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Originally published In Press as doi:10.1074/jbc.M307344200 on October 21, 2003

J. Biol. Chem., Vol. 279, Issue 1, 401-406, January 2, 2004
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Polycomb Group Suppressor of Zeste 12 Links Heterochromatin Protein 1{alpha} and Enhancer of Zeste 2*

Ken Yamamoto{ddagger}§, Miki Sonoda{ddagger}, Junichi Inokuchi¶, Senji Shirasawa¶, and Takehiko Sasazuki||**

From the {ddagger}Division of Molecular Population Genetics, Department of Molecular Genetics, Medical Institute of Bioregulation, and Kyushu University Center of Excellence Program on Lifestyle-Related Diseases, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan and the Department of Pathology, ||Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan

Received for publication, July 9, 2003 , and in revised form, October 1, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Drosophila suppressor of zeste 12 (Su(z)12) is a Polycomb group (PcG) transcriptional repressor and is present in E(z)-ESC, a multiprotein complex with methylation activity specific for lysine 9 and 27 of histone H3. Although PcG- and heterochromatin-mediated gene silencing have been considered distinct, mutant flies of Su(z)12 showed not only homeotic transformation but also position effect variegation. We now report that the mammalian SU(Z)12 directly interacts with heterochromatin protein 1{alpha} (HP1{alpha}) and PcG enhancer of zeste 2 (EZH2), the mammalian counterpart of E(z), in vitro and in vivo. Two distinct domains in SU(Z)12 are involved in these interactions, the region between the zinc finger motif and the VEFS (VRN2-EMF2-FIS2-Su(z)12) box for HP1{alpha} (amino acid residues 479–536) and the VEFS box for EZH2 (amino acid residues 600–639), which are not mutually exclusive. Interestingly this region of the VEFS box has been shown to be critical for the phenotype of the Su(z)12 mutant fly. In addition SU(Z)12 represses transcription activity in the presence of HP1{alpha} in a reporter assay. These results provide a molecular explanation for the functional link of these epigenetic silencing processes mediated by Su(z)12.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Heterochromatin is the fraction of the genome that remains condensed throughout the cell cycle, replicates in the late S phase, and is generally associated with pericentric and telomeric regions of chromosomes (1, 2). Euchromatic genes placed into proximity of heterochromatin by chromosomal rearrangement or trans-recruitment will be variably silenced, a phenomenon known as position effect variegation (PEV).1 Variegation is considered to be caused, in part, by spreading of heterochromatin components over euchromatic regions next to heterochromatin (3). Genetic screens in Drosophila have identified dominant suppressors or enhancers of PEV, and an avenue to elucidate the molecular basis of PEV was attained (4, 5). A suppressor of variegation Su(var)2-5 and Su(var)3-9 encode proteins that are associated with heterochromatin. Recent studies revealed that Su(var)3-9 is a histone methyltransferase specific for lysine residue 9 of histone H3 (H3-K9) (6), thus creating a binding site on the nucleosome for Su(var)2-5, heterochromatin protein 1 (HP1) (7, 8). Because Su(var)3-9 directly interacts with HP1, it is postulated that the Su(var)3-9-HP1 system plays an important role in initiation and/or maintenance of heterochromatin (3, 9).

Homeotic genes are spatially regulated during development, and maintenance of the transcriptional state of these genes is required for concerted action of the Polycomb (PcG) and Trithorax group proteins (10, 11). The PcG-mediated gene silencing has often been compared with the heterochromatin-mediated gene silencing because some conserved domains observed in PcG proteins are shared with HP1 (chromodomain) and Su(var)3-9 (chromodomain and Su(var)3–9, E(z) and Trithorax (SET) domain) and also because PcG proteins, like modifiers of PEV, are dosage-sensitive. However, PcG- and heterochromatin-mediated gene silencing require two distinct sets of protein. Su(var) mutants show no PcG phenotypes, and most PcG mutations do not suppress PEV except for enhancer of zeste (E(z)) and suppressor of zeste 12 (Su(z)12) (12, 13). Extra copies of E(z) or its human homologue EZH2 in transgenic flies enhanced PEV, and E(z) mutation weakly suppressed PEV (12). It was reported that the Su(z)12 mutant fly showed not only homeotic transformation but also suppression of PEV (13). These observations suggest that molecular mechanisms underlying heterochromatin- and PcG-mediated gene silencing might, in part, be common; however, it has remained unclear whether effects on PEV of these genes are direct.

PcG proteins are present in multiple protein complexes (10, 11). In Drosophila there are at least two characterized PcG complexes, a 2-MDa Polycomb repressive complex 1 and a 600-kDa or 1-MDa ESC-E(z) complex. The ESC-E(z) complex and its human counterpart, the EED-EZH2 complex, have been well characterized, and Su(z)12/SU(Z)12 was found to be a component of the ESC-E(z)/EED-EZH2 complex (14–18). The purified ESC-E(z)/EED-EZH2 complex contains histone methyltransferase activity specific for H3-K9 and H3-K27, and this enzymatic activity depends on the SET domain of E(z)/EZH2. Evidence for the presence of Su(z)12 and E(z) in the same complex suggests that both are cooperatively involved in heterochromatin-mediated gene silencing as well as in PcG-mediated gene silencing.

In attempts to elucidate molecular mechanisms involved in the dual functions of Su(z)12, we examined interactions between Su(z)12 and HP1 family proteins and found that SU(Z)12 directly interacts with HP1{alpha} in vitro and in vivo. SU(Z)12 showed trans-repression activity in the presence of HP1{alpha} in a reporter assay. In addition, EZH2 was isolated as a SU(Z)12-interacting protein by yeast two-hybrid screening, and direct interaction was confirmed in vitro. The interaction sites were mapped to different regions in SU(Z)12, the region between the zinc finger and the VEFS (VRN2-EMF2-FIS2-Su(z)12) box and the VEFS box for HP1{alpha} and EZH2, respectively. Notably the carboxyl-terminal half of the VEFS box, the region reported to be critical for the phenotype of the Su(z)12 mutant, is essential for the interaction with EZH2. SU(Z)12-HP1{alpha} and SU(Z)12-EZH2 interactions are not mutually exclusive. Taken together, our findings suggest a role for SU(Z)12 in the maintenance of heterochromatin- and/or HP1-mediated transcriptional repression of euchromatic genes by recruiting EZH2 or EED-EZH2 complex onto HP1{alpha}-bound nucleosomes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Plasmid Constructs—Details on individual plasmid constructs, which were all verified by sequencing, are available upon request. Mouse cDNAs of HP1{alpha}/{beta}/{gamma} and EZH2 were cloned by PCR from mouse thymocyte and embryo cDNAs, respectively. Human cDNA of SU(Z)12 was generously provided by Kazusa Research Institute (Kazusa, Saitama, Japan). The 165I/Y168 mutant of mouse HP1{alpha} was as described previously (9). For in vitro binding assays, the indicated cDNAs were fused to GST in pGEX-2T vector (Amersham Biosciences). For in vitro coupled transcription/translation, the indicated cDNAs were fused to HA tag in pGAD-T7 vector (Clontech). Constructs for mammalian transfection were based on pCMV-HA and pCMV-myc (Clontech) to express HA- and Myc-tagged proteins. The cDNA of HP1{alpha} was subcloned into pG4MpolyII (19) (a gift from R. Losson and P. Chambon, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France) for GAL4DBD fusion in a reporter assay. Four repeats of GAL4DBD binding sequence (17m4) were inserted into SacI and BglII sites of pGL3-pro (Promega) to generate a reporter plasmid (pGL3–17m4-pro) for luciferase assay. For yeast two-hybrid screening, the indicated cDNA of SU(Z)12 was fused to GAL4DBD in pGBK-T7 vector (Clontech).

In Vitro Binding Assays—Preparation of GST fusion proteins and GST pull-down experiments were done as described previously (9). In vitro translated proteins were prepared using the TNT quick coupled transcription/translation system (Promega) according to the protocol provided by the manufacturer. In brief, approximately 10 µg of the indicated GST fusion proteins immobilized on glutathione-Sepharose was incubated with 10 µlof in vitro translated proteins in 100 µl of GST pull-down buffer (GPB; 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 10% glycerol, 0.1% Nonidet P-40, 0.5 mM dithiothreitol) for 2 h at 4 °C. The beads were washed three times with GPB, and the bound proteins were eluted with 2x SDS sample buffer by boiling for 10 min. For the peptide competition assay, various amounts of wild type (KVPVVVLEDILAT) or mutant (KVAVAVAEDILAT) peptides derived from the mouse the p150 subunit of chromatin assembly factor 1 (CAF1) (amino acids 220–232) were added to the binding reaction. Proteins were separated by 5–20% gradient SDS-PAGE and transferred onto nitrocellulose membrane, and Western blots were prepared using an anti-HA tag monoclonal antibody (Santa Cruz Biotechnology) and a standard protocol.

Transient Transfection and Co-immunoprecipitation Assay—COS-7 cells were transiently transfected with 10 µg of the indicated plasmids using a standard calcium phosphate method. After 60 h, cells were washed with cold phosphate-buffered saline and harvested in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1.0% Nonidet P-40, 0.5% sodium deoxycholate). MgCl2 and RNase-free DNase I (Roche Applied Science) were added to final concentrations of 5 mM and 100 units/ml, respectively, and cells were incubated for 1 h at 4 °C. After centrifugation at 12,000 rpm for 15 min at 4 °C, the resulting whole cell extracts were transferred into new tubes and incubated with 10 µl of anti-Myc monoclonal antibody-coupled protein G beads (Santa Cruz Biotechnology) for 2 h at 4 °C. The immunoprecipitates were washed three times with cold RIPA buffer, and final pellets were resuspended in 2x SDS sample buffer. The immunoprecipitates were separated by 5–20% gradient SDS-PAGE and transferred onto nitrocellulose membrane, and Western blots were prepared using an anti-HA or anti-Myc tag monoclonal antibody (Santa Cruz Biotechnology) and a standard protocol. To examine the co-immunoprecipitation of endogenous SU(Z)12 with HP1{alpha}, HeLa cells were transfected with 10 µg of pCMV-myc-HP1{alpha} or vector alone, and nuclei were prepared in buffer N as described previously (20) after 60 h of transfection. Nuclei were treated with RNase-free DNase I at 100 units/ml for 1 h at 4 °C, and then NaCl and glycerol were added to a final concentration of 450 mM and 25%, respectively. Nuclei were incubated for 1 h at 4 °C, and nuclear extracts were obtained by centrifugation at 12,000 rpm for 20 min at 4 °C. The extracts were incubated with anti-Myc monoclonal antibody-coupled protein G beads, and the following steps were done as described above but using an anti-SU(Z)12 polyclonal antibody (Upstate Biotechnology) for the Western blots.

Immunofluorescent Staining—Twenty-four hours after co-transfection with HA-tagged HP1{alpha} and Myc-tagged SU(Z)12, COS-7 cells were fixed with 3.7% paraformaldehyde in phosphate-buffered saline for 10 min at room temperature. After blocking with 1.4% milk in phosphate-buffered saline containing 0.1% saponin, cells were incubated with anti-HA antibody (Roche Applied Science) or anti-Myc antibody (Santa Cruz Biotechnology) in phosphate-buffered saline containing 0.1% bovine serum albumin and 0.1% saponin for 1 h at room temperature followed by incubation with Alexa Fluor 488 anti-rat IgG (highly cross-adsorbed) and Alexa Fluor 546 anti-mouse IgG (highly cross-adsorbed) (Molecular Probes) at a dilution of 1:2000 for 1 h at room temperature. For counterstaining of nucleic acid using propidium iodide (Molecular Probes), cells were incubated with 100 µg/ml DNase-free RNase in 2x SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) followed by incubation with a 500 nM solution of propidium iodide for 5 min at 37 °C. Cells were covered with a drop of Gel/Mount (Biomeda) and viewed by confocal laser scanning microscopy (Fluoview FV500).

Luciferase Assay—COS-7 cells were transiently transfected with 0.5 µg of pGL3–17m4-pro reporter, 1 µg of phRL-CMV as an internal control, and the indicated amounts of pG4M-polyII (expressing GAL4DBD), pG4M-polyII-HP1{alpha} (expressing GAL4DBD-HP1{alpha}), pCMV-myc, and pCMV-myc-SU(Z)12. All samples contained the same amount of pG4M-polyII- and pCMV-derived plasmids. Cells were harvested 36 h posttransfection, and preparation of cell extracts and luciferase assay were done using the Dual-Luciferase® reporter assay system (Promega) according to the protocol provided by the manufacturer. The reporter activities were normalized to internal control (Renilla luciferase activities), and relative luciferase activities were expressed relative to the activity in the presence of unfused GAL4DBD expression vector and pCMV.

Yeast Two-hybrid Screening—Yeast two-hybrid screening was done using MATCHMAKER GAL4 Two-Hybrid System 3 kits and pretransformed MATCHMAKER cDNA Library (Clontech) according to the protocol provided by the manufacturer. cDNA corresponding to the amino acid residues 403–739 of SU(Z)12 was fused to the GAL4DBD in pGBK vector (TRP1) as bait and introduced into the yeast AH109 strain containing integrated His3 and Ade2 reporters driven by GAL1- and GAL2-UAS, respectively, by the standard lithium acetate method. A mouse embryonic cDNA library fused to GAL4 transactivating domain in pACT2 vector (LEU2) that was pretransformed in the yeast strain Y187 was used for the screening. Approximately 2 x 108 independent transformants were spread on the culture plate with selection medium (adenine-, His-, Leu-, and Trp-) containing 20 µg/ml of 5-bromo-4-chloro-3-indolyl-{alpha}-D-galactopyranoside (X-{alpha}-gal) for double selection. Positive blue colonies were spread on selection plates, and the plasmids were extracted using YEASTMAKERTM yeast plasmid isolation kits (Clontech). Each plasmid was introduced into Escherichia coli for amplification, purified, and then sequenced to identify the inserted cDNA.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Interaction between SU(Z)12 and HP1 Family Proteins in Vitro and in Vivo—HP1 is an abundant component of heterochromatin, and genetic and biochemical analyses demonstrated that this molecule plays a key role in heterochromatin-mediated gene silencing (3). As HP1 associates with various transcription regulatory proteins, we examined the interactions with SU(Z)12. The mammalian HP1 family is composed of three closely related proteins, HP1{alpha}, HP1{beta}, and HP1{gamma} (21). We introduced Myc-tagged full-length SU(Z)12 into COS-7 cells by transient transfection together with HA-tagged full-length mouse HP1{alpha}, HP1{beta}, or HP1{gamma}, and a co-immunoprecipitation experiment was done. HP1{alpha} but not HP1{beta} or HP1{gamma} was coimmunoprecipitated with SU(Z)12 (Fig. 1A). The association was further examined using endogenous materials. When nuclear extracts were prepared from HeLa cells that were transfected with Myc-tagged HP1{alpha} and immunoprecipitates were analyzed for the presence of endogenous SU(Z)12, it was evident that endogenous SU(Z)12 was co-immunoprecipitated with HP1{alpha} (Fig. 1B). Previous immunostaining studies have shown a spatially distinct localization pattern for each of the HP1s (21). While HP1{alpha} is found in heterochromatin, HP1{beta} localizes to both heterochromatin and euchromatin, and HP1{gamma} localizes mostly to euchromatin. Consistent with the data from the co-immunoprecipitation experiment, our immunostaining showed that SU(Z)12 is a nuclear protein, and its subnuclear distribution pattern closely matches that of HP1{alpha} in transfected cells (Fig. 1C). Thus, an association between SU(Z)12 and HP1{alpha} in cells was revealed.



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  		FIG. 1.
Interaction between SU(Z)12 and HP1 in vivo. A, HP1{alpha}-specific association of SU(Z)12 in transfected cells. Myc-tagged SU(Z)12 was co-transfected with HA-tagged HP1{alpha}, HP1{beta}, or HP1{gamma} into COS-7 cells. Cell extracts were prepared in RIPA buffer in the presence of MgCl2 and RNase-free DNase I and incubated with anti-Myc antibody-coupled protein G beads. The final immunoprecipitates were separated by 5–20% gradient SDS-PAGE. Western blots using an anti-HA or anti-Myc tag monoclonal antibody indicate co-immunoprecipitation of HP1{alpha} with SU(Z)12 (lane 4). B, endogenous SU(Z)12 is associated with transfected HP1{alpha} in HeLa cells. Myc-tagged HP1{alpha} was transfected into HeLa cells, and nuclear extracts were prepared as described under "Experimental Procedures." Nuclear extracts were then incubated with anti-Myc antibody-coupled protein G beads, and the final immunoprecipitates were subjected to Western blot using anti-SU(Z)12 antibody. C, nuclear localization of SU(Z)12 and HP1{alpha} in transiently transfected cells. COS-7 cells were co-transfected with Myc-tagged SU(Z)12 and HA-tagged HP1{alpha}. Anti-Myc and anti-HA double labeling indicates colocalization of SU(Z)12 and HP1{alpha} in large nuclear blocks of transfected COS-7 cells (lower panel, Merged image). IP, immunoprecipitation.

 
To examine direct interactions, SU(Z)12 proteins were produced by in vitro coupled transcription/translation, and GST pull-down experiments were done using GST fusion full-length HP1{alpha}, HP1{beta}, and HP1{gamma} (Fig. 2A). Interestingly full-length SU(Z)12 bound to HP1{alpha} and HP1{gamma} but not to HP1{beta} in vitro. The amount of pulled down SU(Z)12 by HP1{gamma} was higher than that by HP1{alpha}, whereas an almost equal amount of control HP1{alpha} was pulled down. Despite a structural similarity, HP1 family proteins showed a selective molecular interaction with some proteins in vitro. Histone H1 and brahma-related gene I (BRGI) preferentially interact with HP1{alpha} (21, 22), whereas TAFII130 interacts with HP1{alpha} and HP1{gamma} but not with HP1{beta} (23). SU(Z)12 exhibited the same specificity as TAFII130 in vitro (see below).



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  		FIG. 2.
Mapping of interaction sites in SU(Z)12 and HP1{alpha} A, interaction of SU(Z)12 with HP1{alpha} and HP1{gamma} in vitro. In vitro translated HA-tagged full-length SU(Z)12, SU(Z)12-N (amino acids 1–412), SU(Z)12-Z (amino acids 403–536), SU(Z)12-C (amino acids 479–739), and full-length HP1{alpha} (positive control) were tested for interaction with full-length GST-HP1{alpha}, -HP1{beta}, or -HP1{gamma}. A summary of interactions is given in the panel on the right. B, the chromo shadow domain in HP1{alpha} is involved in interaction with SU(Z)12. In vitro translated HA-tagged SU(Z)12-C (amino acids 479–739) was tested for interaction with the indicated portion of GST-HP1{alpha} proteins. The GST pull-down experiments were done as described under "Experimental Procedures." Pulled down SU(Z)12-C was detected in Western blots using an anti-HA tag antibody. These HP1{alpha} proteins include the chromodomain, the hinge region, and/or the chromo shadow domain. The IY165/168EE mutant (HP1{alpha}-4) does not form a dimer structure (9, 31). On the right of the schematic representation of HP1{alpha} constructs is a summary of the result of interactions. C, fine mapping of the HP1{alpha} interaction site in SU(Z)12. In vitro translated HA-tagged SU(Z)12-C1 and SU(Z)12-C2 were tested for interaction with the GST-HP1{alpha} chromo shadow domain (GST-HP1{alpha}-3). The VEFS box is not involved in interaction with HP1{alpha}. Full, full-length.

 
To search for the interaction site in SU(Z)12, three deletion mutants were generated, and in vitro binding assays were done. Because there are two distinct domains in SU(Z)12, a single C2H2 type of zinc finger and a VEFS box whose functions were unclear (13), constructs were designed to determine the domain responsible for the HP1 interaction. As shown in Fig. 2A, two truncated proteins, SU(Z)12-Z (amino acids 403–536) and SU(Z)12-C (amino acids 479–739), definitely bound to HP1{alpha} and HP1{gamma} in vitro, indicating that the 58-amino acid stretch between the zinc finger motif and VEFS box (amino acids 479–536) is responsible for this interaction. The interaction of this small region with HP1{alpha} was confirmed (Fig. 2C). These results suggest that the zinc finger motif and VEFS box may have functional roles other than HP1 interactions.

Self-association of HP1{alpha} Is Required for Direct Interaction with SU(Z)12—The HP1 family proteins are one class of chromodomain proteins containing an amino-terminal chromodomain and a structurally related carboxyl-terminal chromo shadow domain linked by a poorly conserved hinge region, and several lines of evidence implicate these domains in HP1 functions (24). Regarding protein-protein interaction, the chromodomain binds to methylated H3-K9 that is created by histone methyltransferases such as SUV39H1 (7, 8). The hinge region of HP1{alpha} is involved in interactions with histone H1 (21). In contrast to these domains, the chromo shadow domain can complex a variety of proteins including SUV39H1, Su(var)3-7, TIF1-{alpha}, TIF1-{beta}, TAFII130, BRG1, CAF1p150, the nuclear autoantigen SP100, and the inner nuclear membrane protein lamin B receptor (9, 22, 23, 25–30).

To identify the domains in HP1{alpha} responsible for direct interaction with SU(Z)12, several mutants of HP1{alpha} were generated as GST fusions, and GST pull-down experiments were done using in vitro translated SU(Z)12-C (Fig. 2B). Similar to most of the HP1-interacting proteins, SU(Z)12 interacts with the chromo shadow domain of HP1{alpha}. In addition, the IY165/168EE mutation in this domain, which abrogates dimerization of HP1{alpha} (9, 31), did not interact with SU(Z)12, hence self-interaction of HP1{alpha} is required for the interaction. Most proteins interacting with HP1 via this domain are characterized by conservation of a pentapeptide consensus sequence, PXVXL (32), and peptide phage display experiments revealed that peptides of (P/L)(W/R/Y)V(M/I/L)(M/L/V) sequence can bind the chromo shadow domain (33). In addition, the dimer formation via chromo shadow domains creates a molecular surface to which the pentapeptide motif can bind. Indeed a peptide containing this motif competes for interactions between HP1 and CAF1, TIF-1{beta}, and SUV39H1 (9, 31). To determine whether interaction of SU(Z)12 with HP1{alpha} is in the same mode of protein-protein interaction as these proteins, a peptide competition assay for in vitro binding was done (Fig. 3). The direct interaction of SU(Z)12 was competed for by increasing the amount of the wild type CAF1p150-derived 13-mer peptide KVPVVVLEDILAT (consensus sequence is in bold) but not the mutant peptide KVAVAVAEDILAT where Pro, Val, and Val of the consensus are substituted for Ala (underlined). This result indicates that SU(Z)12 shares an interaction site formed by chromo shadow domain-mediated dimerization in HP1{alpha} with other HP1{alpha}-interacting proteins that contain the pentapeptide consensus. Differing from data obtained in previous studies, the pentapeptide consensus is absent in the HP1{alpha}-interacting site in SU(Z)12. We reported that the amino-terminal 39-amino acid stretch of SUV39H1 is sufficient for direct interaction with the chromo shadow domain of HP1{alpha}, which is also inhibited by the CAF1p150-derived peptide used in this study (9). The pentapeptide consensus is not found in this sequence of SUV39H1 as in the case of SU(Z)12, hence the binding sequence to dimerized HP1 chromo shadow domain is varied.



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  		FIG. 3.
Dimer surface formed by chromo shadow domain of HP1{alpha} is required for direct binding to SU(Z)12. Amino acid sequence of mouse CAF1p150-derived 13-mer peptide containing the conserved pentapeptide sequence PXVXL is indicated. Mutation of proline 220, valine 224, and leucine 226 to alanines are underlined. The interaction between in vitro translated HA-tagged SU(Z)12-C and GSTHP1{alpha} chromo shadow domain (GST-HP1{alpha}-3) was examined in the presence of increasing amounts of wild type or mutant peptide competitor. The result indicates that the CAF1p150-derived peptide containing the pentapeptide consensus competed for interaction between SU(Z)12 and HP1{alpha}.

 
The molecular basis of selective interaction of SU(Z)12 with HP1 family proteins in vitro is not clear. Histone H1 specifically interacts with HP1{alpha} via a poorly conserved hinge region (21). BRG1 also exhibits HP1{alpha}-specific interaction, and the chromo shadow domain is involved (22). However, the amino acid residues in the chromo shadow domain critical for this interaction are not conserved among HP1 family, and mutation of these residues does not affect dimer formation. TAFII130 exhibits selective interaction with HP1{alpha} and HP1{gamma} as does SU(Z)12 in vitro (23). TAFII130 contains a pentapeptide consensus and interacts with the chromo shadow domain of HP1. It might be that amino acid residues located on the dimer surface formed by the chromo shadow domain, which are conserved between HP1{alpha} and HP1{gamma} but not HP1{beta}, are critical for interaction with SU(Z)12 and TAFII130.

SU(Z)12 Represses Transcriptional Activity in the Presence of HP1{alpha}—The PcG and heterochromatin proteins are known to be involved in transcriptional repression. To investigate whether SU(Z)12 represses transcription via interacting with HP1, we measured transcriptional activity of SU(Z)12 in the presence of HP1 by transient transfection and a luciferase assay in COS-7 cells. The coding sequence of HP1{alpha} was fused to GAL4DBD, and reporter plasmid containing four GAL4DBD-binding sites upstream of SV40 promoter was constructed (Fig. 4A). We initially examined the effects of GAL4DBD-HP1{alpha} on reporter activity and found that HP1{alpha} repressed transcription in a dosedependent manner as described before (data not shown) (32). Various amounts of SU(Z)12 were co-transfected with GAL4DBD or GAL4DBD-HP1{alpha} in a condition where GAL4DBD-HP1{alpha} moderately repressed reporter activity (0.2 µg). In the absence of GAL4DBD-HP1{alpha}, SU(Z)12 did not show any effects on transcription activity of reporter gene (Fig. 4B, lanes 1–3). The GAL4DBD-HP1{alpha} repressed luciferase activity to ~43% (compare lanes 1 and 4). When SU(Z)12 was expressed together with GAL4DBD-HP1{alpha}, it was evident that the levels of repression were enhanced in a dose-dependent manner (compare lane 4 (43%), lane 5 (27%), and lane 6 (15%)). Thus, these results suggest that SU(Z)12 can repress transcription when targeted to a reporter gene by HP1{alpha}. We also examined whether SU(Z)12 represses transcription when directly targeted to a reporter by GAL4DBD fusion; however, it did not show any effect (data not shown). It might be possible that the GAL4 fusion masked a portion of SU(Z)12 that is involved in repression function and could not repress the SV40 promoter or that SU(Z)12 cooperates with other factors that are recruited to the promoter by HP1{alpha} in the repression of reporter activity.



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  		FIG. 4.
Transcriptional repression by SU(Z)12 in the presence of HP1{alpha} A, schematic representation of the luciferase reporter construct containing four repeats of GAL4DBD-binding sites and SV40 promoter, which is based on pGL3-pro (Promega). B, effects of SU(Z)12 expression on reporter gene activity. COS-7 cells were transfected with 0.5 µg of pGL3–17m4-pro, 0.1 µg of phRL-CMV, and 0.2 µg of pG4M-polyII (expressing GAL4DBD, lanes 1–3) or pG4M-polyII-HP1{alpha} (expressing GAL4DBD-HP1{alpha}, lanes 4–6) in combination with the indicated amount of expression vector for SU(Z)12 and empty vector. Transfection efficiencies were normalized to internal control (Renilla luciferase activities), and relative luciferase activities were expressed relative to the activity in the presence of unfused GAL4DBD expression vector and pCMV (lane 1). The data represent the average and S.D. of three independent experiments

 
SU(Z)12 Interacts with EZH2 in Vitro and in Vivo—To gain further insight into the interaction between SU(Z)12 and HP1{alpha}, proteins that might be recruited to HP1{alpha}-bound nucleosomes via interacting with SU(Z)12 were searched for by doing yeast two-hybrid screening. cDNA corresponding to amino acid residues 403–739 of SU(Z)12 was fused to the GAL4 DNA binding domain and served as bait (Fig. 5A). This region was selected because it contains two functionally unknown structures, a zinc finger motif and a VEFS box, that could be a module of protein-protein interactions. A mouse embryonic library was screened, and two clones encoding amino acid residues 238–746 of mouse EZH2 were isolated (Fig. 5A). The interaction between SU(Z)12 and EZH2 in mammalian cells was confirmed in co-immunoprecipitation experiments using transiently transfected COS-7 cells with Myc-tagged fulllength SU(Z)12 and HA-tagged full-length mouse EZH2 (Fig. 5B). To identify the interaction site in SU(Z)12 with EZH2, deletion mutants of SU(Z)12 were generated as GST fusion proteins, and in vitro binding assays were done (Fig. 5C). We found that EZH2 interacts with the VEFS box of SU(Z)12 (lanes 4, 5, and 7), the region distinct from the HP1{alpha} interaction site. In addition, the region of amino acid residues 600–639 in the VEFS box was found to be critical for this interaction (compare lanes 5 and 6). An isoleucine at position 600 in the VEFS box (Fig. 5C, upper panel, black triangle) is conserved between Drosophila and human, and a previous genetic study reported that the mutant fly showing suppression of PEV has a P-element insertion into this position (13). Therefore, the possible molecular mechanism of the phenotype of this strain would be that the truncated Su(z) protein, which can interact with HP1{alpha} or other targets but not with E(z), competes with wild-type Su(z)12 protein for interacting with HP1{alpha} or other targets, but it does not function.



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  		FIG. 5.
SU(Z)12 directly interacts with EZH2. A, schematic representation of bait for two-hybrid screening in yeast and an isolated clone encoding truncated mouse EZH2 from the embryonic cDNA library. B, association between SU(Z)12 and EZH2 in vivo. Myc-tagged SU(Z)12 was co-transfected with HA-tagged EZH2 into COS-7 cells. Cell extracts were incubated with anti-Myc antibody-coupled protein G beads, and the final immunoprecipitates were separated by 5–20% gradient SDS-PAGE. Western blot using an anti-HA tag antibody indicates co-immunoprecipitation of EZH2 with SU(Z)12 (lane 4). C, mapping of interaction site with EZH2 in SU(Z)12. In vitro translated HA-tagged full-length EZH2 was tested for interaction with the indicated portion of GST-SU(Z)12 proteins. The black triangle shown in the VEFS box of SU(Z)12 of the construct scheme in the upper panel indicates an isoleucine residue at position 600 where the insertion of a P-element was observed in a mutant strain of Su(z)12 of Drosophila (13). GST pull-down experiments were done as described under "Experimental Procedures." Pulled down EZH2 was detected by Western blot using an anti-HA tag antibody. On the right of a schematic representation of SU(Z)12 constructs is a summary of interactions. The carboxylterminal half of the VEFS box is critical for the interaction. IP, immunoprecipitation.

 
Because the binding sites in SU(Z)12 for HP1{alpha} and EZH2 were mapped to distinct regions, it was expected that SU(Z)12 can link between HP1{alpha} and EZH2 by physical interactions. To examine this possibility, full-length HA-HP1{alpha}, HA-SU(Z)12, and Myc-EZH2 were co-expressed in COS-7 cells, and co-immunoprecipitation experiments were done using an anti-Myc antibody. As shown in Fig. 6A, both SU(Z)12 and HP1{alpha} were co-immunoprecipitated with EZH2 (lane 5). In contrast, HP1{alpha} was not coimmunoprecipitated with EZH2 in the absence of SU(Z)12 (lane 3). Interaction between HP1{alpha} and SU(Z)12 was nil in the GST pull-down experiment using full-length GST-HP1{alpha} and in vitro translated full-length EZH2 (data not shown). Thus, HP1{alpha}-SU(Z)12 and SU(Z)12-EZH2 interactions are not mutually exclusive, and SU(Z)12 may recruit EZH2 to HP1{alpha}-bound nucleosomes (Fig. 6B).



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  		FIG. 6.
Association of SU(Z)12 with HP1{alpha} and EZH2 in vivo. A, interaction of SU(Z)12 with HP1{alpha} and EZH2 is not mutually exclusive. Myc-tagged EZH2, HA-tagged SU(Z)12, and HP1{alpha} were co-expressed in COS-7 cells. Cell extracts were prepared in RIPA buffer in the presence of MgCl2 and RNase-free DNase I and incubated with anti-Myc antibody-coupled protein G beads. The final immunoprecipitates were separated by 5–20% gradient SDS-PAGE. Western blots using an anti-HA monoclonal antibody indicate co-immunoprecipitation of HP1{alpha} with EZH2 in the presence of SU(Z)12 (lane 5). B, schematic representation of the interaction sites in SU(Z)12 for HP1{alpha} and EZH2. The region between the zinc finger motif and the VEFS box and the VEFS box in SU(Z)12 is required for interaction with HP1{alpha} and EZH2, respectively. IP, immunoprecipitation.

 
The results presented here implicate each of SU(Z)12 and EZH2 in heterochromatin-mediated gene silencing. SU(Z)12 may bridge EZH2 histone methyltransferase to the HP1-coated nucleosomes to methylate H3-K9, thus providing a binding site for HP1 as in the SUV39H1-HP1 system. We do not know whether these systems additively or exclusively function in heterochromatin maintenance in cell. According to a study analyzing Suv39h-deficient cells, H3-K9 methylation at pericentric heterochromatin specifically depends on this histone methyltransferase (34), hence the HP1-SU(Z)12-EZH2 system may function at a non-pericentric heterochromatin. Alternatively this system may function at a particular stage during cell differentiation because expression of EZH2 is up-regulated in lymphocytes in a proliferation status (35), and EZH2 deficiency is embryonically lethal in mice at an early stage of development (36), whereas the Suv39h-deficient mouse fetus has a normal development until day 12.5 (34). The results presented here also suggest that HP1-SU(Z)12-EZH2 might be involved in the euchromatic gene repression mediated by HP1 (37, 38).


   FOOTNOTES
 
* This work was supported by a grant-in-aid for scientific research (C) from the Japan Society for the Promotion of Science, by a grant-in-aid for scientific research on priority areas "Genome Biology," and by a grant from the 21st Center of Excellence Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** Director-General of Research Institute, International Medical Center of Japan. Back

§ To whom correspondence should be addressed. Fax: 81-92-642-4614; E-mail: kyama{at}bioreg.kyushu-u.ac.jp.

1 The abbreviations used are: PEV, position effect variegation; EZH2, enhancer of zeste 2; GAL4DBD, Gal4 DNA binding domain; GST, glutathione S-transferase; HA, hemagglutinin; HP1, heterochromatin protein 1; PcG, Polycomb group; Su(var), suppressor of variegation; SU(Z)12, suppressor of zeste 12; VEFS, VRN2-EMF2-FIS2-Su(z)12; CAF1, chromatin assembly factor 1; SET, Su(var)3-9-E(z)-Trithorax; ESC, extra sex combs; EED, embryonic ectoderm development; TIF, transcriptional intermediatory factor; TAFII130, TATA-binding protein-associated factor, RNA polymerase II, 130 kDa; SUV39H1, suppressor of variegation 3–9 homolog 1. Back


   ACKNOWLEDGMENTS
 
We thank R. Losson and P. Chambon for a gift of plasmid pG4M-polyII, S. Ohishi and J. Nakai for technical assistance, N. Furuno for manuscript preparation, and M. Ohara for comments.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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