Zinc Finger Protein Wiz Links G9a/GLP Histone Methyltransferases to the Co-repressor Molecule CtBP
2006; Elsevier BV; Volume: 281; Issue: 29 Linguagem: Inglês
10.1074/jbc.m603087200
ISSN1083-351X
AutoresJun Ueda, Makoto Tachibana, Tsuyoshi Ikura, Yoichi Shinkai,
Tópico(s)Kruppel-like factors research
ResumoG9a is a SET-domain mammalian histone methyltransferase responsible for mono- and dimethylation of lysine 9 in histone H3 (H3K9) at euchromatic regions. Recently we reported that G9a forms a stoichiometric heteromeric complex with another SET-domain-containing molecule, GLP/Eu-HMTase1. Although G9a and GLP can independently methylate H3K9 in vitro, G9a/GLP heteromeric formation seems to be essential for their function as a euchromatic H3K9 methyltransferase in vivo. To further elucidate how G9a/GLP-mediated histone methylation and transcriptional regulation are controlled, we purified and characterized G9a complexes from mouse embryonic stem cells. We identified a novel G9a/GLP-associating zinc finger molecule named Wiz that can interact with G9a and GLP independently but is more stable in the G9a/GLP heteromeric complexes. Interestingly, Wiz small inhibitory RNA knocks down not only Wiz but also G9a. GLP deficiency also decreases G9a levels, suggesting that the Wiz/G9a/GLP tri-complex may protect G9a from degradation and that Wiz plays a major role in G9a/GLP heterodimer formation. Furthermore, amino acid sequence analysis of Wiz predicted two potential CtBP binding sites, and indeed CtBP binding to Wiz and association of CtBP with the Wiz/G9a/GLP complex was observed. These data indicate that Wiz not only contributes to the stability of G9a but also links the G9a/GLP heteromeric complex to the CtBP co-repressor machinery. G9a is a SET-domain mammalian histone methyltransferase responsible for mono- and dimethylation of lysine 9 in histone H3 (H3K9) at euchromatic regions. Recently we reported that G9a forms a stoichiometric heteromeric complex with another SET-domain-containing molecule, GLP/Eu-HMTase1. Although G9a and GLP can independently methylate H3K9 in vitro, G9a/GLP heteromeric formation seems to be essential for their function as a euchromatic H3K9 methyltransferase in vivo. To further elucidate how G9a/GLP-mediated histone methylation and transcriptional regulation are controlled, we purified and characterized G9a complexes from mouse embryonic stem cells. We identified a novel G9a/GLP-associating zinc finger molecule named Wiz that can interact with G9a and GLP independently but is more stable in the G9a/GLP heteromeric complexes. Interestingly, Wiz small inhibitory RNA knocks down not only Wiz but also G9a. GLP deficiency also decreases G9a levels, suggesting that the Wiz/G9a/GLP tri-complex may protect G9a from degradation and that Wiz plays a major role in G9a/GLP heterodimer formation. Furthermore, amino acid sequence analysis of Wiz predicted two potential CtBP binding sites, and indeed CtBP binding to Wiz and association of CtBP with the Wiz/G9a/GLP complex was observed. These data indicate that Wiz not only contributes to the stability of G9a but also links the G9a/GLP heteromeric complex to the CtBP co-repressor machinery. In eukaryotes, DNA is wrapped around core histones to form nucleosome particles and condensed chromatin structures with various nuclear molecules. Therefore, regulation of chromatin structure and dynamics is a very critical step for genomic functions. Covalent histone modifications play critical roles in regulating these processes (1.Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (538) Google Scholar). 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The function of H3K9 methylation was initially defined based upon the first described histone lysine methyltransferases (HMTases), 2The abbreviations used are: HMTase, histone lysine methytransferase; ES, embryonic stem; CtBP, C-terminal binding protein; Wiz, widely interspaced zinc finger motifs; DAPI, 4′,6-diamino-2-phenylindole; GFP, green fluorescent protein; TRF, telomeric repeat binding factor; EGFP, enhanced green fluorescent protein; siRNA, small inhibitory RNA; CID, ctbp interaction domain; ZF, zinc finger motif. mouse Suv39h1 and its Drosophila and yeast counterparts Su(var)3–9 and Clr4 (17.Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2186) Google Scholar, 18.Czermin B. Schotta G. Hulsmann B.B. Brehm A. Becker P.B. Reuter G. Imhof A. EMBO Rep. 2001; 2: 915-919Crossref PubMed Scopus (144) Google Scholar, 19.Nakayama J. Rice J.C. Strahl B.D. Allis C.D. Grewal S.I. Science. 2001; 292: 110-113Crossref PubMed Scopus (1379) Google Scholar). These HMTases are members of SET-domain-containing molecules and specifically methylate H3K9, which plays a crucial role in heterochromatin formation and heterochromatic gene silencing (19.Nakayama J. Rice J.C. Strahl B.D. Allis C.D. Grewal S.I. Science. 2001; 292: 110-113Crossref PubMed Scopus (1379) Google Scholar, 20.Elgin S.C. Grewal S.I. Curr. Biol. 2003; 13: R895-R898Abstract Full Text Full Text PDF PubMed Google Scholar). H3K9 methylation has also been shown to control DNA methylation, typically in fungus Neurospora crassa and plant Arabidopsis thaliana (21.Tamaru H. Selker E.U. Nature. 2001; 414: 277-283Crossref PubMed Scopus (856) Google Scholar, 22.Jackson J.P. Lindroth A.M. Cao X. Jacobsen S.E. Nature. 2002; 416: 556-560Crossref PubMed Scopus (1019) Google Scholar). Methylated H3K9 allows the binding of the chromodomain of heterochromatin protein 1 (HP1), a step that is crucial for most of the chromatin functions regulated by H3K9 methylation (4.Bannister 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 (2184) Google Scholar, 6.Lachner M. O'Carroll D. Rea S. Mechtler K. Jenuwein T. Nature. 2001; 410: 116-120Crossref PubMed Scopus (2177) Google Scholar, 19.Nakayama J. Rice J.C. Strahl B.D. Allis C.D. Grewal S.I. Science. 2001; 292: 110-113Crossref PubMed Scopus (1379) Google Scholar). In transcriptional regulation within euchromatin, H3K9 methylation is generally associated with transcriptional silencing (1.Lachner M. O'Sullivan R.J. Jenuwein T. J. Cell Sci. 2003; 116: 2117-2124Crossref PubMed Scopus (538) Google Scholar); however, recent reports suggest that H3K9 methylation is also associated with transcriptional activation (elongation) (23.Vakoc C.R. Mandat S.A. Olenchock B.A. Blobel G.A. Mol. Cell. 2005; 19: 381-391Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 24.Lee D.Y. Northrop J.P. Kuo M.H. Stallcup M.R. J. Biol. Chem. 2006; 281: 8476-8485Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Therefore, the roles of H3K9 methylation on transcriptional regulation remain controversial. G9a is also a SET-domain-containing molecule and a major mammalian histone methyltransferase responsible for mono- and dimethylation of H3K9 at euchromatic regions (25.Tachibana M. Sugimoto K. Nozaki M. Ueda J. Ohta T. Ohki M. Fukuda M. Takeda N. Niida H. Kato H. Shinkai Y. Genes Dev. 2002; 16: 1779-1791Crossref PubMed Scopus (975) Google Scholar, 26.Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). Recently, we described that G9a forms a stoichiometric heteromeric complex with another SET-domain-containing molecule, GLP/Eu-HMTase1. Although G9a and GLP can independently methylate H3K9 in vitro, G9a/GLP heteromeric formation seems to be essential for exerting their function as a euchromatic H3K9 methyltransferase (27.Tachibana M. Ueda J. Fukuda M. Takeda N. Ohta T. Iwanari H. Sakihama T. Kodama T. Hamakubo T. Shinkai Y. Genes Dev. 2005; 19: 815-826Crossref PubMed Scopus (611) Google Scholar). It has been reported that both G9a and GLP are components of several transcriptional repression complexes, such as those involving E2F6, CtBP1, and CDP/cut (28.Ogawa H. Ishiguro K. Gaubatz S. Livingston D.M. Nakatani Y. Science. 2002; 296: 1132-1136Crossref PubMed Scopus (629) Google Scholar, 29.Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (640) Google Scholar, 30.Nishio H. Walsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11257-11262Crossref PubMed Scopus (122) Google Scholar). We have also revealed that G9a exists as ∼1-megadalton complexes in mouse embryonic stem (ES) cells. To elucidate how G9a/GLP HMTases regulate H3K9 methylation, how this transcriptional regulation is controlled, and the significance of the G9a/GLP heteromeric complex, we isolated the G9a complex from mouse ES cells and identified and characterized one of the novel G9a-associating molecules, Wiz. Cell Culture—Mouse ES cells were maintained in 10% fetal calf serum and LIF (500 units/ml)-containing medium. Generation of Stable Cell Lines—G9a–/– ES cells (clone #22-10, (25.Tachibana M. Sugimoto K. Nozaki M. Ueda J. Ohta T. Ohki M. Fukuda M. Takeda N. Niida H. Kato H. Shinkai Y. Genes Dev. 2002; 16: 1779-1791Crossref PubMed Scopus (975) Google Scholar)) were co-transfected with a cDNA encoding human G9a (GenBank™ accession number NM_006709) tagged with His-FLAG epitopes at the carboxyl terminus in the pCAGGS expression plasmid and a vector conferring Hygromycin B resistancy (pGK-hygroB) by the use of Lipofectamine 2000 reagent (Invitrogen). Resistant cells were selected in ES cell medium containing 150 μg/ml hygromycin B and checked for the expression of human G9a by anti-G9a or anti-FLAG antibody by Western blotting. One of the positive clones (termed as HF7) was used for G9a complex purification. G9a Complex Purification—The G9a complexes were purified from a nuclear extract prepared from HF7 cells. As a control, mock purification was performed from a nuclear extract prepared from the G9a–/– ES cells expressing mouse G9a-L without tags (clone #15-3). After removing the cytoplasmic fraction with buffer A (10 mm HEPES-KOH at pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride), nuclear pellets were suspended and lysed with buffer D (20 mm HEPES-KOH at pH 7.9, 1.5 mm MgCl2, 420 mm NaCl, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 0.1% Nonidet P-40, and 20% glycerol) for 30 min on ice. The insoluble nuclear fraction was removed by centrifugation. The nuclear extract was first applied to nickel-nitrilotriacetic acid-agarose beads (Qiagen) and bound materials were eluted with buffer D containing 0.2 m immidazole. The eluates were further incubated with anti-FLAG mouse monoclonal antibody (M2)-conjugated agarose beads (Sigma) and then eluted with FLAG peptide (Sigma). The purified proteins were resolved by 4–20% gradient SDS-PAGE and silver-stained. The polypeptides specific to HF7 cells were excised and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Aliquots of the purified G9a complexes were also loaded onto a 10-ml 10–30% glycerol gradient in buffer D, centrifuged at 32,000 rpm for 12 h, and fractionated. Each fraction was concentrated with trichloroacetic acid, resolved by SDS-PAGE, and visualized by silver staining or Western blotting. Immunofluorescence Analysis—By the use of TransIT-LT1 (Mirus), pEGFP-C2-Wiz was transfected to NIH3T3 cells. After 2 days in culture, cells were collected, cytospun, and fixed with 4% paraformaldehyde for 10 min. Then the cells were permeabilized with 0.1% Triton X-100 for 10 min and incubated with anti-G9a (#8620) at 37 °C for 40 min. Anti-mouse IgG conjugated with Zenon Alexa Fluor 568 (Molecular Probes) was used for detection. The nuclei were counterstained with DAPI, observed under fluorescence microscopy, and analyzed with AxioVision software (Zeiss). Immunoprecipitation and Western Blot Analysis—For immunoprecipitation of endogenous G9a, GLP, and Wiz, ES cells were harvested with phosphate-buffered saline containing trypsin (0.05%) and EDTA (0.2 mm). Nuclear extracts were prepared as described under “G9a Complex Purification.” Nuclear extracts from 107 ES cells were incubated with 2 μg of antibodies overnight and immune complexes were collected with 20 μl of protein G slurry (1:1 ratio) for 1 h. The immune complexes were washed twice with 300 μl of buffer D. For the stringent immunoprecipitation experiments, RIPA buffer (phosphate-buffered saline containing 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and protease inhibitor; Nakalai) was used for nuclear extraction and following immunoprecipitation steps. For transient G9a, GLP, Wiz, and CtBP interaction analyses, HEK 293T cells were transfected using TransIT-LT1, with combinations of FLAG-tagged cDNAs driven by the pcDNA3 vector (Invitrogen) or the pM vector (Clontech) and the corresponding cDNAs subcloned into the pEGFP-C vectors (Clontech). After 2 days in culture, whole cell extracts were prepared with buffer D and used for immunoprecipitation as described above. For Western blot analysis, immunoprecipitated molecules or total cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked with 5% milk, and probed with the specific antibodies described below. Antibodies—For detection of mouse and human G9a, mouse monoclonal antibody #8620 (Perseus Proteomics Inc.) and hamster monoclonal antibody #14-1 (Medical & Biological Laboratories Co., Ltd.) were used, respectively. For detection of mouse and human GLP, mouse monoclonal antibody #422 (Perseus Proteomics Inc.) and hamster monoclonal antibody C7–5 (Medical & Biological Laboratories Co., Ltd.) were used, respectively. For detection of epitope-tagged protein, rabbit anti-GFP (Code No.598, MBL), anti-FLAG M2 (code number F3165, Sigma) and anti-GAL4-DNA-binding domain (RK5C1, Santa Cruz Biotechnology) were used. For specificity control in two sequential immunodepletions analyses, anti-TRF1 was used (31.Iwano T. Tachibana M. Reth M. Shinkai Y. J. Biol. Chem. 2004; 279: 1442-1448Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). For the negative control of immunoprecipitation experiments, anti-Myc (9E10) antibody was used. To control for protein loading, anti-tubulin (CP06, Oncogene) was used. For CtBP detection, mouse monoclonal antibodies against CtBP1 and CtBP2 (BD Biosciences) were used. Plasmid Constructions—For the construction of FLAG-mWiz expression vector, mWiz cDNA (GenBank™ accession number AB255388) was inserted into XhoI site of the pcDNA3 vector, which has FLAG-tag sequence between HindIII/BamHI sites. For the construction of EGFP-mWiz expression vector, mWiz cDNA was inserted into BamHI/XbaI sites of pEGFP-C2 vector. For the construction of GAL4-mWiz expression vector, mWiz cDNA was inserted into BamHI/SalI sites of pm vector (Clontech). ΔZF6 version of Wiz was made by removal of cDNA coding amino acid positions from 853 to 956 (downstream of PstI site). The ZF6 version of Wiz (amino acid positions from 851 to 956) was made by inserting mWiz cDNA corresponding to this region to PstI/BamHI sites of pEGFP-C1 vector. For the construction of EGFP-tagged mCtBP1 and mCtBP2 expression vectors, mCtBP1 cDNA was inserted into EcoRI site of pEGFP-C2 vector and mCtBP2 cDNA was inserted into KpnI/XbaI sites of pEGFP-C3 vector. FLAG-mCtBP2 was constructed by inserting FLAG-tag sequence into KpnI site of pcDNA3.1-His-CtBP2 vector (kind gift from Dr. Shimotohno). All the mG9a, mGLP, and mSuv39h1 expression vectors used were described previously (25.Tachibana M. Sugimoto K. Nozaki M. Ueda J. Ohta T. Ohki M. Fukuda M. Takeda N. Niida H. Kato H. Shinkai Y. Genes Dev. 2002; 16: 1779-1791Crossref PubMed Scopus (975) Google Scholar, 26.Tachibana M. Sugimoto K. Fukushima T. Shinkai Y. J. Biol. Chem. 2001; 276: 25309-25317Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 27.Tachibana M. Ueda J. Fukuda M. Takeda N. Ohta T. Iwanari H. Sakihama T. Kodama T. Hamakubo T. Shinkai Y. Genes Dev. 2005; 19: 815-826Crossref PubMed Scopus (611) Google Scholar). Anti-Wiz Antibody Production—mWiz cDNA was subcloned into the bacterial expression vector pGEX-4T-3 (Amersham Biosciences). Recombinant fusion molecules (amino acid positions from 300 to 956 of the Wiz polypeptide) were used to immunize rabbits. Anti-Wiz antibody was affinity-purified using glutathione S-transferase-fused Wiz (amino acid positions from 300 to 514 of mWiz). siRNA—Double-stranded RNA oligonucleotides (21 nucleotides) homologous to G9a, GLP, and Wiz were designed as follows: G9a, 5′-CCAUG CUGUC AACUA CCAUG G (forward) and 5′-AUGGU AGUUG ACAGC AUGGA G (reverse); GLP, 5′-GCGUG GUCAA GUAUG AGCUG A (forward) and 5′-AGCUC AUACU UGACC ACGCU G (reverse); Wiz #1, 5′-GAGCA AGCCA AAACC UCAAA C (forward) and 5′-UUGAG GUUUU GGCUU GCUCC U (reverse); Wiz #2, 5′-GAAUA AGGAA CGUGG AUCUU U (forward) and 5′-AGAUC CACGU UCCUU AUUCU C (reverse); Wiz #3, 5′-GCAGA ACAUC AACAA AUUUG A (forward) and 5′-AAAUU UGUUG AUGUU CUGCC G (reverse); Control siRNA, 5′-GGAAG GCUCU UGAUG AAAUG G (forward); 5′-AUUUC AUCAA GAGCC UUCCA U (reverse). Cells were treated with annealed siRNAs at a final concentration of 25 nm by the use of Oligofectamine (Invitrogen). As a control, unrelated nuclear protein siRNA was used. Northern Blot Analysis—Eight micrograms of total RNAs were separated by 1% agarose-formaldehyde gel electrophoresis, transferred to a nylon membrane, and hybridized with 32P-labeled cDNA probes. Purification of the G9a Complex—The G9a complex was purified from G9a–/– ES cells stably expressing human G9a tagged with carboxyl-terminal His-FLAG epitopes (clone HF7). Nuclear extracts from HF7 cells were first incubated with nickel-nitrilotriacetic acid-agarose, and the bound polypeptides were eluted with buffer containing 0.2 m immidazole. The G9a complexes were further purified with agarose beads conjugated with anti-FLAG antibody. Control mock purification was performed in parallel using G9a–/– ES cells stably expressing mouse G9a-L without tags (clone 15-3). The second affinity purification step with anti-FLAG antibody resulted in the identification of several polypeptides that associated with G9a in the HF7 but not the 15-3 extracts (Fig. 1A). It is of note that G9a–/– ES cell phenotypes such as Mage-a genes expression were rescued in HF7 cells (data not shown). The components of the G9a complex were identified by mass spectrometry. As reported in previous studies (27.Tachibana M. Ueda J. Fukuda M. Takeda N. Ohta T. Iwanari H. Sakihama T. Kodama T. Hamakubo T. Shinkai Y. Genes Dev. 2005; 19: 815-826Crossref PubMed Scopus (611) Google Scholar, 28.Ogawa H. Ishiguro K. Gaubatz S. Livingston D.M. Nakatani Y. Science. 2002; 296: 1132-1136Crossref PubMed Scopus (629) Google Scholar, 29.Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Shi Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (640) Google Scholar, 30.Nishio H. Walsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11257-11262Crossref PubMed Scopus (122) Google Scholar), GLP was present in the complex. In addition to GLP, we identified a protein named Wiz (widely interspaced zinc finger motifs), previously reported as a factor containing five Kruppel (C2H2)-type zinc finger motifs in a widely interspaced manner (32.Matsumoto K. Ishii N. Yoshida S. Shiosaka S. Wanaka A. Tohyama M. Brain Res. Mol. Brain Res. 1998; 61: 179-189Crossref PubMed Scopus (12) Google Scholar) (Fig. 1B). Although brain specificity has been attributed to Wiz, our Northern blot analysis showed that Wiz is ubiquitously expressed in various mouse tissues (Fig. 1C), suggesting a more general function of Wiz. To confirm the interaction between G9a and Wiz, we performed co-immunoprecipitation experiments. HEK 293T cells were transfected with vectors expressing EGFP-Wiz and/or FLAG-tagged G9a molecules (FLAG-G9a). Lysates prepared from the transfected cells were incubated with anti-FLAG antibody and the immunoprecipitates subjected to Western blots analyses. Use of anti-FLAG antibodies clearly showed that EGFP-Wiz co-immunoprecipitated with FLAG-tagged G9a (Fig. 1D, lane 4). Similarly, when we immunoprecipitated EGFP-Wiz with anti-GFP, FLAG-tagged G9a was co-precipitated (lane 8). Since in previous studies we have shown that nuclear-targeted EGFP alone does not bind to FLAG-tagged G9a (27.Tachibana M. Ueda J. Fukuda M. Takeda N. Ohta T. Iwanari H. Sakihama T. Kodama T. Hamakubo T. Shinkai Y. Genes Dev. 2005; 19: 815-826Crossref PubMed Scopus (611) Google Scholar), we conclude that G9a can stably associate with Wiz in cells. Wiz Is a Nuclear Protein That Co-localizes with G9a—Next, to examine the sub-cellular localization of Wiz, NIH3T3 cells were transiently transfected with a EGFP-Wiz expression vector and stained with anti-G9a antibody. As shown in Fig. 1E, Wiz (EGFP-Wiz) was detected only in the nucleus but largely excluded from nucleoli (arrowhead) and, importantly, co-localized with G9a. These data further support the association of Wiz with G9a. Endogenous Wiz Is in the G9a/GLP Complex—To confirm that this association exists between endogenously expressed G9a/GLP and Wiz, we generated specific antibody against Wiz. Western blot analysis revealed that our anti-Wiz antibody specifically detected two distinct bands in TT2 mouse ES cells (about 120 and 130 kDa, arrows) and human cell lines (about 125 kDa and 135 kDa in HeLa and HEK 293), as well as the 120-kDa protein (arrowhead) expressed from mWiz cDNA (Fig. 1F, left panel). Furthermore, addition of Wiz siRNA decreased the intensity of signals detected by this antibody in human cells (Fig. 4 and not shown). Therefore, we concluded that our antibody recognizes both mouse and human Wiz proteins. We then performed co-immunoprecipitation experiments using anti-G9a, anti-GLP, and anti-Wiz antibodies. We clearly showed that anti-G9a or anti-GLP antibodies co-immunoprecipitated not only GLP and G9a but also Wiz in TT2 mouse ES cells; in turn, anti-Wiz antibody co-immunoprecipitated G9a and GLP molecules (Fig. 1F, right panel). Furthermore, this G9a-GLP/Wiz interaction was observed in several mouse and human cell lines (Fig. 5C and supplemental Fig. S1). These results indicate that association of endogenous Wiz and the G9a/GLP complex exists widely.FIGURE 5Association of CtBPs with Wiz and the G9a/GLP/Wiz/CtBPs complex formation. A, EGFP-CtBP1 or CtBP2 were co-expressed in HEK 293T cells, either with FLAG-G9a, GLP, or Wiz, specified at the top of the figure. Immunoprecipitates were collected with control anti-Myc antibody or anti-FLAG antibody. FLAG-Wiz, but not FLAG-G9a nor FLAG-GLP, significantly precipitated both CtBP1 and CtBP2. B, Wiz contains two CtBP interaction domains (termed CID) that are located close to ZF3 and ZF4, respectively. For the CID mutation analysis, CID1, CID2, or both were mutated to the ASASA sequence (left panel). FLAG-CtBP2 was co-expressed with Wiz CID mutants, specified at the top of the figure. Immunoprecipitates were collected with control anti-Myc antibody or anti-FLAG antibody. This experiment indicated that Wiz contains two CID domains, and most of the association is carried through CID2 (right panel). C, nuclear extracts from HeLa cells were prepared for precipitation of endogenous Wiz, CtBP1, and CtBP2. Antibodies used for immunoprecipitation are specified at the top of the figure. All of the antibodies used precipitated partner proteins. D, nuclear extracts from mouse ES cells (TT2) were prepared for precipitation of endogenous G9a, GLP, Wiz, CtBP1, and CtBP2. Antibodies used for immunoprecipitation are specified at the top of the figure. Only CtBP2 was detected in the α-G9a/GLP/Wiz triple complexes. IP, immunoprecipitation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition, we determined the homogeneity of the G9a complex described in the legend to Fig. 1A. The purified materials were separated on a 10–30% glycerol gradient by ultracentrifugation and each fraction was analyzed by silver staining (data not shown) and Western blot (Fig. 1G). G9a appears in at least three different forms (fractions 2–4, 5–9, and 12), and both GLP and Wiz co-precipitate with all forms. We next performed sequential immunodepletion analysis of Wiz with anti-GLP antibody (Fig. 1H). Two consecutive immunoprecipitations with anti-GLP resulted in an efficient depletion of not only G9a but also Wiz. From these observations, we conclude that Wiz is a major component of the G9a complex, at least in mouse ES cells. Both G9a and GLP Can Interact with Wiz—To determine the domain that is responsible for the interaction between G9a and Wiz, we constructed several deletion mutants of G9a and performed co-immunoprecipitation assays with Wiz (Fig. 2A and data not shown). Interestingly, Wiz was unable to interact with the G9a mutant missing a SET-domain (G9aΔSET) (Fig. 2A, lanes 3 and 6). Since the SET-domains of G9a and GLP are highly homologous, this suggested that Wiz might also interact with GLP. Co-expression analysis of FLAG-Wiz with EGFP-GLP or EGFP-GLPΔSET in HEK 293T cells revealed that EGFP-GLP, but not EGFP-GLPΔSET, clearly co-immunoprecipitated with FLAG-Wiz (Fig. 2A, lanes 9 and 12). However, another SET-domain-containing molecule, Suv39h1, failed to bind Wiz (lane 15). Therefore, this SET-domain-dependent Wiz interaction appears specific to G9a and GLP. ZF6 of Wiz Is Essential for the Interaction with Both G9a and GLP—We also analyzed which domain of Wiz is important for G9a and GLP association. As shown in Fig. 2B, the last zinc finger motif, ZF6, which has a larger interval between C2 and H2 (Fig. 1B, bottom) and not recognized as the zinc finger motif in the original report (32.Matsumoto K. Ishii N. Yoshida S. Shiosaka S. Wanaka A. Tohyama M. Brain Res. Mol. Brain Res. 1998; 61: 179-189Crossref PubMed Scopus (12) Google Scholar), was required for the interaction between both G9a and GLP (Fig. 2B, lanes 3, 6, 9, and 12). To further evaluate whether the SET-domain of G9a or GLP and the ZF6 of Wiz are directly involved in their interactions, we performed co-immunoprecipitation assays using such domain-only molecules. As shown in Fig. 2C, EGFP-tagged Wiz ZF6 specifically interacted with the FLAG-tagged SET-domains of G9a or GLP, whereas EGFP alone did not associate with either of them (Fig. 2C, lanes 3, 6, 9, and 12). Finally, we introduced two types of point mutations into ZF6 that should impair zinc ion binding (33.Simpson R.J. Cram E.D. Czolij R. Matthews J.M. Crossley M. Mackay J.P. J. Biol. Chem. 2003; 278: 28011-28018Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), designat
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