Association of Transcription Factor YY1 with the High Molecular Weight Notch Complex Suppresses the Transactivation Activity of Notch

Notch receptors are evolutionarily conserved from Drosophila to human and play important roles in cell fate decisions. After ligand binding, Notch receptors are cleaved to release their intracellular domains. The intracellular domains, the activated form of Notch receptors, are then translocated into the nucleus where they interact with other transcriptional machinery to regulate the expression of cellular genes. To dissect the molecular mechanisms of Notch signaling, the cellular targets that interact with Notch1 receptor intracellular domain (N1IC) were screened. In this study, we found that endogenous transcription factor Ying Yang 1 (YY1) was associated with exogenous N1IC in human K562 erythroleukemic cells. The ankyrin (ANK) domain of N1IC and zinc finger domains of YY1 were essential for the association of N1IC and YY1 according to the pull-down assay of glutathione S-transferase fusion proteins. Furthermore, both YY1 and N1IC were present in a large complex of the nucleus to suppress the luciferase reporter activity transactivated by Notch signaling. The transcription factor YY1 indirectly regulated the transcriptional activity of the wild-type CBF1-response elements via the direct interaction of N1IC and CBF1. We also demonstrated the association between endogenous N1IC and intrinsic YY1 in human acute T-cell lymphoblastic leukemia cell lines. Taken together, these results indicate that transcription factor YY1 may modulate Notch signaling via association with the high molecular weight Notch complex. Notch receptors are evolutionarily conserved from Drosophila to human and play important roles in cell fate decisions. After ligand binding, Notch receptors are cleaved to release their intracellular domains. The intracellular domains, the activated form of Notch receptors, are then translocated into the nucleus where they interact with other transcriptional machinery to regulate the expression of cellular genes. To dissect the molecular mechanisms of Notch signaling, the cellular targets that interact with Notch1 receptor intracellular domain (N1IC) were screened. In this study, we found that endogenous transcription factor Ying Yang 1 (YY1) was associated with exogenous N1IC in human K562 erythroleukemic cells. The ankyrin (ANK) domain of N1IC and zinc finger domains of YY1 were essential for the association of N1IC and YY1 according to the pull-down assay of glutathione S-transferase fusion proteins. Furthermore, both YY1 and N1IC were present in a large complex of the nucleus to suppress the luciferase reporter activity transactivated by Notch signaling. The transcription factor YY1 indirectly regulated the transcriptional activity of the wild-type CBF1-response elements via the direct interaction of N1IC and CBF1. We also demonstrated the association between endogenous N1IC and intrinsic YY1 in human acute T-cell lymphoblastic leukemia cell lines. Taken together, these results indicate that transcription factor YY1 may modulate Notch signaling via association with the high molecular weight Notch complex. Notch genes encode evolutionarily conserved receptors that are utilized to control cell fate decisions during development. Notch signaling participates in several cellular functions such as proliferation, apoptosis, and differentiation, depending upon the cellular context of Notch activation (reviewed in Refs. 1Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4951) Google Scholar, 2Miele L. Osborne B. J. Cell. Physiol. 1999; 181: 393-409Crossref PubMed Scopus (204) Google Scholar, 3Kopan R. J. Cell Sci. 2002; 115: 1095-1097Crossref PubMed Google Scholar). Human Notch was first identified as a gene involved in the t(7;9)(q34; q34.3) chromosomal translocation detected in some T-cell acute lymphoblastic leukemias. This gene was shown to generate Notch1 receptor intracellular domain (N1IC), 1The abbreviations used are: N1IC, Notch1 receptor intracellular domain; YY1, Ying Yang 1; RAM, RAM23 domain; ANK, ankyrin repeats; PEST, proline-glutamate-serine-threonine-rich; TAD, transcriptional activator domain; GST, glutathione S-transferase; HA, hemagglutinin; ChIP, chromatin immunoprecipitation; CAT, chloramphenicol acetyltransferase. an activated form of Notch1 receptor (4Ellisen L.W. Bird J. West D.C. Soreng A.L. Reynolds T.C. Smith S.D. Sklar J. Cell. 1991; 66: 649-661Abstract Full Text PDF PubMed Scopus (1454) Google Scholar). Further studies showed that the human Notch receptor family consists of four members (Notch1–4), located on chromosomes 9q34, 1p13-p11, 19p13.2-p13.1, and 6p21.3, respectively (5Larsson C. Lardelli M. White I. Lendahl U. Genomics. 1994; 24: 253-258Crossref PubMed Scopus (84) Google Scholar, 6Sugaya K. Sasanuma S. Nohata J. Kimura T. Fukagawa T. Nakamura Y. Ando A. Inoko H. Ikemura T. Mita K. Gene (Amst.). 1997; 189: 235-244Crossref PubMed Scopus (22) Google Scholar). Notch receptors are single-span transmembrane proteins with several functional domains. In the extracellular domain, there are several epidermal growth factor repeats and three Lin/Notch repeats. The Notch intracellular domain consists of a RAM domain, ankyrin repeats (ANK), nuclear localization signal, a transcriptional activator domain (TAD), and a proline-glutamate-serine-threonine-rich (PEST) domain. The RAM domain is the primary binding site of activated Notch receptor with C promoter binding factor-1 (CBF1)/recombination signal binding protein-Jκ (RBP-Jκ) (7Tamura 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 (403) Google Scholar), a human homolog of Drosophila Su(H). The ANK repeat domain can also associate with CBF1 to modulate the interaction (8Fortini M.E. Artavanis-Tsakonas S. Cell. 1994; 79: 273-282Abstract Full Text PDF PubMed Scopus (460) Google Scholar). In the prevailing model of Notch signaling, Notch receptors are activated through binding with ligands on neighboring cells. The Notch intracellular domains are released and translocated into the nucleus after proteolytic cleavages triggered by ligand binding. Then Notch intracellular domains activate the expression of their target genes via both CBF1-dependent and -independent pathways (reviewed in Ref. 9Baron M. Semin. Cell Dev. Biol. 2003; 14: 113-119Crossref PubMed Scopus (275) Google Scholar). The control of Notch signaling is very complicated and not yet fully understood. So far, there are several N1IC-associated cellular factors that have been identified to both positively and negatively modulate Notch signaling. These identified N1IC-associated cellular factors include Numb (10Zhong W. Feder J.N. Jiang M.M. Jan L.Y. Jan Y.N. Neuron. 1996; 17: 43-53Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 11Guo M. Jan L.Y. Jan Y.N. Neuron. 1996; 17: 27-41Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar), CIR (12Hsieh J.J. Zhou S. Chen L. Young D.B. Hayward S.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 23-28Crossref PubMed Scopus (251) Google Scholar), SKIP (13Zhou S. Fujimuro M. Hsieh J.J. Chen L. Miyamoto A. Weinmaster G. Hayward S.D. Mol. Cell. Biol. 2000; 20: 2400-2410Crossref PubMed Scopus (216) Google Scholar), MAML1/LAG3 (14Wu L. Aster J.C. Blacklow S.C. 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Although the members of Notch-associated factors and the downstream gene targets are expanding, the underlying molecular mechanisms of Notch signaling in diverse developmental systems remain unresolved. To further dissect Notch signaling, we screened and characterized activated Notch1 receptor-associated proteins in a hematopoietic system. The transcription factor Ying Yang 1 (YY1) was identified to be associated with the activated form of Notch1 receptor in a high molecular weight complex in the nucleus, and this association modulated the CBF1-dependent gene expression. Plasmids and Plasmid Construction—The cDNA of Notch1 receptor intracellular domain was cloned by RT-PCR from the total RNA of HL-60 cells. The expression construct of pcDNA-HA-N1IC, a derivative of the mammalian cell expression vector pcDNA3-HA2, contains the cDNA-encoded amino acid of human Notch1 receptor with an the The fusion (28Oswald F. Täuber B. Dobner T. Bourteele S. Kostezka U. Adler G. Liptay S. Schmid R.M. Mol. Cell. Biol. 2001; 21: 7761-7774Crossref PubMed Scopus (233) Google Scholar), and direct the expression of fusion proteins with amino acid and of human Notch1 Plasmids and encode of YY1 with were used for pull-down and four of wild-type CBF1-response elements in of a luciferase J.J. T. P. E. Hayward S.D. Mol. Cell. Biol. 1996; 16: PubMed Scopus Google Scholar). The reporter contains to the promoter and gene H. Y. 2000; PubMed Scopus Google Scholar). Cell and human cell K562 and acute T-cell lymphoblastic leukemia and were in with were in with the of K562 cell K562 were with a expression of by a gene after were to in and with The from were screened for the expression of fusion by with and the the vector was also into K562 to a cell the reporter in were with of of reporter and of expression in the To for of was used as an were in the reporter after activity was as H. Y. 2000; PubMed Scopus Google Scholar). the of luciferase reporter K562 were and the wild-type CBF1-response elements were with and their control after luciferase from both and and luciferase proteins were the reporter assay luciferase activity was then used to for chromatin the were with of reporter and were after were in and and and was with to a of and of of were into of and then for 1 to the of was by the of cell and C for 1 Chen J. 1996; PubMed Google Scholar). was to the and for and then by The was with C and fusion expression were for the of and fusion proteins. After by for were and with and and 1 for Then was to cell to a of and the were for The were a of and the were for The proteins were after and the was with several of and were in as To of of a of with of fusion proteins was and then for After to the the of was by three and in for and the nuclear of and the cell were in a 1 and and for After for the of were in a high 1 1 and and then on for The after were as nuclear of were on the of of a in The were to in a for The were into from the and of were to for the of N1IC and YY1 proteins. The were in high and on a The were for to the of the on the with wild-type of were with their for as by F. A. K. M. S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). were with for to and and the was by to a of were in of with and After the of cell the was with for After the were with and the were with of by Then of of and were to a of The of and in the was by for After was by and in of and control of were used as a for the and and of were and on a and then by 2002; 26: Scopus Google Scholar). of the of Notch1 with and of Notch1 receptor intracellular domain were into to CBF1-dependent by the reporter The and N1IC, not the ANK domain, were to the reporter gene through the endogenous CBF1 with the activation by N1IC of the assay the that the RAM domain is the primary binding domain for ANK repeats interact with and is important for activity H. K. Honjo T. 1998; 26: PubMed Scopus Google Scholar). To into the of Notch signaling, we used a and pull-down assay with to cellular factors associated with the Notch1 The transcription factor YY1 is of the identified of N1IC-associated proteins. To the association between YY1 and N1IC, was to K562 cell lines. to the the expression of the endogenous and exogenous N1IC were to be detected in K562 L.T. C. Norton J. Capobianco A.J. Bresnick E.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The domain of Notch1 receptor to and of N1IC by the (26Gupta-Rossi N. Bail O.L. Gonen H. Brou C. Logeat F. Six E. Ciechanover A. Israël A. J. Biol. Chem. 2001; 276: 34371-34378Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). the domain of Notch1 receptor was from this in to N1IC The fusion of was from by N1IC After the fusion was further by factor YY1 was also detected after and this with the fusion was also with cellular YY1 by These results showed that N1IC was associated with endogenous YY1 by the of the of N1IC and YY1 in by the dissect the essential for the association of YY1 and N1IC, the in binding of these proteins were by the pull-down and fusion proteins of N1IC and YY1 were by and of and were for the pull-down assay by and fusion proteins. fusion proteins of and were associated with endogenous YY1 of This that the ANK domain of Notch1 receptor was to associate with N1IC was associated with the YY1 fusion not with other YY1 fusion and the of amino acid the zinc finger domain of YY1 is essential for the association with To further the of amino acid of YY1 is to associate with N1IC, and fusion proteins were used to pull-down N1IC in the of cells. N1IC was associated with the YY1 fusion not with fusion These results that the zinc finger domain of YY1 is not for the association with The Notch1 with YY1 in a in the intracellular domain of Notch1 receptor been demonstrated to be associated with the transcription factor CBF1 and in an high molecular weight complex in the nucleus S. Capobianco A.J. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). To the association between the activated Notch1 receptor and YY1 in the nucleus, were to nuclear from and cells. the were by N1IC was detected in and After and with transcription factor YY1 was detected in and also in were three of transcription factor YY1 in the and The showed that N1IC and YY1 in both molecular weight and molecular weight In the of N1IC, YY1 showed the as that of cells. The of N1IC not the of endogenous YY1 in the To the associated complex of N1IC and YY1 is present in these and were and with YY1 was with N1IC in and not in This that N1IC is associated with YY1 as both a molecular weight complex and a molecular weight complex in the of by Notch1 and of the of Notch receptor-associated proteins into Notch signaling. To the of the association between N1IC and YY1 in Notch signaling, we used a reporter gene K562 were with a luciferase reporter four of CBF1-response elements and and their control after were and for luciferase N1IC can the expression of the reporter gene CBF1-response there was a of the luciferase activity by N1IC with the YY1 expression this of luciferase activity was by to This of YY1 was not in the luciferase reporter four of the CBF1-response The the zinc finger domains, not suppress the luciferase activity transactivated by N1IC, also the of the zinc finger domains in association between N1IC and YY1 as shown in This that YY1 the luciferase reporter activity transactivated by Notch1 N1IC with YY1 in and been that the activated form of Notch1 receptor is and can a very L.T. C. Norton J. Capobianco A.J. Bresnick E.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (84) Google Scholar). is that the of N1IC may the association between N1IC and endogenous To this we the association between N1IC and YY1 in by The intrinsic N1IC be with endogenous YY1 endogenous YY1 also be with intrinsic N1IC not The intrinsic YY1 also be with endogenous N1IC in These results showed that intrinsic N1IC be associated with endogenous YY1 in cells. The on the CBF1-response on the that of Notch1 signaling by YY1 was not an from in signaling was to the interaction between the complex and In intrinsic N1IC and endogenous YY1 be with the wild-type CBF1-response not the YY1 not be with the wild-type CBF1-response in K562 not the assay was also used to the association of transcription factor YY1 and N1IC with in the context of cells. were with luciferase reporter with wild-type CBF1-response after the were for the assay The of was present in the from wild-type CBF1-response was not present in with the These data that transcription factor YY1 indirectly on the wild-type CBF1-response via with N1IC with CBF1. was also for the assay cells. In this study, we the between the intracellular domain of human Notch1 receptor and transcription factor We that N1IC is associated with YY1 in the nucleus and that YY1 the CBF1-dependent luciferase reporter activity transactivated by Notch1 This is the first the of the transcription factor YY1 in CBF1-dependent Notch signaling. Jeffries S. Capobianco A.J. Mol. Cell. Biol. 2002; PubMed Scopus Google showed that a high molecular weight Notch complex of was present in the nucleus of and in a human T-cell leukemia cell We that YY1 is associated with N1IC as both and large in the nucleus and the CBF1-dependent luciferase reporter activity transactivated by YY1 may modulate Notch signaling via the large high molecular weight Notch complex. In the of YY1 in the nucleus not N1IC is This may be to the of The transcription factor YY1 several roles in the transcription of target genes. a of YY1 may be involved in the of Notch signaling in the the of YY1 in the of exogenous N1IC in the nucleus was the as that in the of YY1 is a complex that may as a transcriptional K. M.L. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar, Y. E. T. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar, J.R. E. K. Mol. Cell. Biol. PubMed Scopus Google Scholar), a transcriptional activator S. Y. K. Mol. Cell. Biol. PubMed Scopus Google Scholar), a transcriptional E. Y. T. 1991; PubMed Scopus (342) Google Scholar). expression of the YY1 in both transcription of genes and cell R.J. Mol. 1997; Google Scholar). the association of N1IC with this transcription factor YY1 may be to the of Notch signaling. These results that YY1 may regulate cell by via the control of Notch signaling. YY1 is a in both K562 and cells. Although of the YY1 expression showed in the expression of the YY1 in the luciferase reporter assay not the of exogenous YY1 showed of CBF1-dependent luciferase activity transactivated by of the is that a of exogenous YY1 is to suppress Notch YY1 may from endogenous YY1 in the exogenous YY1, not the endogenous YY1, with the high molecular weight Notch complex Notch In this study, YY1 the activation of CBF1-dependent luciferase activity transactivated by N1IC from to the YY1 not the activity of the reporter gene by the of exogenous YY1 was to N1IC, and a very of N1IC can functions L.T. C. Norton J. Capobianco A.J. Bresnick E.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In the reporter gene of the transcription factor not activate the CBF1-response This with the to YY1 with the wild-type CBF1-response in K562 by not YY1 be with the wild-type CBF1-response in and K562 cells. The luciferase reporter with the wild-type CBF1-response also be with and in the assay transcription factor YY1 regulated the transcriptional activity of the wild-type CBF1-response via an association with the high molecular weight Notch complex CBF1. The zinc finger domains, binding domains of the transcription are for the association of YY1 with N1IC according to the fusion pull-down finger domains are also important for the interaction of YY1 with several other cellular including and (reviewed in Ref. M.J. E. Gene (Amst.). 1999; PubMed Scopus Google Scholar). In the binding of the activated Notch1 the ANK domain of N1IC was associated with endogenous YY1 The ANK domain been detected in such as signal and transcriptional developmental and P. V. Cell Biol. Full Text PDF PubMed Scopus Google Scholar, P. Genet. 17: PubMed Scopus Google Scholar). This functional is a for the interaction between proteins N. M. N. B.M. Mol. Cell. Biol. 2001; 21: PubMed Scopus Google Scholar, Sci. 1999; 24: Full Text Full Text PDF PubMed Scopus Google Scholar). Although the ANK domain of N1IC is to associate with YY1, CBF1-dependent Notch signaling The p300 with N1IC and as a transcriptional (28Oswald F. Täuber B. Dobner T. Bourteele S. Kostezka U. Adler G. Liptay S. Schmid R.M. Mol. Cell. Biol. 2001; 21: 7761-7774Crossref PubMed Scopus (233) Google Scholar). The association between p300 and N1IC the of and of the domain, a of amino acid to the ANK domain, N1IC the interaction with p300 in (28Oswald F. Täuber B. Dobner T. Bourteele S. Kostezka U. Adler G. Liptay S. Schmid R.M. Mol. Cell. Biol. 2001; 21: 7761-7774Crossref PubMed Scopus (233) Google Scholar). The p300 also with YY1 through the of p300 that domain J. Cell Sci. 2001; PubMed Google Scholar). The of the four zinc and the of YY1 are for and association with respectively M.J. E. Gene (Amst.). 1999; PubMed Scopus Google Scholar). The association between N1IC and YY1 the ANK domain of N1IC and the four zinc of YY1 of domain, not the association of ANK domain of N1IC with This may the that N1IC with YY1 through p300 and the of YY1 on activity of We Wu for and expression of of fusion proteins and H. Chen for the reporter We also S. Hayward for the of reporter and