Involvement of the Histone Deacetylase SIRT1 in Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF)-interacting Protein 2-mediated Transcriptional Repression

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 (CTIP1 and CTIP2) enhance transcriptional repression mediated by COUP-TF II and have been implicated in hematopoietic cell development and malignancies. CTIP1 and CTIP2 are also sequence-specific DNA-binding proteins that repress transcription through direct, COUP-TF-in-dependent binding to a GC-rich response element. CTIP1- and CTIP2-mediated transcriptional repression is insensitive to trichostatin A, an inhibitor of known class I and II histone deacetylases. However, chromatin immunoprecipitation assays revealed that expression of CTIP2 in mammalian cells resulted in deacetylation of histones H3 and/or H4 that were associated with the promoter region of a reporter gene. CTIP2-mediated transcriptional repression, as well as deacetylation of promoter-associated histones H3/H4 in CTIP2-transfected cells, was reversed by nicotinamide, an inhibitor of class III histone deacetylases such as the mammalian homologs of yeast Silent Information Regulator 2 (Sir2). The human homolog of yeast Sir2, SIRT1, was found to interact directly with CTIP2 and was recruited to the promoter template in a CTIP2-dependent manner. Moreover, SIRT1 enhanced the deacetylation of template-associated histones H3/H4 in CTIP2-transfected cells, and stimulated CTIP2-dependent transcriptional repression. Finally, endogenous SIRT1 and CTIP2 co-purified from Jurkat cell nuclear extracts in the context of a large (1–2 mDa) complex. These findings implicate SIRT1 as a histone H3/H4 deacetylase in mammalian cells and in transcriptional repression mediated by CTIP2. Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 (CTIP1 and CTIP2) enhance transcriptional repression mediated by COUP-TF II and have been implicated in hematopoietic cell development and malignancies. CTIP1 and CTIP2 are also sequence-specific DNA-binding proteins that repress transcription through direct, COUP-TF-in-dependent binding to a GC-rich response element. CTIP1- and CTIP2-mediated transcriptional repression is insensitive to trichostatin A, an inhibitor of known class I and II histone deacetylases. However, chromatin immunoprecipitation assays revealed that expression of CTIP2 in mammalian cells resulted in deacetylation of histones H3 and/or H4 that were associated with the promoter region of a reporter gene. CTIP2-mediated transcriptional repression, as well as deacetylation of promoter-associated histones H3/H4 in CTIP2-transfected cells, was reversed by nicotinamide, an inhibitor of class III histone deacetylases such as the mammalian homologs of yeast Silent Information Regulator 2 (Sir2). The human homolog of yeast Sir2, SIRT1, was found to interact directly with CTIP2 and was recruited to the promoter template in a CTIP2-dependent manner. Moreover, SIRT1 enhanced the deacetylation of template-associated histones H3/H4 in CTIP2-transfected cells, and stimulated CTIP2-dependent transcriptional repression. Finally, endogenous SIRT1 and CTIP2 co-purified from Jurkat cell nuclear extracts in the context of a large (1–2 mDa) complex. These findings implicate SIRT1 as a histone H3/H4 deacetylase in mammalian cells and in transcriptional repression mediated by CTIP2. CTIP1 1The abbreviations used are: CTIP 1 and 2, COUP-TF-interacting proteins 1 and 2; COUP-TF, chicken ovalbumin upstream promoter transcription factor; BCL, B cell leukemia; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; HA, hemagglutinin; HDAC, histone deacetylase; HEK293, human embryonic kidney 293 cells; mDa, megadalton; Sir2, silent information regulator 2; SIRT1, Sir2-like protein 1 or sirtuin 1; TSA, trichostatin A; ChIP, chromatin immunoprecipitation; DBD, DNA binding domain; KRAB, Kruppel-associated box; HP1, heterochromatin protein 1. (EVI9 or BCL11A) and CTIP2 (BCL11B) are two related C2H2 zinc finger proteins that were originally isolated and identified as COUP-TF-interacting proteins (1Avram D. Fields A. Pretty On Top K. Nevrivy D. Ishmael J.E. Leid M. J. Biol. Chem. 2000; 275: 10315-10322Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). CTIP1 2D. Avram and M. Leid, unpublished results. (1Avram D. Fields A. Pretty On Top K. Nevrivy D. Ishmael J.E. Leid M. J. Biol. Chem. 2000; 275: 10315-10322Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) and CTIP2 both enhance COUP-TFII-mediated transcriptional repression in transfected cells independently of trichostatin A (TSA)-sensitive histone deacetylation. Both CTIPs are expressed in hematopoietic cells of lymphoid origin, 3D. Shepherd and M. Leid, manuscript in preparation. which is of interest because lymphoid-derived cells are devoid of transcripts encoding COUP-TF family members. 3D. Shepherd and M. Leid, manuscript in preparation. Thus, it is likely that CTIPs either function with other nuclear receptors in cells of lymphoid origin or act as COUP-TF-independent transcription factors in these cells. The latter appears to be true as CTIP1 and CTIP2 have been demonstrated to repress expression of a reporter gene through direct binding to a recently identified binding site, 5′-GGCCGGAGG-3′ (upper strand) (2Avram D. Fields A. Senawong T. Topark-Ngarm A. Leid M. Biochem. J. 2002; 368: 555-563Crossref PubMed Scopus (119) Google Scholar). This repression was observed in the absence of cotransfected COUP-TF proteins and was insensitive to reversal by TSA (2Avram D. Fields A. Senawong T. Topark-Ngarm A. Leid M. Biochem. J. 2002; 368: 555-563Crossref PubMed Scopus (119) Google Scholar). These findings indicate that CTIP proteins may regulate transcription independently of COUP-TF proteins and TSA-sensitive HDACs in some cell types and/or promoter contexts. CTIP1 and CTIP2 have been implicated in the etiology of both myeloid and lymphoid malignancies. Overexpression of the CTIP1 gene following proviral integration in murine hematopoietic cells results in the generation of myeloid leukemia (3Nakamura T. Yamazaki Y. Saiki Y. Moriyama M. Largaespada D.A. Jenkins N.A. Copeland N.G. Mol. Cell. Biol. 2000; 20: 3178-3186Crossref PubMed Scopus (86) Google Scholar). Similarly, dysregulation of the human CTIP1 locus (2p13) either by amplification or translocation appears to result in B cell chronic lymphocytic leukemia and immunocytoma (4Satterwhite E. Sonoki T. Willis T.G. Harder L. Nowak R. Arriola E.L. Liu H. Price H.P. Gesk S. Steinemann D. Schlegelberger B. Oscier D.G. Siebert R. Tucker P.W. Dyer M.J. Blood. 2001; 98: 3413-3420Crossref PubMed Scopus (259) Google Scholar). The human CTIP2 locus (14q32) has been associated with a translocation event, t(5;14)(q35;q32), which results in acute T lymphoblastic leukemia (5Bernard O.A. Busson-LeConiat M. Ballerini P. Mauchauffe M. Della Valle V. Monni R. Nguyen Khac F. Mercher T. Penard-Lacronique V. Pasturaud P. Gressin L. Heilig R. Daniel M.T. Lessard M. Berger R. Leukemia. 2001; 15: 1495-1504Crossref PubMed Scopus (199) Google Scholar). More recently, CTIP2 loss of function mutations were found to contribute to mouse lymphoma genesis, leading the authors to speculate that CTIP2 may function as a tumor suppressor protein (6Wakabayashi Y. Inoue J. Takahashi Y. Matsuki A. Kosugi-Okano H. Shinbo T. Mishima Y. Niwa O. Kominami R. Biochem. Biophys. Res. Commun. 2003; 301: 598-603Crossref PubMed Scopus (101) Google Scholar). The analysis of both CTIP1-(7) and CTIP2-(8) null animals has demonstrated a role for each in hematopoiesis and postnatal development. Although human CTIP1 and CTIP2 are expressed in early, multipotent hemopoietic progenitors, neither the physiological function(s) nor the contribution of these proteins to neoplastic processes in these cells is known (5Bernard O.A. Busson-LeConiat M. Ballerini P. Mauchauffe M. Della Valle V. Monni R. Nguyen Khac F. Mercher T. Penard-Lacronique V. Pasturaud P. Gressin L. Heilig R. Daniel M.T. Lessard M. Berger R. 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These findings a role for the histone deacetylase SIRT1 in the transcriptional repression of CTIP2 in mammalian cells. reporter was a from of was by amplification of the CTIP2 (1Avram D. Fields A. Pretty On Top K. Nevrivy D. Ishmael J.E. Leid M. J. Biol. Chem. 2000; 275: 10315-10322Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) with and into The was by amplification with by into a and E. M. M. S. T. J. 2002; PubMed Scopus Google Scholar) were from T. of encoding proteins were by amplification of by into The used for proteins were by amplification with for into or were by DNA was E. M. M. S. T. J. 2002; PubMed Scopus Google Scholar). with human and H3 and H4 were from and were from and The was a protein by the for of has revealed that the an the the of the and M. Leid, unpublished results. assays were on transfected cells as by the Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) with cells were with of the of of and/or the the cells were with and with in were with and in The cells were in and a inhibitor The were by and with and a inhibitor of the was as an to the of reporter in transfected cells for of the were used for immunoprecipitation with and the of histone and histone H4 of each immunoprecipitation were with and were with the The and the were to an reversal of by with for 1 DNA was by a and a upstream of the and a of the promoter upstream of the transcriptional The was by and were three to and cells were transfected and as TSA and were and cells were A expression was cotransfected as an and was used to as P. Ishmael J.E. Avram D. Nevrivy Leid M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells were transfected as with each of expression encoding and/or the cells were in 1 and a inhibitor by for a were by and as Avram D. Ishmael J.E. P. Leid M. J. Biol. 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PubMed Scopus Google Scholar) with the following of and was with and CTIP2 was found in the nuclear Jurkat nuclear was to a that been in and The was with the A to and in a with and The A of the was to to each CTIP2 and SIRT1 were in the by with and The the of was and a of A that been with the the was with and in a and the were by as The the which both CTIP2 and SIRT1 was to 1 of a and a that was a of The been with and with protein from were for the of CTIP2 and SIRT1 by CTIP2-mediated by an of that transcriptional repression was of TSA-sensitive histone deacetylation (1Avram D. Fields A. Pretty On Top K. Nevrivy D. Ishmael J.E. Leid M. J. Biol. Chem. 2000; 275: 10315-10322Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, D. Fields A. Senawong T. Topark-Ngarm A. Leid M. Biochem. 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Full Text Full Text PDF PubMed Scopus Google Scholar) have in transfected cells and used to and transcriptional repression mediated by the Thus, a was in the the of histone deacetylation may CTIP2-mediated transcriptional repression in mammalian cells. in the of histone H3/H4 associated with the template were observed the of transfected (upper of with However, of of the of histone H3/H4 associated with the template (upper of and and was by of the transfected cells with TSA of the of However, the deacetylation of template-associated histones observed in CTIP2-transfected cells was reversed by of the cells with of the and of Although of the deacetylation of promoter-associated histone H3 and/or H4 by of the CTIP2-transfected cells with was reversal was observed and of the of This may be because of the of of as an inhibitor of deacetylases and/or of in cells. of to result in cellular the of of these which are with transcriptional repression that histone deacetylation may the of CTIP2-mediated transcriptional repression in transfected cells. CTIP2 with and SIRT1 to the in Mammalian results indicate that a of class III HDACs may be associated with and the transcriptional repression of CTIP2 in mammalian cells. were extracts from cells transfected with an expression encoding CTIP2 was with endogenous SIRT1 by by This that transfected CTIP2 directly or with endogenous SIRT1 in cells. were to endogenous SIRT1 was recruited to the promoter template in cells transfected with a expression SIRT1 was associated with the template in cells transfected with However, of endogenous SIRT1 to the template was in cells These CTIP2-dependent of endogenous SIRT1 to the promoter template in cells, and a role for histone deacetylase in the transcriptional repression of CTIP2. SIRT1 CTIP2-mediated gene assays were in transfected cells to SIRT1 CTIP2-mediated transcriptional repression. the of the proteins was by and was by the was and of Similarly, was with a SIRT1 J. S. D. F. A. L. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, E. M. M. S. T. J. 2002; PubMed Scopus Google to an to that of the protein of These which are with results in 2, A and that transfected CTIP2 with both SIRT1 and a SIRT1 in mammalian cells. The of these was in cells cotransfected with expression encoding the and either or SIRT1 Both SIRT1 and SIRT1 reporter gene expression in a in the absence of and Although the for of SIRT1 to the template these is these which are with E. M. M. S. T. J. 2002; PubMed Scopus Google that the of the is for repression of the of reporter gene the of SIRT1 was for of CTIP2-mediated transcriptional repression. of SIRT1 stimulated transcriptional repression mediated by in a SIRT1 enhance CTIP2-mediated repression These findings that the cotransfected SIRT1 the transcriptional repression of CTIP2 and the of the assays were to histone deacetylation may the for the observed of CTIP2-mediated transcriptional repression by The of transfected expression used in these were to which neither CTIP2 (upper of nor SIRT1 (upper independently the of promoter-associated histone H3/H4 to an This was as both and SIRT1 of histone H3/H4 transfected the of and SIRT1 resulted in a large in the of histone H3/H4 that was associated with the reporter gene template and was that observed by of either expression and of the of The SIRT1 enhance deacetylation of promoter-associated histones H3/H4 in cells transfected with and of the of These findings that histone deacetylation may in the of the to CTIP2-mediated transcriptional repression. CTIP2 with SIRT1 in and that CTIP2 and SIRT1 with in transfected cells. However, these are of direct and from with a a of Thus, in were to CTIP2 directly with CTIP2 was found to interact with SIRT1 to with These results that CTIP2 and SIRT1 in a direct, The of a of CTIP2 revealed that the SIRT1 is CTIP2 of CTIP2 region with of A, and it or of and Thus, a region that is in devoid of C2H2 zinc finger (1Avram D. Fields A. Pretty On Top K. Nevrivy D. Ishmael J.E. Leid M. J. Biol. Chem. 2000; 275: 10315-10322Abstract Full Text Full Text PDF PubMed Scopus (165) Google appears to be for with SIRT1 in The CTIP2 of SIRT1 was by on the of Biol. 2001; PubMed Scopus Google three of SIRT1 the the sirtuin and the of which were to for in in with the region of SIRT1 interact with the However, with the SIRT1 sirtuin the sirtuin were to the CTIP2 and was found to interact with and with Thus, these results that CTIP2 with a of the SIRT1 sirtuin that with the of the these results that CTIP2 and SIRT1 interact directly in and the region of CTIP2 and the of the sirtuin of CTIP2 and SIRT1 of a in Jurkat were transfected cells CTIP2. However, it is important to that endogenous CTIP2 and SIRT1 interact in the cellular in the absence of nuclear extracts were from Jurkat cells, which both CTIP2 and SIRT1 human CTIP2 was with SIRT1 by both the and by an or results were the and were reversed These findings that endogenous CTIP2 and SIRT1 with in the of mammalian cells expressed physiological Jurkat cell nuclear extracts were to the of CTIP2 Jurkat cell nuclear was to a which was in a with CTIP2 was in the of the of CTIP2 be in the with of the the of the SIRT1 in the of the 2 of the These findings that of SIRT1 in Jurkat cell CTIP2 to the in This be with the that SIRT1 cellular of which to be of CTIP2. a of SIRT1 with CTIP2 the the and was and to a to the CTIP2 Both CTIP2 (upper and SIRT1 in the the The was to a to the of CTIP2 and SIRT1 CTIP2 was from the as a with a 1 and 2 and of SIRT1 was as a 2 and of These results CTIP2 the of the protein and for the CTIP2 SIRT1 to be in some of which to CTIP2 of that and of which the of the SIRT1 both CTIP2 and SIRT1 were and with the SIRT1 The was by with the CTIP2 to that CTIP2 and SIRT1 a in preparation. CTIP2 was found to with the SIRT1 the that was observed in Jurkat cell nuclear extracts was through a and three The of and has been to the of transcriptional from yeast to histone and are to of chromatin and have been implicated in the of of transcriptional T. 2001; PubMed Scopus Google Scholar). The transcriptional of histone and of in the of histones H3 and is and either H. R. J. J. T. 2002; PubMed Scopus Google Scholar) or repression M. T. Biol. 2002; PubMed Scopus Google Scholar, M. D. S. K. T. 2001; PubMed Scopus Google Scholar) in a manner. transcriptional nuclear receptors and of other transcription factor have been to TSA-sensitive HDACs to the in of chromatin and transcriptional K. PubMed Scopus Google Scholar). of these the HDACs recruited to the template of by transcriptional have been or to be either class I or II HDACs O. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). have observed that CTIP1 (1Avram D. Fields A. Pretty On Top K. Nevrivy D. Ishmael J.E. Leid M. J. Biol. Chem. 2000; 275: 10315-10322Abstract Full Text Full Text PDF PubMed Scopus (165) Google a of a family of C2H2 zinc finger proteins, transcription of a reporter gene in a that was to reversal by findings were for CTIP2 However, CTIP2-mediated transcriptional repression was found to be by nicotinamide, an inhibitor of the class III of the with these expression of CTIP2 in cells resulted in of SIRT1 to the promoter and deacetylation of template-associated histones H3/H4 in a that was by TSA reversed by Moreover, of SIRT1, a was found to enhance CTIP2-mediated transcriptional repression in transfected cells. The that SIRT1 a role in CTIP2-mediated transcriptional repression was by the that the two proteins directly and from extracts from both and transfected cells, as well as Jurkat cells. Finally, SIRT1 co-purified with CTIP2 three and the of CTIP2 was to be 1 and 2 mDa, the of other the results of assays the cellular that SIRT1 is of histones H3 and histones that a role in transcriptional Y. D. 2001; 15: PubMed Scopus Google Scholar, L. 2000; PubMed Google Scholar). Although the chromatin of transfected DNA may from that of their J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the of TSA on and the observed in histone H3/H4 as by and that the transfected reporter template some of chromatin This is also by Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. J. PubMed Scopus Google Scholar, R. B. Res. PubMed Scopus Google Scholar). The region of which an transcriptional repression Avram and M. Leid, unpublished results. was found to interact with the sirtuin of SIRT1 B and the sirtuin is proteins, it is that other of the sirtuin family may also as an is may that other proteins may a in CTIP2-mediated transcriptional repression, in cellular and/or promoter contexts. Similarly, the region of CTIP2 is also and in CTIP1 and may also be to interact with have also observed that CTIP1 directly with SIRT1 in and in transfected C2H2 zinc finger proteins, such as the Kruppel-associated protein have also been to repress transcription in a P. D. Biol. Chem. 2001; PubMed Scopus Google Scholar, S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). repression by zinc finger proteins with the which in may of heterochromatin protein 1 family to the template Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar, K. Mol. Cell. Biol. PubMed Scopus Google Scholar). 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CTIPs may to SIRT1 other family to a either by direct DNA binding or to a COUP-TF family recruited to the SIRT1 may histone leading to heterochromatin and gene of of of the proteins of the CTIP to the promoter of the results that CTIP2-mediated transcriptional repression the of SIRT1 to the which the histone deacetylase deacetylation of promoter-associated histones H3 and/or of deacetylation may be chromatin and or these deacetylation may result in of to a either of SIRT1 to the DNA template by CTIP2 be to result in gene This be for and of the the of CTIP proteins in cells of the hematopoietic and/or the T. and for M. for the and and L. for with the are to M. for