Nomura S. Tamura K. Honjo T. Development. 1997; 124: 4133-4141Crossref PubMed Google Scholar, 21.Hsieh J.J.-D. Henkel T. Salmon P. Robey E. Peterson M.G. Hayward S.D. Mol. Cell. Biol. 1996; 16: 952-959Crossref PubMed Scopus (393) Google Scholar, 23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar, 27.Henkel T. Ling P.D. Hayward S.D. Peterson M.G. Science. 1994; 265: 92-95Crossref PubMed Scopus (364) Google Scholar, 29.Waltzer L. Bourillot P.Y. Sergeant A. Manet E. Nucleic Acids Res. 1995; 23: 4939-4945Crossref PubMed Scopus (69) Google Scholar). It has been demonstrated that hypoacetylated histones accumulate within transcriptionally silenced genes, while hyperacetylated histones accumulate within transcriptionally active genes (30.Wade P.A. Pruss D. Wollfe A.P. Trends Biochem. Sci. 1997; 22: 128-132Abstract Full Text PDF PubMed Scopus (397) Google Scholar, 31.Kornberg R.D. Lorch Y. Cell. 1999; 98: 285-294Abstract Full Text Full Text PDF PubMed Scopus (1400) Google Scholar). In fact, many transcriptional repressors and corepressors have been shown to associate with histone deacetylase (HDAC) activity that generates a repressed chromatin structure with decreased accessibility to the transcription machinery (31.Kornberg R.D. Lorch Y. Cell. 1999; 98: 285-294Abstract Full Text Full Text PDF PubMed Scopus (1400) Google Scholar, 32.Kouzarides T. Curr. Opin. Genet. Dev. 1999; 9: 40-48Crossref PubMed Scopus (581) Google Scholar). Recently, Kao et al. (33.Kao H.-Y. Ordentlich P. Koyano-Nakagawa N. Tang Z. Downes M. Kintner C.R. Evans R.M. Kadesch T. Genes Dev. 1998; 12: 2269-2277Crossref PubMed Scopus (481) Google Scholar) demonstrated that RBP-J interacts with a corepressor complex including the SMRT/NCoR family of nuclear receptor corepressors and histone deacetylase 1 (HDAC1) (34.Xu L. Glass C.K. Rosenfeld M.G. Curr. Opin. Genet. Dev. 1999; 9: 140-147Crossref PubMed Scopus (801) Google Scholar). They showed that SMRT/NCoR competed with TAN-1 (human Notch1 RAMIC) for RBP-J-binding and antagonized the RBP-J-mediated transactivation activity of TAN-1. It was also shown that the RBP-J/CBF1-interacting corepressor, CIR can interact with the similar HDAC complex including HDAC2 and SAP30 (35.Hsieh J.J.-D. Zhou S. Chen L. Young D.B. Hayward S.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 23-28Crossref PubMed Scopus (250) Google Scholar). On the other hand, a number of transcriptional activators have been shown to associate with histone acetyltransferases (HATs) such as the global coactivator P300/CBP (36.Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2338) Google Scholar) and the prototype HAT PCAF, which was originally isolated in human as a P300/CBP-associated factor by virtue of its sequence similarity to yeast GCN5 (37.Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1304) Google Scholar). Here we describe the functional interaction between mouse Notch1 RAMIC and two conserved HATs, mouse PCAF and GCN5 (38.Xu W. Edmondson D.G. Roth S.Y. Mol. Cell. Biol. 1998; 18: 5659-5669Crossref PubMed Scopus (134) Google Scholar). We show that the interaction is conserved across species and RAMIC can recruit each of the HATs to RBP-J. Furthermore, we show that the RBP-J-mediated transactivation activity of RAMIC is repressed by two HAT inhibitor proteins, E1A and Twist, which are structurally unrelated to each other. Our results suggest that HATs including PCAF and GCN5 may be involved in the RBP-J-mediated transactivation by Notch RAMIC. A luciferase reporter plasmid pGa981-6 contains the hexamerized 50-base pair EBNA2RE of the EBV TP-1 promoter in front of the minimal β-globin promoter of pGa50-7 (18.Kato H. Taniguchi Y. Kurooka H. Minoguchi S. Sakai T. Okazaki-Nomura S. Tamura K. Honjo T. Development. 1997; 124: 4133-4141Crossref PubMed Google Scholar, 23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar, 39.Minoguchi S. Taniguchi Y. Kato H. Okazaki T. Strobl L.J. Zimber-Strobl U. Bornkamm G.W. Honjo T. Mol. Cell. Biol. 1997; 17: 2679-2687Crossref PubMed Google Scholar). ptk-HES1 is a thymidine kinase (tk)-luciferase reporter plasmid containing the −87 to −51 promoter region of the mouseHES-1 gene (5.Kuroda K. Tani S. Tamura K. Minoguchi S. Kurooka H. Honjo T. J. Biol. Chem. 1999; 274: 7238-7244Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). TK-MH:100×4-luciferase reporter plasmid carries four copies of the GAL4-binding site upstream of the tk promoter (18.Kato H. Taniguchi Y. Kurooka H. Minoguchi S. Sakai T. Okazaki-Nomura S. Tamura K. Honjo T. Development. 1997; 124: 4133-4141Crossref PubMed Google Scholar, 23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar, 39.Minoguchi S. Taniguchi Y. Kato H. Okazaki T. Strobl L.J. Zimber-Strobl U. Bornkamm G.W. Honjo T. Mol. Cell. Biol. 1997; 17: 2679-2687Crossref PubMed Google Scholar) and pGL3-G5B-luciferase reporter plasmid carries five copies of the GAL4-binding site upstream of the E1b TATA promoter (40.Agata Y. Matsuda E. Shimizu A. J. Biol. Chem. 1999; 274: 16412-1642240Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). For the normalization of luciferase activities, pCMX-LacZ (23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar) containing the β-galactosidase gene driven by the CMV promoter was employed. pSG5-Flag-RBP-J (human RBP3) and -Flag-RBP-J-ICΔANK (mouse Notch1, amino acids 2098–2531) (23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar), pEFBOSneo-6×Myc-RA- MIC(1747–2531), −6×Myc-RAMICΔC-(1747–2193), and −6×Myc-IC-(1810–2531) (18 19, 23), pEFBOSneo-GAL4-RBP-J (mouse RBP2) (39.Minoguchi S. Taniguchi Y. Kato H. Okazaki T. Strobl L.J. Zimber-Strobl U. Bornkamm G.W. Honjo T. Mol. Cell. Biol. 1997; 17: 2679-2687Crossref PubMed Google Scholar), pEFBOSneo-GAL4-IC′-(1848–2531) and -GAL4-TAD-(2194–2398) (23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar), pEFBOSneo-GAL4-IC′ΔC-(1848–2170) and -GAL4-IC′ΔC-(1848–2170; M1) (18.Kato H. Taniguchi Y. Kurooka H. Minoguchi S. Sakai T. Okazaki-Nomura S. Tamura K. Honjo T. Development. 1997; 124: 4133-4141Crossref PubMed Google Scholar) were described previously. For the generation of pCMX-VP16-RAMIC deletion constructs, various regions of mouse Notch1 RAMIC derived from pBluescript II KS(−) (Stratagene)-RAMIC-(1747–2531), pCS2+6MT-mNotchIC-(1810–2531) (17.Kopan R. Nye J.S. Weintraub H. Development. 1994; 120: 2385-2396Crossref PubMed Google Scholar), or pBluescript II KS(+)-GAL4-IC-(1848–2531) (23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar) were digested with appropriate restriction enzymes and subcloned into the pCMX-VP16 vector (23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar), in-frame to the C terminus of the VP16 TAD. To target VP16 fusion proteins lacking the intrinsic NLSs of RAMIC into the nucleus, pCMX-VP16 harboring the NLS of the SV40 large T antigen (pCMX-VP16-NLS, Ref. 40.Agata Y. Matsuda E. Shimizu A. J. Biol. Chem. 1999; 274: 16412-1642240Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) was used for subcloning. pCMX-VP16-Drosophila Notch IC-(1783–2703) was generated by subcloning the ApaLI (blunted)-BglII fragment excised from pGAD424 (CLONTECH)-Drosophila Notch IC into theEcoRV/BamHI sites of pCMX-VP16. To generate pFlag-CMV2-PCAF and -GCN5, theBamHI-KpnI fragment of pRSETB-mPCAF (38.Xu W. Edmondson D.G. Roth S.Y. Mol. Cell. Biol. 1998; 18: 5659-5669Crossref PubMed Scopus (134) Google Scholar) and theKpnI (blunted)-SalI fragment of pRSETB-mGCN5 were subcloned into the BglII/KpnI sites and theEcoRV/SalI sites of pFlag-CMV2 vector (Eastman Kodak), respectively. pCMX-VP16-PCAF and -GCN5 were constructed by inserting the NotI (blunted)-BamHI fragment of pFlag-CMV2-PCAF and the EcoRI-SalI fragment of pFlag-CMV2-GCN5, into the EcoRV/BamHI sites and the EcoRI/SalI sites of pCMX-VP16, respectively. The yeast GAL4 DNA-binding domain (DBD) fusion vector, pCMX-GAL4, was constructed in two steps. The HindIII-PstI fragment containing the GAL4 TAD of pGAD424 was replaced by theHindIII-PstI fragment containing the GAL4 DBD of pGBT9 (CLONTECH), and the insert containing the GAL4 DBD and the multiple restriction enzyme sites was excised withHindIII. Then, the HindIII fragment of pCMX-VP16 containing the VP16 TAD was replaced by the excised insert. TheNotI (blunted)-BamHI fragment of pFlag-CMV2-PCAF and the KpnI (blunted)-SalI fragment of pRSETB-mGCN5 were subcloned into the SmaI/BamHI sites and the SmaI/SalI sites of pCMX-GAL4 in-frame to the C terminus of the GAL4 DBD, respectively, resulting in pCMX-GAL4-PCAF and -GAL4-GCN5. Deletion constructs of GAL4-PCAF and GAL4-GCN5 were generated by excising the inserts from pFlag-CMV2-PCAF, the full-length pCMX-GAL4-PCAF, or the full-length pCMX-GAL4-GCN5 with appropriate restriction enzymes and subcloning them into pCMX-GAL4. Two fragments of PCAF (1–192 and 193–424) were amplified by polymerase chain reaction and cloned into pGEM-T vector-Easy (Promega). After the sequences of the cloned fragments were confirmed on both strands, they were subcloned into pCMX-GAL4. The StuI-SnaBI fragment of pCS2+6MT-mNotchIC was replaced by the blunt endedHindIII-BamHI fragment derived from pFlag-CMV2-PCAF, resulting in pCS2+6MT-PCAF. pCMX-E1A was constructed by subcloning theHindIII-BglII fragment excised from pBJ9ΩAd5-E1A12S (41.Reid J.L. Bannister A.J. Zegerman P. Martinez-Balbas M.A. Kouzarides T. EMBO J. 1998; 17: 4469-4477Crossref PubMed Scopus (108) Google Scholar) into the HindIII/BamHI sites of pCMX. The coding region of mouse Twist was obtained by polymerase chain reaction amplification with specific primers, 5′-ATGATGCAGGACGTGTCCAGCTCG-3′ (forward) and 5′-TGCTAGTGGGACGCGGACATGGAC-3′ (reverse), using AKR mouse genomic DNA as template. The amplified fragment was cloned into pGEM-T vector-Easy and the sequence of both strands was confirmed. TheEcoRI-FspI and FspI-SalI fragments were excised from the plasmid, and the two fragments were simultaneously ligated to the EcoRI/SalI sites of pFlag-CMV2, resulting in pFlag-CMV2-Twist. SV40 transformed monkey kidney COS7 cells, human embryonic kidney 293T cells, and murine fibroblast NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Murine myoblast C2C12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum. The C2C12-derived cell line L9, which contains the pGa981-6 reporter construct with the blasticidin S resistance gene, was maintained in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum and 10 μg/ml blasticidin S (Kaken Seiyaku). COS7, L9, and NIH3T3 cells were seeded on 6-well plates at 2 × 105 cells per well 18 h before transfection and transiently transfected with various combinations of expression plasmid DNA indicated in each figure legend, using LipofectAMINE reagent (Life Technologies, Inc.). The total amount of plasmid DNAs was kept constant (1–2 μg) by adding the same expression plasmids without inserts. Transfected cells were harvested 48 h after transfection and luciferase activities were measured according to the manufacturer's instructions (TOYO INK) in a Berthold luminometer, LumatLB9501. Ethanol or 0–100 ng/ml trichostatin A (Sigma) was added to the culture medium at 24 h before harvesting. In all the experiments, the relative luciferase activity was normalized to the β-galactosidase activity obtained by transfection with 50 ng of pCMX-LacZ. Each experiment was repeated more than two times and the representative results are shown as the average of triplicate values with standard deviations. Anti-Myc monoclonal antibody (mAb), 9E10 was prepared from hybridoma. Anti-Flag mAb, M2 was purchased from Eastman Kodak. Anti-E1A mAb, M73, and anti-GAL4 DBD mAb, RK5C1, were purchased from Santa Cruz Biotechnology. Anti-VP16 polyclonal antibody was purchased from CLONTECH. Anti-PCAF polyclonal antibody was provided by Y. Nakatani (37.Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1304) Google Scholar). As secondary antibodies, horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) (Southern Biotechnology) and anti-rabbit IgG (Jackson ImmunoResearch Laboratories) were used. For Western analysis, whole cell lysates of transfected NIH3T3 cells or nuclear extracts of transfected COS7 cells were mixed with SDS sample buffer, boiled for 5 min, and separated by SDS-PAGE. Separated proteins were transferred onto a nitrocellulose filter (Amersham Pharmacia Biotech) by Semi-Dry (Bio-Rad). After blocking with phosphate-buffered saline containing 2% skim milk, the filter was incubated with the primary antibody for 1 h at room temperature, washed with phosphate-buffered saline containing 0.05% Tween 20, and incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL Plus detection system, Amersham Pharmacia Biotech). For immunoprecipitation, 293T cells seeded on 6-cm dishes at 5 × 105 cells per plate 18 h prior to transfection were transiently transfected using the CellPhect transfection kit (Amersham Pharmacia Biotech). After 40 h of incubation, cells were washed, scraped in phosphate-buffered saline, and lysed in IP buffer (25 mm HEPES, pH 7.9, 150 mm KCl, 0.1% Nonidet P-40, 1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, 0.1 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 μg/ml pepstatin A). After the cell lysates were rotated for 30 min at 4 °C, the cell debris was removed by centrifugation and the supernatant was used as whole cell extract. The whole cell extracts were incubated with anti-Flag M2-affinity gel (Sigma) by rotating for 3 h at 4 °C. Immunoprecipitates were washed three times with ice-cold IP buffer. Bound proteins were eluted in SDS sample buffer and subjected to Western blot analysis. Preparation of nuclear extracts from COS7 cells and EMSA for GAL4 fusion proteins were performed as described previously (23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar). The binding reaction contained 20 mm HEPES (pH 7.9), 50 mm NaCl, 5 mm MgCl2, 10 μmZnSO4, 10% glycerol, 0.1 mg/ml bovine serum albumin, 1 μg poly(dI-dC), 0.5 ng of 32P-labeled oligonucleotide probe MH100, and 4 μg of nuclear extracts. The reaction mixture in a final volume of 30 μl was incubated for 30 min at 20 °C and loaded onto a native 4% polyacrylamide gel in a buffer (pH 7.5) containing 6.7 mm Tris, 3.3 mm NaOAc, and 1 mmEDTA. After electrophoresis at 130 V for 2.5 h at 4 °C, the gels were dried and analyzed using an Imaging Analyzer BAS1500 (Fuji Film). Previously we reported that the fusion protein between RBP-J and the C-terminal portion of mouse Notch1 RAMIC (RBP-J-ICΔANK, Fig.1) failed to activate transcription of the pGa981-6 luciferase reporter gene carrying 12 canonical RBP-J-binding sites (23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar), although the C terminus of mouse Notch1 activates transcription autonomously when fused to the yeast GAL4 DBD (Ref. 23.Kurooka H. Kuroda K. Honjo T. Nucleic Acids Res. 1998; 26: 5448-5455Crossref PubMed Scopus (160) Google Scholar and see Fig. 5). We also showed that the repressed transactivation activity of the fusion protein was relieved by coexpression of either the RAM domain or the ANK repeats of RAMIC, suggesting that a putative corepressor(s) would associate with the RBP-J-ICΔANK fusion protein and might be displaced by the coexpressed RBP-J-binding domains. Since RBP-J interacts with HDAC corepressor complexes (33.Kao H.-Y. Ordentlich P. Koyano-Nakagawa N. Tang Z. Downes M. Kintner C.R. Evans R.M. Kadesch T. Genes Dev. 1998; 12: 2269-2277Crossref PubMed Scopus (481) Google Scholar, 35.Hsieh J.J.-D. Zhou S. Chen L. Young D.B. Hayward S.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 23-28Crossref PubMed Scopus (250) Google Scholar), we asked whether the fusion protein between RBP-J and the C-terminal portion of mouse Notch1 RAMIC is repressed by the HDAC activity. The reporter plasmid pGa981-6 or pGa50-7 (without RBP-J-binding sites) was transfected into COS7 cells with the plasmid encoding RBP-J or RBP-J-ICΔANK, and the cells were treated with increasing concentrations (0–100 ng/ml) of a histone deacetylase-specific inhibitor, trichostatin A (TSA; Ref. 42.Yoshida M. Kijima M. Akita M. Beppu T. J. Biol. Chem. 1990; 265: 17174-17179Abstract Full Text PDF PubMed Google Scholar). Transcription of pGa981-6 was activated by TSA in a dose-dependent manner (7-fold with 100 ng/ml TSA) when RBP-J-ICΔANK, but not when the non-chimeric RBP-J, was transfected (Fig.2 A). This activation was dependent on the presence of RBP-J-binding sites as well as on the fused C terminus of RAMIC, arguing against the general effect of TSA on transcription. Next we investigated whether the effect of TSA was restricted to episomal DNA or it was also applicable to chromosomally integrated DNA. As shown in Fig. 2 B, transcription was markedly activated by TSA (45-fold with 100 ng/ml TSA) when RBP-J-ICΔANK was transiently transfected into L9, a line of mouse myoblast C2C12 cells that was established by stable transfection with the pGa981-6 reporter. When RBP-J alone was transfected into L9, weak transactivation was observed by high concentration of TSA (2.7-fold with 100 ng/ml). Thus, inhibition of HDAC activity results in derepression of the transactivation activity of the fusion protein, either with the transient" @default.
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• W2058733797 date "2000-06-01" @default.
• W2058733797 modified "2023-10-08" @default.
• W2058733797 title "Functional Interaction between the Mouse Notch1 Intracellular Region and Histone Acetyltransferases PCAF and GCN5" @default.
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