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• W2006975907 abstract "The identities of signal transducer proteins that integrate histone hypoacetylation and transcriptional repression are largely unknown. Here we demonstrate that THAP7, an uncharacterized member of the recently identified THAP (Thanatos-associated protein) family of proteins, is ubiquitously expressed, associates with chromatin, and represses transcription. THAP7 binds preferentially to hypoacetylated (un-, mono-, and diacetylated) histone H4 tails in vitro via its C-terminal 77 amino acids. Deletion of this domain, or treatment of cells with the histone deacetylase inhibitor TSA, which leads to histone hyperacetylation, partially disrupts THAP7/chromatin association in living cells. THAP7 coimmunoprecipitates with histone deacetylase 3 (HDAC3) and the nuclear hormone receptor corepressor (NCoR) and represses transcription as a Gal4 fusion protein. Chromatin immunoprecipitation assays demonstrate that these corepressors are recruited to promoters in a THAP7 dependent manner and promote histone H3 hypoacetylation. The conserved THAP domain is a key determinant for full HDAC3 association in vitro, and both the THAP domain and the histone interaction domain are important for the repressive properties of THAP7. Full repression mediated by THAP7 is also dependent on NCoR expression. We hypothesize that THAP7 is a dual function repressor protein that actively targets deacetylation of histone H3 necessary to establish transcriptional repression and functions as a signal transducer of the repressive mark of hypoacetylated histone H4. This is the first demonstration of the transcriptional regulatory properties of a human THAP domain protein, and a critical identification of a potential transducer of the repressive signal of hypoacetylated histone H4 in higher eukaryotes. The identities of signal transducer proteins that integrate histone hypoacetylation and transcriptional repression are largely unknown. Here we demonstrate that THAP7, an uncharacterized member of the recently identified THAP (Thanatos-associated protein) family of proteins, is ubiquitously expressed, associates with chromatin, and represses transcription. THAP7 binds preferentially to hypoacetylated (un-, mono-, and diacetylated) histone H4 tails in vitro via its C-terminal 77 amino acids. Deletion of this domain, or treatment of cells with the histone deacetylase inhibitor TSA, which leads to histone hyperacetylation, partially disrupts THAP7/chromatin association in living cells. THAP7 coimmunoprecipitates with histone deacetylase 3 (HDAC3) and the nuclear hormone receptor corepressor (NCoR) and represses transcription as a Gal4 fusion protein. Chromatin immunoprecipitation assays demonstrate that these corepressors are recruited to promoters in a THAP7 dependent manner and promote histone H3 hypoacetylation. The conserved THAP domain is a key determinant for full HDAC3 association in vitro, and both the THAP domain and the histone interaction domain are important for the repressive properties of THAP7. Full repression mediated by THAP7 is also dependent on NCoR expression. We hypothesize that THAP7 is a dual function repressor protein that actively targets deacetylation of histone H3 necessary to establish transcriptional repression and functions as a signal transducer of the repressive mark of hypoacetylated histone H4. This is the first demonstration of the transcriptional regulatory properties of a human THAP domain protein, and a critical identification of a potential transducer of the repressive signal of hypoacetylated histone H4 in higher eukaryotes. The dynamic nature of chromatin modification clearly remains at the core of the process of transcription. The acetylation of histone H3 and H4 tails is strongly associated with transcriptional activation, whereas hypoacetylated histones are a mark of transcriptionally silent heterochromatin (1Wu J. Grunstein M. Trends Biochem. Sci. 2000; 25: 619-623Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 2Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2352) Google Scholar). Consistent with this, proteins that acetylate histones (histone acetyltransferases) are generally involved in transcriptional activation, whereas enzymes that remove acetyl groups from histones (histone deacetylases (HDACs)) 1The abbreviations used are: HDAC, histone deacetylase; CBP, CREB-binding protein; DBD, DNA binding domain; HID, histone interacting domain; INHAT, inhibitor of acetyltransferases; GST, glutathione S-transferase; TK, thymidine kinase; THAP, Thanatos-associated protein; siRNA, small interfering RNA; luc, luciferase; TAF-Iα, template-activating factor-Iα; TAF-Iβ, template-activating factor-Iβ; TSA, trichostatin A. 1The abbreviations used are: HDAC, histone deacetylase; CBP, CREB-binding protein; DBD, DNA binding domain; HID, histone interacting domain; INHAT, inhibitor of acetyltransferases; GST, glutathione S-transferase; TK, thymidine kinase; THAP, Thanatos-associated protein; siRNA, small interfering RNA; luc, luciferase; TAF-Iα, template-activating factor-Iα; TAF-Iβ, template-activating factor-Iβ; TSA, trichostatin A. function in transcriptional repression. In contrast, methylation of histone tails can have both positive and negative effects on transcription (3Zhang Y. Reinberg D. Genes Dev. 2001; 15: 2343-2360Crossref PubMed Scopus (1216) Google Scholar). The number, variety, and interdependence of histone modifications has led to the histone code hypothesis that predicts that modifications acting in combinatorial or sequential fashion on histone tails specify unique downstream functions (4Fischle W. Wang Y. Allis C.D. Curr. Opin. Cell Biol. 2003; 15: 172-183Crossref PubMed Scopus (970) Google Scholar, 5Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6448) Google Scholar, 6Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7468) Google Scholar). More recently, a signaling network model of chromatin has been proposed that suggests that multiple chromatin modifications combine to confer bistability, robustness, and adaptability to transcriptional signaling networks (7Schreiber S.L. Bernstein B.E. Cell. 2002; 111: 771-778Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). Both models predict the existence of cellular proteins that would either bind or be released from histones based on their modification status. Consistent with both models, histone H3 methylated at lysine 9 recruits heterochromatin protein 1 (8Bannister A.J. Zegerman P. Partridge J.F. Miska E.A. Thomas J.O. Allshire R.C. Kouzarides T. Nature. 2001; 410: 120-124Crossref PubMed Scopus (2139) Google Scholar, 9Lachner M. O'Carroll D. Rea S. Mechtler K. Jenuwein T. Nature. 2001; 410: 116-120Crossref PubMed Scopus (2145) Google Scholar), which plays an essential role in the establishment and maintenance of transcriptionally silent heterochromatin. Acetylated histones can also serve as specific docking sites for proteins. Acetylated histone H3 and H4 are able to recruit bromodomain-containing proteins, including the chromatin remodeling enzymes Swi/Snf and the transcriptional coactivators TAF(II) 250 and GCN5 to localized regions of chromatin (10Agalioti T. Chen G. Thanos D. Cell. 2002; 111: 381-392Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, 11Hassan A.H. Neely K.E. Workman J.L. Cell. 2001; 104: 817-827Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 12Hudson B.P. Martinez-Yamout M.A. Dyson H.J. Wright P.E. J. Mol. Biol. 2000; 304: 355-370Crossref PubMed Scopus (130) Google Scholar, 13Jacobson R.H. Ladurner A.G. King D.S. Tjian R. Science. 2000; 288: 1422-1425Crossref PubMed Scopus (674) Google Scholar, 14Owen D.J. Ornaghi P. Yang J.C. Lowe N. Evans P.R. Ballario P. Neuhaus D. Filetici P. Travers A.A. EMBO J. 2000; 19: 6141-6149Crossref PubMed Scopus (407) Google Scholar, 15Zeng L. Zhou M.M. FEBS Lett. 2002; 513: 124-128Crossref PubMed Scopus (541) Google Scholar). This is considered to be a critical mechanism whereby acetylated histones are translated into transcriptionally active domains. Histone acetylation also has proposed roles in addition to the signaling role, such as reducing higher order chromatin structure by neutralizing the positive charge on histone tails and preventing their association with the negatively charged phosphate backbone of DNA and altered interaction with neighboring nucleosomes (16Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6725) Google Scholar, 17Luger K. Curr. Opin. Genet. Dev. 2003; 13: 127-135Crossref PubMed Scopus (226) Google Scholar). Sir3 and Tup1 are two yeast proteins that specifically bind hypoacetylated histones and have been linked to transcriptional repression. In yeast, at telomeres and the silent mating-type loci, Sir2 functions along with Sir3, Sir4, and Rap1 to mediate silencing (18Moretti P. Freeman K. Coodly L. Shore D. Genes Dev. 1994; 8: 2257-2269Crossref PubMed Scopus (459) Google Scholar, 19Moazed D. Kistler A. Axelrod A. Rine J. Johnson A.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2186-2191Crossref PubMed Scopus (176) Google Scholar, 20Strahl-Bolsinger S. Hecht A. Luo K. Grunstein M. Genes Dev. 1997; 11: 83-93Crossref PubMed Scopus (591) Google Scholar). Sir3 interacts more efficiently with histone H4 tails that are hypoacetylated by Sir2 (21Carmen A.A. Milne L. Grunstein M. J. Biol. Chem. 2002; 277: 4778-4781Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). In addition, the yeast corepressor Tup1 interacts directly with histone H3 and H4 tails via a domain that is also required for transcriptional repression (22Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Scopus (405) Google Scholar). This interaction is weakened by high levels of histone acetylation, suggesting a mechanism whereby repression can be relieved. The Ssn6-Tup1 corepressor complex also interacts with class I histone deacetylases that are required for its function (23Watson A.D. Edmondson D.G. Bone J.R. Mukai Y. Yu Y. Du W. Stillman D.J. Roth S.Y. Genes Dev. 2000; 14: 2737-2744Crossref PubMed Scopus (129) Google Scholar). In higher eukaryotes, however, the mechanisms whereby un- or hypoacetylated histones mediate transcriptional repression are largely unknown. We have previously shown that INHAT subunits pp32, TAF-Iα, and TAF-Iβ bind to histones and consequently prevent them from serving as substrates for the histone acetyltransferase coactivators p300/CBP and p300/CBP-associated factor, a process we refer to as histone masking (24Seo S.-b. McNamara P. Heo S. Turner A. Lane W.S. Chakravarti D. Cell. 2001; 104: 119-130Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 25Seo S.B. Macfarlan T. McNamara P. Hong R. Mukai Y. Heo S. Chakravarti D. J. Biol. Chem. 2002; 277: 14005-14010Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Recently we and others have shown that TAF-Iβ and pp32 associate with histone deacetylases and can bind to histone tails, but not if lysines are hyperacetylated, making them candidate human proteins that can translate the transcriptionally repressive marks of unacetylated histones (26Kutney S.N. Hong R. Macfarlan T. Chakravarti D. J. Biol. Chem. 2004; 279: 30850-30855Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 27Schneider R. Bannister A.J. Weise C. Kouzarides T. J. Biol. Chem. 2004; 279: 23859-23862Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In this work we characterize THAP7, a novel human protein we isolated in an interaction screen with TAF-Iβ. This protein is a member of a large class of proteins containing the novel THAP domain, a C2-CH signature (Cys-Xaa2–4-Cys-Xaa35–50-Cys-Xaa2-His) zinc finger domain sharing significant similarity to the site-specific DNA binding domain of the Drosophila P element transposase (28Roussigne M. Kossida S. Lavigne A.C. Clouaire T. Ecochard V. Glories A. Amalric F. Girard J.P. Trends Biochem. Sci. 2003; 28: 66-69Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). There are 12 distinct human proteins that contain the THAP domain, and all share a similar N-terminal location of the domain as well as its size of ∼90 residues. To date, only two members of this family have been characterized; THAP0 (DAP4/p52rIPK), a protein that was isolated in a genetic screen for genes involved in interferon-γ-induced apoptosis in HeLa cells (29Deiss L.P. Feinstein E. Berissi H. Cohen O. Kimchi A. Genes Dev. 1995; 9: 15-30Crossref PubMed Scopus (528) Google Scholar) and in a screen for activators of the interferon-induced protein kinase R (30Gale Jr., M. Blakely C.M. Hopkins D.A. Melville M.W. Wambach M. Romano P.R. Katze M.G. Mol. Cell. Biol. 1998; 18: 859-871Crossref PubMed Scopus (77) Google Scholar), and THAP1, a protein that potentiates both serum withdrawal and tumor necrosis factor α-induced apoptosis (31Roussigne M. Cayrol C. Clouaire T. Amalric F. Girard J.P. Oncogene. 2003; 22: 2432-2442Crossref PubMed Scopus (115) Google Scholar). Genetic evidence in Caenorhabditis elegans and Drosophila indicates that THAP domain proteins may have roles in chromatin-based processes, including transcription (28Roussigne M. Kossida S. Lavigne A.C. Clouaire T. Ecochard V. Glories A. Amalric F. Girard J.P. Trends Biochem. Sci. 2003; 28: 66-69Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 32Boxem M. van den Heuvel S. Curr. Biol. 2002; 12: 906-911Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 33Reddy K.C. Villeneuve A.M. Cell. 2004; 118: 439-452Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). However, no transcriptional regulatory properties of any member of the THAP domain family have been reported. Here we demonstrate that THAP7 binds to histones and nucleosomes in a tail-dependent manner and binds to histone H4 in an acetylation sensitive manner. Transfected THAP7 associates with chromatin in living cells and promoter-targeted THAP7 represses transcription of transiently transfected and chromosomally integrated reporters and associates with HDAC3 and NCoR. This is the first demonstration of chromatin targeting and transcriptional regulatory function of a human THAP family protein. Yeast Two-hybrid Screen—TAF-Iβ was cloned into the yeast pBD-Gal4 (Stratagene) vector and transformed into YRG-2 His-yeast. These yeast were then transformed with a cDNA library from human vascular smooth muscle cells cloned into the yeast pAD-Gal4 (Stratagene) vector and plated onto SD-Trp-Leu-His plates (34McNamara P. Seo S.-b. Rudic R.D. Sehgal A. Chakravarti D. FitzGerald G.A. Cell. 2001; 105: 877-889Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Approximately 100 yeast colonies grew and these yeast cells were spread onto one plate and then replica plated onto SD-Trp-His plates containing 5 mm 3-aminotriazole. Only 20 colonies still grew and plasmids were isolated from these yeast and sequenced. Half of the sequenced clones were the novel human protein THAP7. Northern Analysis—A 32P-THAP7 probe was generated by high prime DNA labeling, and incubated with a poly(A) mRNA multiple tissue Northern Array (normalized using six different housekeeping genes) and a multiple tissue Northern blot (Clontech). After extensive washing, the array and blot were analyzed by a phosphorimaging device. Plasmids and Proteins—THAP7 and its truncation mutants were PCR amplified from the yeast pAD-Gal4–2.1 (Stratagene) THAP7 clone and inserted into the pCMX vector, the pCDNA3.1myc His vector (Invitrogen), the pCMX Gal4 (amino acids 1–147) vector, the pEGFP C2 vector (Clontech), and pGEX-4T2. GST fusion proteins were expressed in BL21 cells, and purified utilizing glutathione-Sepharose beads. Sequences of recombinant DNA were verified by automated sequencing. In Vitro Interaction Assays—For histone binding assays, 10 μg of histones purified from HeLa cells were preincubated with a pan-monoclonal anti-histone antibody (Roche) in the presence of protein G-Sepharose beads and in vitro translated [35S]methionine-labeled proteins generated using the TnT in vitro translation system (Promega). For GST pull-down assays, in vitro translated [35S]methionine-labeled proteins or purified histones (Roche) were mixed with GST fusion proteins purified from BL21 cells in buffer containing 150 mm KCl, 20 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 1 mm dithiothreitol, 10 μg/ml bovine serum albumin, protease inhibitor mixture, and 0.5% Nonidet P-40 for 30 min, washed extensively, and bound proteins were collected on glutathione-Sepharose beads and resolved by SDS-PAGE. Gels were fixed, dried, and analyzed by a phosphorimaging device. For nucleosome and histone H3 binding assays, 270 mm KCl and 1% Nonidet P-40 were used. Cell Culture and Transfections—293T cells or 293T cells containing a chromosomally integrated luciferase gene with Gal4 DNA binding sites under control of an SV40 or TK promoter were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum with penicillin/strepomycin and puromycin where necessary. Cells were seeded and transfected with Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). For coimmunoprecipitations, cells were harvested 24 h after transfection. Luciferase assays were carried out 48 h after transfection according to the manufacturer's instructions (Promega). TSA treatment for 16 h (200 nm or 1 μm) was performed 24 h after transfection. Coimmunoprecipitations—THAP7-transfected 293T cells were lysed in hypotonic buffer A on ice for 5 min (10 mm HEPES-KOH, pH 7.4, 1.5 mm MgCl2, 10 mm KCl, 1 mm phenylmethylsulfonyl fluoride, plus protease inhibitor mixture). Nuclei were isolated by centrifugation at 2000 rpm and resuspended in CoIP buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 1 mm phenylmethylsulfonyl fluoride, protease inhibitors, plus 1% Nonidet P-40) and sonicated. Nuclear lysates or whole cell lysates generated by harvesting cells directly in CoIP buffer and sonicated as above were incubated at a 1:1000 dilution with monoclonal anti-myc antibody (Cell Signaling), anti-FLAG antibody (Santa Cruz), or anti-NCoR antibody (Santa Cruz) overnight, pulled down with protein G dynabeads (Dynal) and washed with CoIP buffer. Bound proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were then immunoblotted with anti-myc antibody, anti-FLAG antibody, or anti-NCoR antibody as indicated. Histone Peptide Binding Assays—Biotinylated histone peptides from Upstate Biotech or Synpep were quantified by dot blot onto nitrocellulose membrane and probed with streptavidin-horseradish peroxidase to ensure equal loading. Three μg of the indicated peptide was incubated with in vitro translated THAP7 in buffer containing 150 mm KCl, 20 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 1 mm dithiothreitol, 10 μg/ml bovine serum albumin, protease inhibitor mixture, and 0.5% Nonidet P-40. Peptides were pulled down with streptavidin-agarose beads, washed, and bound proteins were separated by SDS-PAGE and analyzed by a phosphorimaging device. Nucleosome Preparation/Chromatin Association Assays—Nucleosomes (chromatin fraction) were prepared essentially as described by Nielsen et al. (35Nielsen A.L. Oulad-Abdelghani M. Ortiz J.A. Remboutsika E. Chambon P. Losson R. Mol. Cell. 2001; 7: 729-739Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). In short, ∼107 293T cells were lysed in NIB buffer (15 mm Tris-HCl, pH 7.4, 60 mm KCl, 15 mm MgCl2, 15 mm NaCl, 1 mm CaCl2, protease inhibitor mixture, 1 mm phenylmethylsulfonyl fluoride) plus 0.3% Nonidet P-40, some of which was saved (whole cell extract). Following centrifugation of nuclei at 2000 rpm, supernatant was saved (cytoplasmic extract) and nuclei were washed with NIB plus 0.6% Nonidet P-40 buffer (wash 1). Nuclear lysates were then treated with micrococcal nuclease for 8 min and spun at 10,000 rpm. The supernatant was saved (wash 2) and the pellet was resuspended in ice-cold 2 mm EDTA, pH 8.0. After centrifugation at 10,000 rpm, the supernatant containing solubilized nucleosomes was collected (chromatin fraction). The presence of nucleosomes in the chromatin fraction was confirmed by DNA agarose gel electrophoresis and SDS-PAGE followed by Coomassie staining. All collected fractions were then subjected to immunoblot analysis with anti-myc (Cell Signaling), anti-Hp1α (Upstate), and histone H1 (Upstate) antibodies as indicated or were subjected to trypsin digestion for 10 min followed by addition of ×20 soybean trypsin inhibitor where indicated. Alternatively, transfected 293T cells were lysed in buffer A (described above) containing 0.5% Nonidet P-40 on ice for 10 min. Nuclei were spun down for 10 min at 2000 rpm. The nuclei were then extracted with buffer A with increasing amounts of NaCl to solubilize chromatin-associated proteins. Extracted proteins were then subjected to SDS-PAGE and immunoblot analysis as described above. Reporter Gene Studies—293T cells or 293T cells containing an integrated SV40 or TK luciferase reporter gene containing upstream Gal4 DNA binding sites (Gal4 SV40-luc and Gal4 TK-luc) were seeded into 48-well plates to be 50–70% confluent the next day. 293T cells were transfected with Gal4 TK-Luc reporter and pRL Renilla luciferase (Promega), Gal4 DNA-binding protein (DBD) alone, Gal4 DBD-THAP7, or the indicated Gal4 DBD-THAP7 truncation mutant. Cells were lysed after 48 h and luciferase activity was measured according to the manufacturer's instructions (Promega). Western blots utilizing anti-Gal4 antibody (Santa Cruz) were used to verify expression of mutant constructs. For the NCoR siRNA experiment, cells were first transfected with siRNA for 24 h, followed by transfection with reporter constructs. For dose response and repressor mapping, the data from three independent experiments were averaged. Chromatin Immunoprecipitation Assay—293T cells were fixed with 1% formaldehyde for 15 min followed by addition of 0.125 m glycine for 5 min at room temperature. Cells were then washed in phosphate-buffered saline, spun down at 1,000 rpm, and resuspended in chromatin immunoprecipitation lysis buffer (1% SDS, 1 mm EDTA, 50 mm Tris-HCl, pH 8.0). Cells were then sonicated for a total of 30 s at level 3. After a 14,000 rpm spin, the supernatant was diluted 10-fold in chromatin immunoprecipitation dilution buffer (0.1% SDS, 1.1% Triton X-100), 1 mm EDTA, 150 mm NaCl, 20 mm Tris-HCl, pH 8.0) and blocked with protein G-Sepharose beads and 20 μg of sonicated salmon sperm DNA. After spinning down the beads at 3,000 rpm for 1 min, the supernatant was incubated overnight with anti-acetylated histone H3, anti-acetylated histone H4 (Upstate), anti-Gal4, anti-NCoR, anti-HDAC3, or anti-HDAC4 antibodies (Santa Cruz). The IP material was pulled down by incubating with protein G-Sepharose for 3 h and beads were washed once with wash buffer 1 (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 150 mm NaCl, 20 mm Tris-HCl, pH 8.0), again with wash buffer 1 plus 350 mm NaCl, once with wash buffer 3 (0.25 m LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mm EDTA, 10 mm Tris-HCl, pH 8.0), and twice with TE, pH 8.0. Immune complexes were eluted in 200 μl of elution buffer (1% SDS, 0.1 m sodium bicarbonate). Cross-links were reversed after addition of 200 mm NaCl by incubation overnight at 65 °C. DNA was then isolated using Qiagen spin columns and analyzed by PCR utilizing primers generated against the promoter of the MH100 TK-luc plasmid. THAP7 Identification, Homologues, and Expression—To search for novel TAF-Iβ interactors and potential histone-binding proteins, we performed a yeast two-hybrid screen using TAF-Iβ as the bait and a human vascular smooth muscle cell library. Of the 20 strongest interacting clones, half were the hypothetical human protein THAP7. Human THAP7 is 309 amino acids long and contains a proline-rich region and a basic stretch at its C terminus (Fig. 1A). It also contains a newly discovered and highly conserved THAP zinc finger domain that is found in at least 12 distinct human proteins, and also in mouse, rat, pig, cow, chicken, Xenopus, zebrafish, C. elegans, and Drosophila proteins (28Roussigne M. Kossida S. Lavigne A.C. Clouaire T. Ecochard V. Glories A. Amalric F. Girard J.P. Trends Biochem. Sci. 2003; 28: 66-69Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The mouse homologue of human THAP7 has 93.2% identity at the amino acid sequence throughout the entire length of the protein, whereas the rat homologue contains 92.2% identity (Fig. 1B). However, the rat homologue of THAP7 has an extended N terminus that contains a RanGAP domain, containing a leucine-rich repeat domain as well as a tropomodulin domain within it. The rat homologue also contains a tubulin domain not present in the human and mouse proteins (Fig. 1A). It is possible that this extended sequence of the rat homologue represents a chromosomal translocation that resulted in a fusion protein during evolution. None of these proteins has been characterized to date. THAP7 poly(A)+ mRNA is expressed in all human tissues examined and is expressed as three distinct transcripts (approximately 1.2, 1.3, and 1.6 Kb) (Fig. 1, C and D). It is hyper-expressed in testes and up-regulated in the leukemic cell line K562. The implication of this expression pattern is currently unknown. THAP7 Associates with Histone Tails in Vitro—We isolated THAP7 in a yeast two-hybrid screen with the INHAT subunit TAF-Iβ. Because very little is known about the function of the recently identified novel THAP domain proteins, we set out to extensively characterize the THAP7 protein. Because TAF-Iβ binds to histones, we set out to determine whether THAP7 could also associate with histones in vitro. In vitro translated 35S-labeled THAP7 was incubated with histones purified from HeLa cells and immunoprecipitated with anti-histone antibody. THAP7 was pulled down only in the presence of histones (Fig. 2A), whereas under the same conditions, the transcription factor and nuclear hormone receptor RARα was not. To determine whether THAP7 could associate directly with histones, we incubated individually purified histones with GST or GST-THAP7. GST proteins used in binding assays are shown in Fig. 2B. GST-THAP7 bound to both histone H3 and histone H4, but not to histone H2A or histone H2B (Fig. 2C). These data indicate that THAP7 binds directly to histones, and that the interaction is specific for histones H3 and H4. To further analyze if this interaction was dependent upon histone tails, we used recombinant GST-histone H3 fusions containing only the histone tail (amino acids 1–46) or a mutant lacking the tail (Fig. 2D, left panel). 35S-Labeled THAP7 bound to GST-histone H3 and to the GST-histone H3 tail, but binding was almost completely absent to histone H3 that lacked the tail (Fig. 2D, right panel). These results demonstrate that the histone H3 tail is necessary and sufficient for THAP7 interaction in vitro. In vivo, histones primarily exist in the form of histone-DNA complexes termed nucleosomes. To examine if THAP7 could also bind to intact nucleosomes isolated from cells and to determine whether the binding was indeed dependent upon histone tails, we purified nucleosomes from 293T cells using published protocols (35Nielsen A.L. Oulad-Abdelghani M. Ortiz J.A. Remboutsika E. Chambon P. Losson R. Mol. Cell. 2001; 7: 729-739Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar), trypsinized the nucleosomes as previously reported (36Guyon J.R. Narlikar G.J. Sif S. Kingston R.E. Mol. Cell. Biol. 1999; 19: 2088-2097Crossref PubMed Scopus (55) Google Scholar), and tested their ability to bind GST-THAP7 (Fig. 2E). After trypsinization, nucleosomes run at a characteristically smaller size on SDS-PAGE gels (Fig. 2E, left panel) because of the removal of exposed tails (36Guyon J.R. Narlikar G.J. Sif S. Kingston R.E. Mol. Cell. Biol. 1999; 19: 2088-2097Crossref PubMed Scopus (55) Google Scholar). Intact nucleosomes bound to GST-THAP7 but not GST (Fig. 2E, middle panel). Because histone H2A and histone H2B exist in a tight complex with histones H3 and H4 in nucleosomes, GST-THAP7 as expected pulled down all the core histones present in the nucleosome. Importantly, tail-less nucleosomes did not bind to GST-THAP7 (Fig. 2E, right panel). These results together indicate that histone tails are essential for THAP7-histone/nucleosome interactions. To further examine whether the histone binding properties of THAP7 are mediated by histone tails, we utilized histone H3 and H4 N-terminal tail peptides conjugated to biotin. These peptides were incubated with in vitro translated THAP7, washed, and pulled down with streptavidin-agarose. Full-length THAP7-(1–309) bound to both histone H3 and H4 tails (Fig. 3B, middle panel). We next mapped the histone interaction domain (HID) of THAP7. For this purpose, we incubated a series of in vitro translated and radiolabeled THAP7 mutants (Fig. 3B, left panel, schematic shown in Fig. 3A) with biotinylated histone H3 or H4 tails in vitro. All of the THAP7 mutants that contained the C-terminal 77 amino acids, rich in positively charged residues, could bind to histone H3 and histone H4 tails, but a mutant (1–231) lacking this region could not (Fig. 3B, middle and right panels). These binding results (summarized in Fig. 3A) indicate that the THAP domain is not necessary and that the C-terminal 77 amino acids of THAP7 are necessary and sufficient for histone H3 and H4 tail binding in vitro. This region is thus referred to as the HID. THAP7 Associates with Chromatin in Intact Cells—Because THAP7 associated with nucleosomes, total histones, and histone tails in vitro, it was important to demonstrate that THAP7 does indeed associate with chr" @default.
• W2006975907 created "2016-06-24" @default.
• W2006975907 creator A5021649597 @default.
• W2006975907 creator A5037795026 @default.
• W2006975907 creator A5047281260 @default.
• W2006975907 creator A5068363917 @default.
• W2006975907 creator A5079265940 @default.
• W2006975907 creator A5086507952 @default.
• W2006975907 date "2005-02-01" @default.
• W2006975907 modified "2023-10-18" @default.
• W2006975907 title "Human THAP7 Is a Chromatin-associated, Histone Tail-binding Protein That Represses Transcription via Recruitment of HDAC3 and Nuclear Hormone Receptor Corepressor" @default.
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