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Genes to Cells (2007) 12, 877-888. doi:10.1111/j.1365-2443.2007.01098.x
© 2007 Blackwell Publishing or its licensors
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Identification of ZNF200 as a novel binding partner of histone H3 methyltransferase G9a

 Miki Nishida1,2, Masaki Kato1,3,a, Yasuko Kato1,2, Nobuhiro Sasai1,3, Jun Ueda4, Makoto Tachibana4, Yoichi Shinkai4 and Masamitsu Yamaguchi1,2,*

1 Department of Applied Biology, and
2 Insect Biomedical Research Center, and
3 Venture Laboratory, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
4 Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
G9a belongs to the subfamily of histone H3 lysine 9 (H3-K9)-specific methyltransferases. On amino acid sequence alignment of human and Drosophila G9a, we found that the N-terminal region from amino acids 532–605 to be evolutionarily conserved and named this the G9a homology domain (GHD). Using the GHD of human G9a (hG9a) as a bait, we isolated cDNA encoding a zinc finger protein 200 (ZNF200), which contains five C2H2-type zinc finger domains in tandem arrays. Interaction between G9a and ZNF200 could be demonstrated by in vitro binding assays and immunoprecipitation experiments using cultured human HEK293 cell extracts. GST pull-down assays using deletion derivatives of ZNF200 revealed that the interaction is through a region encompassing three of the five zinc finger domains. Furthermore, ZNF200 appear to co-localize with G9a in the nucleoplasm of HEK293 cells as discrete speckles. These results demonstrate that ZNF200 is a novel binding partner of G9a.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Many lines of evidence indicate that post-translational modifications of histone tails, including acetylation, phosphorylation and methylation, play important roles in regulating replication, transcription and chromatin structure (Grunstein 1997; Wei et al. 1999; Zhang & Reinberg 2001; Aggarwal & Calvi 2004). It is reported that methylation of histone H3 is linked to distinct effects on transcription, depending on the enzyme and modified residues (Chen et al. 1999; Wang et al. 2001; Nishioka et al. 2002). One of the histone methyltransferases (HMTases), SUV39H1, the human homologue of Drosophila Su(var)3-9, has been reported to specifically methylate histone H3 at the lysine 9 (H3-K9) residue (Rea et al. 2000). The catalytic domain of SUV39H1 corresponds to the SET domain that is an evolutionarily conserved sequence motif in chromatin proteins which is involved in epigenetic regulation (Jenuwein et al. 1998). The methylation of H3-K9 appears to be recognized by the heterochromatin protein1 (HP1) that is directly associated with DNA methylation and gene silencing (Bannister et al. 2001; Lachner et al. 2001). SUV39H1 is enriched in heterochromatin (Melcher et al. 2000), and appears to play a role in its formation and maintenance in the pericentric region (Peters et al. 2001). G9a and G9a-like protein (GLP), a euchromatic histone methyltrasferase1 are other SET domain-containing proteins carrying HMTase activity (Tachibana et al. 2001; Ogawa et al. 2002), but with functions distinct from SUV39H1. G9a forms a heteromeric complex with GLP in cells and their localization is mostly outside of the pericentric heterochromatin region in the nucleus (Tachibana et al. 2001, 2005). It has also been reported that both G9a and GLP are crucial for methylation of histone H3-K9 in euchromatin and also for mouse early embryogenesis (Tachibana et al. 2002, 2005). Although an involvement of G9a in transcriptional repression has been shown (Tachibana et al. 2002), it has yet to be clarified how G9a/GLP complexes are recruited to target gene promoters. To understand the molecular mechanisms, identification and characterization of novel proteins associated with G9a and/or GLP are necessary.

Here, we found an evolutionarily conserved region (amino acids 532–605) in the N-terminus of G9a, shared with GLP, and named it the G9a homology domain (GHD). In the present study, G9a-interacting proteins were screened by the yeast two-hybrid system using GHD of human G9a (hG9a) as the bait and the zinc finger protein 200 (ZNF200) was identified as an interactant. This protein was found to contain five C2H2-type zinc finger domains in tandem arrays, indicating that it is a member of Krüppel-type family (Bellefroid et al. 1991). The relevance of the observed interaction is supported by the observation that hG9a and ZNF200 at least partially co-localize, in the nucleoplasm. These results suggest that hG9a complexes may be recruited to specific chromosomal loci via interaction between GHD of hG9a and zinc finger domains of ZNF200.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Sequence conservation of Drosophila and hG9a-related proteins

G9a is a lysine-preferring HMTase, first identified in mammals (Tachibana et al. 2001). Based on sequence information, the Drosophila homologue (Drosophila G9a; dG9a) of hG9a was identified. The Flybase gene code is CG2995 with the accession number of FBgn0040372. Sequence comparisons of the 1637 amino acids of dG9a and hG9a indicated overall identities of 28% (Fig. 1A). Human GLP (hGLP) is another H3-K9-specific HMTase (Ogawa et al. 2002). Sequence comparison of the 1267 amino acids of hGLP and hG9a indicate overall identities of 48% (Fig. 1A). Human G9a contains a SET domain flanked by two cysteine-rich regions in its C-terminal region known as pre-SET and post-SET (Fig. 1A). Recent studies suggest that the SET domain requires combination with these two adjacent cysteine-rich regions to exert HMTase activity (Rea et al. 2000). Drosophila G9a and hGLP are devoid of the post-SET domain, but characteristic cysteine residues are conserved.


Figure 1
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  		Figure 1  Sequence comparison of Drosophila and human G9a related proteins. (A) Schematic representation of the relationship between human GLP (hGLP), human G9a (hG9a) and Drosophila G9a (dG9a). The SET domain and cysteine rich regions known as pre-SET and post-SET are shown. The putative HMTase catalytic core region is indicated by scattered dots. The region containing six contiguous ankyrin repeats located upstream of the putative HMTase core region is also shown, along with the GHD (G9a homology domain). (B) Sequence alignment of the GHD among hGLP, hG9a and dG9a using CLUSTALW. Conserved residues are shown in gray, and conserved cysteine residues are marked by arrowheads. The bait used for the two-hybrid screen is the region of hG9a amino acids 454–548 encompassing the GHD.

 
Alignment of all three proteins revealed four highly conserved regions. The most highly related is the 109 amino acid SET domain (54% identity). The N-terminal 99 amino acids of the pre-SET domain show 32% identity. Upstream of the putative catalytic region are 199 amino acid ankyrin repeats (29% identity). The N-terminal 74 amino acid regions of the three proteins are also highly conserved (34% identity), although their functions are undefined. The region was therefore named the GHD (Fig. 1A), containing characteristic cysteine residues, displaying low homology to a RING finger motif (Freemont et al. 1991) (Fig. 1B), and possibly correspond to a protein-specific interaction motif.

Identification of ZNF200 as a protein interacting with hG9a

G9a belongs to the subfamily of HMTases with specificity for lysine 9 of histone H3 (Tachibana et al. 2001). H3-K9 methylation is associated with gene silencing and linked to the formation of heterochromatin (Bannister et al. 2001; Lachner et al. 2001). To generate further insight into the molecular mechanisms of the histone methylation-associated repression, we screened for hG9a-interacting proteins by yeast two-hybrid system using the N-terminal fragment of hG9a (amino acids 454–548, denoted hG9a454–548) contained in GHD as a bait (Fig. 1B). A plasmid library of fusions between the coding sequence for the GAL4 activation domain and cDNAs from human fetal brain cells was used as prey. Screening of 4.56 x 105 clones yielded a cDNA encoding a 238 amino acid protein.

A BLAST search revealed that the isolated cDNA corresponds to a part of human cDNA MGC: 45 293 (accession number BC032575). This cDNA encodes a ZNF200, whose function is yet unknown (Deng et al. 1998). As shown in Fig. 2, the ZNF200 protein contains five C2H2-type zinc finger domains in tandem arrays at its C-terminal region (Fig. 2B), indicating that the protein is a member of the Krüppel-type family (Schuh et al. 1986), which was originally identified with reference to the Xenopus laevis basal transcription factor TFIIIA and Drosophila melanogaster gap gene product Krüppel (Bellefroid et al. 1991). Usually located at the C-terminal region, the C2H2 motif contains two cysteines in an anti-parallel ß-sheet and two histidines in an {alpha}-helix. These four conserved amino acids tetrahedrally bind a zinc ion to form a globular domain (Pabo & Sauer 1992). The classical Krüppel-type C2H2 motif is 21 amino acids long and approximates the form CX2CX3FX8HX3H, where X represents any amino acid (Schuh et al. 1986). The portion of ZNF200 recovered in the yeast two-hybrid screen starts at amino acid residue 157 (pACT2-ZNF200157–395). Although ZNF200 was isolated by two-hybrid screen using GHD as a bait, its orthologues in other species such as mouse and Drosophila have not been identified on the genome database.


Figure 2
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  		Figure 2  Primary structure of ZNF200. (A) Amino acid sequence of human ZNF200. The portion of ZNF200 recovered in a yeast two-hybrid screen starts at amino acid residue 157. Numbers are shown for the amino acid sequence. (B) Alignment of the sequences of the zinc finger domains. ZNF200 carries five C2H2 zinc fingers (ZF1–ZF5) conforming to the C2H2 consensus, (Y/F)XCX2CG(K/R)XFX8HX3H, in which X represents any amino acid.

 
To demonstrate that interactions between hG9a and ZNF200 detected in the two-hybrid screen are specific, pACT2-ZNF200157–395 was reintroduced into AH109 yeast cells with pGBKT7-hG9a454–548. Yeast cells transformed with these plasmids grew in selective medium, indicating that the interaction between ZNF200 and hG9a actually occurs in yeast cells (Fig. 3A). Since hG9a and hGLP show 48% identity in overall amino acid sequences, the interaction between hGLP and ZNF200 was also examined by two hybrid assay. Since yeast cells co-transformed with pACT2-ZNF200157–395 and pGBKT7-hGLP522–612, grew in selective medium (Fig. 3C), the interaction between ZNF200 and hGLP in yeast cells was confirmed.


Figure 3
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  		Figure 3  ZNF200 interacts with hG9a in yeast cells. In the yeast two-hybrid assay, bait (empty pGBKT7, pGBKT7-hG9a454–548 or pGBKT7-hGLP522–612) and target (empty pACT2 or pACT2-ZNF157–395) plasmids were co-transformed into AH109 S. cerevisiae. Colonies were isolated and their ability to grow in the absence (A, C) or presence (B, D) of histidine and adenine was assessed. Human p53 and the Simian Virus 40 large T antigen were used as a positive control (A and B).

 
Specificity of anti-ZNF200 antibody

We have produced polyclonal anti-ZNF200 antibody and its specificity was evaluated by Western immunoblot analysis (Fig. 4A). With the anti-ZNF200 antibody, 50 kDa bands were detected on Western blots of extracts from HEK293 cells transfected with or without the EGFP-expression plasmids (Fig. 4A, lanes 1–3). Based on amino acid sequence, ZNF200 would be expected to migrate at 45 534 Da that is in the similar range of the observed 50 kDa immuno-reactive band. Both 73 and 50 kDa bands were detected in the extracts of HEK293 cells transfected with EGFP-ZNF200-expression plasmids (Fig. 4A, lane 3). The 73 kDa band also reacted with anti-GFP antibody, indicating that it represents exogenously expressed EGFP-ZNF200 (Fig. 4A, lane 6). The 27 kDa GFP protein was detected in extracts from cells transfected with the EGFP-expression plasmids on the same blot (Fig. 4A, lane 5). These results indicate that the anti-ZNF200 antibody can specifically detect the ZNF200 protein in the HEK293 cell extracts.


Figure 4
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  		Figure 4  ZNF200 associates with hG9a in vivo. (A) Western immunoblot analysis. Cell extracts from HEK293 cells transfected with pEGFP-C2 (lanes 2, 5 and 8) or pEGFP-ZNF200 (lanes 3, 6 and 9), or without plasmids (lanes 1, 4 and 7) were immunoblotted. Anti-ZNF200 antibody (lanes 1–3) or anti-GFP antibody (lanes 4 to 5) was used as the primary antibody. Equal loading was confirmed by Western blot with anti-{alpha}–tubulin antibody (lanes 7–9). (B) Immunostaining of HEK293 cells with anti-ZNF200 antibody. Immunostaining analysis of HEK293 cells transiently expressing EGFP-ZNF200 fusion protein (EGFP-ZNF200). DNA was stained with DAPI (a). The images of EGFP fusion proteins (b) and anti-ZNF200 staining signals (c) are shown as green and red signals, respectively, overlaid in panel (d). (C) HEK293 cells were co-transfected with plasmids expressing FLAGHA-tagged hG9a (FLAG-hG9a) and EGFP-fused ZNF200 (EGFP-ZNF200) (+, top of each lane). Proteins were extracted, immunoprecipitated with anti-FLAG antibodies (lanes 5–8 and 13–16) and subjected to immunoblot analysis with anti-FLAG (lanes 5–8) or anti-GFP (lanes 13–16) antibodies. Input, cell lysates before immunoprecipitation; IP: FLAG, immunoprecipitaion with anti-FLAG antibodies. Antibodies used for the immunoblot are shown on the left. Apparent positions of FLAG-hG9a (180 kDa) and EGFP-ZNF200 (73 kDa) are indicated by arrows on the right. (D) Co-immunoprecipitation of the endogenous human G9a and ZNF200 in cell extracts from HEK293 cells. Proteins were extracted from cells and immunoprecipitated with anti-ZNF200 antibody (lanes 3 and 5) or normal rabbit IgG (control IgG, lanes 2 and 4). Immune complexes were subjected to immunoblot analysis with anti-G9a (lanes 1–3) or anti-ZNF200 antibody (lanes 4 and 5). Input, 1 % of cell lysates before immunoprecipitation.

 
To further confirm specificity of the anti-ZNF200 antibody, HEK293 cells transfected with EGFP-ZNF200-expression plasmids were doubly immunostained with anti-GFP antibody and anti-ZNF200 antibody. Both antibodies exclusively stained nuclei (Fig. 4B), although anti-ZNF200 antibody could not detect the endogenous ZNF200 probably due to low level of the protein.

Association of ZNF200 with hG9a in vivo

To examine whether ZNF200 can associate with hG9a in vivo, HEK293 cells were transfected with expression plasmids for FLAGHA-tagged full-length hG9a (pFLAG-hG9a) and/or the full-length ZNF200 fused with EGFP (pEGFP-ZNF200). Whole-cell lysates were prepared and the immunoprecipitation was carried out with anti-FLAG antibodies, then the immunoprecipitates were analyzed for the presence of EGFP-ZNF200 fusion protein by immunoblot analysis with anti-GFP antibodies. Expectedly, FLAG-hG9a was detected with anti-FLAG antibodies in the immunoprecipitates from cells transfected with pFLAG-hG9a (Fig. 4C, lanes 6 and 8). EGFP-ZNF200 was detected with anti-GFP antibodies in the immunoprecipitates only when both FLAG-hG9a and EGFP-ZNF200 were co-expressed (Fig. 4C, lane 16). These results indicate that ZNF200 can associate with hG9a in human cells.

To further examine association of endogenous hG9a and ZNF200 proteins, cell extracts were immunoprecipitated with anti-ZNF200 antibody and then the immunoprecipitates were analyzed for the presence of hG9a by immunoblot analysis with anti-G9a antibody (Fig. 4D). The 180 kDa hG9a band in addition to the 50 kDa ZNF200 band was detected in the immunoprecipitate with the anti-ZNF200 antibody (Fig. 4D, lanes 3 and 5), but not with the control IgG (Fig. 4D, lanes 2 and 4). These results clearly indicate the association of ZNF200 with hG9a in vivo.

Subcellular localization

To further characterize the interaction between hG9a and ZNF200 in intact cells, we examined the subcellular localization of hG9a and ZNF200. Since anti-ZNF200 antibody cannot detect the endogenous ZNF200 in the immunostaining analysis of cells, we examined the subcellular localization of exogenously expressed hG9a and ZNF200 in human cells. Expression plasmids for FLAGHA-hG9a and EGFP-ZNF200 were co-introduced into HEK293 cells. In the interphase nuclei, both ZNF200 (green signals) and hG9a (red signals) was found to be localized in the nucleoplasm as discrete clear speckles (Fig. 5A,B). The subcellular localization of hG9a is consistent with a previous reports for EGFP-hG9a (Tachibana et al. 2001). When the two images were merged, overlapping foci appeared as yellow dots (Fig. 5D). These results suggest that ZNF200 and hG9a co-localize in most areas of nuclei.


Figure 5
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  		Figure 5  Nuclear localization of transiently expressed FLAGHA-tagged hG9a and EGFP-tagged ZNF200 in the HEK293 cells. HEK293 cells were co-transfected with plasmids expressing FLAGHA-tagged hG9a (HA-hG9a) and EGFP-fused ZNF200 (EGFP-ZNF200). The images of EGFP fusion proteins (A) and HA fusion proteins (B) are shown as green and red signals, respectively. (C) DNA from interphase cells stained with 4, 6-diamidino-2-phenylindole (DAPI). Three images are overlaid in panel (D) (merge). The images of EGFP fusion proteins (E) and HA fusion proteins (F) are shown as green and red signals, respectively. (G) DNA (blue) from metaphase cells stained with DAPI. Three images are overlaid in panel H.

 
DAPI-staining of DNA allowed us to identify mitotic chromosomes (Fig. 5G). Interestingly, ZNF200 (green) and hG9a (red) in HEK293 cells during metaphase were dissociated from the condensed mitotic chromosomes stained with DAPI (blue) (Fig. 5E,F). When the three images were merged, regions overlapped with green and red signals were painted yellow, but blue signals of DAPI alone were still observed (Fig. 5H). These results suggest that ZNF200 and hG9a co-localize outside of the mitotic chromosomes at M phase.

At least two of the five zinc finger domains of ZNF200 are required for the interaction with hG9a

To map the region in ZNF200 responsible for binding to hG9a, we performed in vitro protein–protein interaction assays using GST fusion proteins. Five deletion derivatives of ZNF200 were produced and examined for their ability to interact with hG9a (Fig. 6A). HEK293 cells were transfected with pFLAG-hG9a DNA and cell extracts were prepared and subjected to GST pull-down assay using the GST-ZNF200 fusion protein or deletion derivatives. Deletion of the N-terminal region of ZNF200, generating ZNF157–395, exerted no effect on the interaction with hG9a (Fig. 6B, lane 9). Thus, the C-terminal region of ZNF200 that contains the five zinc fingers (ZF1–ZF5) is sufficient for effective interaction with hG9a. The C-terminal deletion derivative ZNF3–363, in which the C-terminal region corresponding to the ZF5 was deleted, effectively interacted with hG9a (Fig. 6B, lane 6), and another derivative ZNF3–335 carrying ZF1–ZF3 still retained the ability to interact with hG9a (Fig. 6B, lane 7). However, further deletion, generating ZNF3–279, in which ZF2–ZF5 were lacking, resulted in abrogation of the interaction with hG9a (Fig. 6B, lane 8). These results indicate that the C-terminal 60 amino acids of ZNF200, containing ZF4 and ZF5, are dispensable for the interaction with hG9a, with the minimal functional domain stretching from amino acids 157 to 335, a region containing ZF1–ZF3.


Figure 6
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  		Figure 6  Mapping of the hG9a-binding region of ZNF200. (A) Bacterially expressed GST fusion proteins, named as GST-ZNF3–395 (ZNF3–395), GST-ZNF3–363 (ZNF3–363), GST-ZNF3–335 (ZNF3–335), GST-ZNF3–279 (ZNF3–279), GST-ZNF157–395 (ZNF157–395). The gray boxes denote the zinc finger domains (ZF1–ZF5). On the right (Binding), the ability to interact with hG9a is shown. (B) HEK293 cells were transfected with plasmids expressing FLAG-tagged hG9a (FLAG-hG9a), then proteins were extracted, subjected to GST pull-downs with GST-ZNF200 or its deletion derivatives and probed for FLAG-hG9a by immunoblotting. Top, GST pull-down assay; bottom, SDS-PAGE analysis of bacterially expressed and purified proteins; *, bands corresponding to intact GST-fusion proteins from each construct. Top of each lane, fusion proteins used in the assay are shown. GST, negative control (GST without a fusion protein); Mock, mock-transfected cell lysates; Input, cell lysates before immunoprecipitation (5% of that used in pull-downs). The 133 kDa FLAG-hG9a and GST bands are indicated by arrows on the right.

 
Possible involvement of ZNF200 in transcriptional regulation was examined using a reporter assay with a human DNA polymerase ß promoter carrying the multiple GAL4-binding sites (Sekimata et al. 2001). Expression of GAL4 DNA-binding domain (GAL4-DBD) fused with full-length mouse G9a strongly repressed transcription of the reporter gene as reported previously (Fig. 7B) (Tachibana et al. 2002). However, expression of GAL4-DBD fused with ZNF200 or its deletion mutants exerted no significant effect on transcription of the reporter gene (Fig. 7A,B). Co-expression of G9a with GAL4-DBD-ZNF200 fusion protein exerted no effect on transcription of the reporter gene, suggesting that ZNF200 cannot recruit G9a in this assay (Fig. 7C). We have also co-expressed the GAL4-DBD-G9a fusion protein and ZNF200 with the reporter plasmid (Fig. 7D). However, neither enhancement nor suppression of G9a-mediated transcriptional repression by ZNF200 was observed. These results suggest that ZNF200 does not play a role in G9a-mediated transcriptional repression and interaction between G9a and ZNF200 may require chromatin structure.


Figure 7
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  		Figure 7  Effect of ZNF200 on promoter activity. (A) Schematic representation of the GAL4-DBD-fused expression plasmids for ZNF200 that were used for luciferase transient expression assays. (B) HeLa cells were transfected with the pGL3-G5polß reporter plasmid, 200 ng each of GAL4-DBD-fused expression plasmids for G9a (pM-G9aL) or ZNF200 as indicated. (C) HeLa cells were transfected with pGL3-G5polß reporter plasmids, GAL4-DBD-fused expression vectors (100 ng) and hG9a expression vectors (100 ng). (D) HeLa cells were transfected with the pGL3-G5polß reporter plasmid, the GAL4-DBD-fused expression plasmids for G9a (50 ng each) and for ZNF200 expression plasmids (0, 50, 100, 150, 200, 250, 300 ng each). All the transient luciferase assays were performed multiple times. Luciferase activity was normalized to Renilla luciferase activity and expressed as activity relative to that with expression plasmid for GAL4-DBD alone that was scored as 100.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The first identified HMTase, SUV39H1, is enriched in heterochromatin (Melcher et al. 2000) and SUV39H deletion results in growth-retarded mice with chromosomal instability and loss of H3-K9 methylation of pericentric heterochromatin (Peters et al. 2001, 2003; Rice et al. 2003). In contrast, G9a localization is mostly outside of the pericentric heterochromatin region of the nucleus (Tachibana et al. 2001). G9a deletion is embryonically lethal and results in loss of euchromatic H3-K9 methylation (Tachibana et al. 2002). These reports suggest that the two HMTases have distinct functions within the cell and that the role of G9a appears to be restricted to euchromatic gene regulation. Despite the key function of H3-K9 methylation in gene regulation, very little is known about how G9a is recruited to specific promoters.

In the present study, we found an evolutionarily conserved GHD domain in the N-terminal region of G9a and showed that amino acids 454–548 containing GHD interact with ZNF200 (amino acids157–395) in a two-hybrid assay (Fig. 3). Association between G9a and ZNF200 was further demonstrated by in vitro binding assays and immunoprecipitation experiments (Figs 4 and 5). Interestingly, the region within GHD of hG9a is also reported to be involved in the interaction with the other transcriptional repressor NRSF/REST (Roopra et al. 2004). Therefore, GHD is considered as a motif that serves to interact with other key regulatory proteins and G9a may be recruited to specific chromosomal loci via interaction between GHD and additional regulatory proteins.

The present study indicated that ZNF200 interacts with hG9a through a region stretching from amino acids 157 and 335, encompassing ZF1–ZF3 (Fig. 6). This region appears to be sufficient for the association with G9a. However, functions for the remaining two zinc fingers are yet to be clarified. Zinc finger domains, generally referred to as nucleic acid-binding or protein-interacting, are present in a large number of eukaryotic proteins, most of which are transcriptional regulators. If the remaining zinc fingers of ZNF200 have DNA-binding activity, ZNF200 may be a sequence-specific transcriptional factor that can recruit G9a to target genes in vivo.

The sub-cellular distribution of FLAGHA-hG9a indicates partial overlapping with EGFP-ZNF200 in the nucleus during interphase (Fig. 5D), ZNF200 being localized diffusely (Fig. 5A) while hG9a appeared in the nucleoplasm as rather discrete clear speckles, known to be regions outside of the heterochromatin domains around centromeric loci (Tachibana et al. 2001). Previous studies indicated that G9a could interact with other transcriptional repressors, such as NRSF/REST (Roopra et al. 2004), PRDI-BF1 (Gyory et al. 2004) and Cut (Nishio & Walsh 2004). Therefore, hG9a may form complexes with these factors in the nuclear region where no overlapping with ZNF200 signals is observed. Since green signals of ZNF200 are still observed when images are merged, it is also possible that ZNF200 plays roles other than recruiting G9a to the chromosome foci.

During metaphase, both FLAGHA-hG9a and EGFP-ZNF200 are dissociated from condensed mitotic chromosomes (Fig. 5). On the other hand, a previous report described endogenous SUV39H1 to specifically accumulate at centromeric positions of human metaphase chromosomes (Aagaard et al. 1999), indicating a contribution to their structural organization. In the present study, FLAGHA-hG9a exhibited a quite different localization pattern in metaphase cells, presumably reflecting differences between G9a and SUV39H1 functions. Since ZNF200 is also dissociated from mitotic chromosomes, it may not be a component of the scaffold. Although the hypothesis needs to be tested with specific antibodies against endogenous molecules, the present findings suggest a possibility that G9a and ZNF200 may not be involved in the organization of higher order chromatin structures during metaphase, but rather contribute to regulation of chromatin structure during interphase. Since co-expression of G9a with GAL4-DBD-ZNF200 fusion protein exerted no effect on transcription of the transfected reporter gene carrying GAL4-binding sites, interaction between G9a and ZNF200 may require chromatin structure. In addition, since both G9a and ZNF200 are highly expressed in testis (Deng et al. 1998; Tachibana et al. 2001), they may contribute to regulation of the testis-specific chromatin structure.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Plasmid construction

All constructs refer to the amino acid sequence as reported in NP_003 445 (National Center for Biotechnology Information). Full-length ZNF200 cDNA was obtained by site-directed mutagenesis of an EST clone (Clone ID: 5 260 695, Open Biosystems) which carries a single nucleotide deletion at the position 271 A. To insert the nucleotide, polymerase chain reactions (PCR) were performed using the EST clone as a template with two synthetic oligonucleotide primers (5'-GTGCATCCTCGTCCCTTGGTGAAGCTTCTGCCCAAAGGAGTCC and 5'-GGACTCCTTTGGGCAGAAGCTTCACCAAGGGACGAGGATGCAC) containing the nucleotide insertions shown in bold letters. Site-directed mutagenesis was performed with a QuickChange Site-Directed Mutagenesis Kit (Stratagene).

For the expression of Aequorea victoria green fluorescent protein (EGFP) fused to full-length ZNF200, ZNF200 cDNA was obtained by PCR amplification using two synthetic oligonucleotide primers (5'-AAGAGATCTTGGCTTCAAAAGTGGTTCCTATG-3' and 5'-TTCGTCGACTTACTTCTGCTTTCGGGTCTTACAG-3'), and inserted between the BglII and SalI sites in pEGFP-C2 (Clontech) to create pEGFP-ZNF200.

For the preparation of glutathione S-transferase (GST) fusion recombinant proteins, cDNA fragments corresponding to ZNF2003–395, ZNF2003–363, ZNF2003–335 and ZNF2003–279 were produced by PCR amplification using appropriate primers (5'-AAGGTCGACTGGCTGCAAAAGTGGTTCC-3' and each primer; 5'-GGTGCGGCCGCTTACTTCTGCTTTCGGGTC-3', 5'-CACATTTTGCGGCCGCTTATGGTCTCTCAGCC-3', 5'-CACATTCTGCGGCCGCTTATATCTTCTCCCTTATATG-3' or 5'-CACAGTGAGCGGCCGCTTAGGGTTTTTCTCC-3', respectively), and cloned into the SalI and NotI sites in pGEX4T-1 (GE Healthcare). pGST-ZNF200157–395 was created by subcloning the BamHI/XhoI insert from pACT2-ZNF200157–395 into pGEX-5X-1 (GE Healthcare).

Full-length hG9a1–1210 tagged with the FLAG and HA epitope sequences (pIRESneo-FLAGHA-hG9a) was described earlier (Tachibana et al. 2005). For protein expression in yeast cells, the DNA fragment corresponding to hG9a454–584 and GLP522–612 were obtained by PCR amplification using synthetic oligonucleotide primers (5'-AGTCGAATTCGAAAAGCTGTCAGGCTGCAAT-3' and 5'-AGTCGGATCCTTAGATGGTCACCTCTTGAGCTTGAGA-3') or (5'-CGAGAATTCCGGTGCACAAACAGCGTGG-3' and 5'-GCCGGATCCTTAAGCTATCGTCACCTCTTTGG-3') and inserted between EcoRI and BamHI sites in pGBKT7 (Clontech) to create pGBKT7-hG9a454–548 and pGBKT7-hGLP522–612, respectively.

Both nucleotide sequencing and restriction enzyme digestion verified the identities of all constructs.

Yeast two-hybrid screen

The yeast two hybrid screen was performed using the pre-transformed MATCHMAKER library system (Clontech). Cells of yeast strain AH109 (MATa, trip1-901, leu2-3, 112, ura3-52, his3-200, gal4{Delta}, gal80{Delta}, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2 URA3::MEL1UAS-MEL1TATA-LacZ MEL1) transformed with pGBKT7-hG9a454–548 were mated with Y187 cells (MAT{alpha}, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4{Delta}, gal80{Delta}, met–, URA3::GAL1UAS-GAL1TATA-lacZ, MEL1) pre-transformed with human fetal brain cDNA/pACT2. Diploid his +, ade +, trp +, leu + transformants were selected on minimal medium lacking histidine, adenine, tryptophan and leucine, and supplemented with 2% glucose and 2.5 mM 3-amino-1,2,4-triazole. They were further confirmed to express ß-galactosidase with 5-bromo-4-chloro-3-indoryl-ß-D-galactopyranoside (X-Gal) as a substrate. Plasmid DNAs, isolated from yeast candidate clones, were transfected to transform E. coli KC8 carrying hisB, leuB and trpC mutations. KC8 cells transformed with the pACT2 plasmid were grown on M9 minimal medium supplemented with essential amino acids and thiamine but lacking leucine.

In the yeast two-hybrid assay, bait (empty pGBKT7, pGBKT7-hG9a454–548 or pGBKT7-hGLP522–612) and target (empty pACT2 or pACT2-ZNF157–395) plasmids were co-transformed into AH109 S. cerevisiae. Colonies were isolated and their ability to grow in the absence or presence of histidine and adenine was assessed.

Cell culture, transfections and luciferase assays

HEK293 cell lines were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/mL of penicillin and 100 µg/mL of streptomycin at 37 °C in a 5% CO2 atmosphere and transfected with plasmid DNAs using FuGene 6 reagent (Roche) according to the manufacturer's instructions.

HeLa cells were seeded into 24-well dishes (1.5 x 104 cells/well) and cultured for 20 h before transfection with 47 ng of pGL3-G5polß (Sekimata et al. 2001), 3 ng of SV40-RL (Promega) and indicated amounts of the expression plasmids. After incubation for 48 h, cell lysates were prepared and assayed for luciferase activity using a luciferase assay kit (Promega). For expression of a GAL4-DBD fused to ZNF200, ZNF200{Delta}Zn, ZNF200-Zn, pM-ZNF200, pM-ZNF200{Delta}Zn and pM-ZNF200-Zn were generated by subcloning a SalI-SpeI fragment of ZNF200 encoding amino acids (aa) 3–395, EcoRI-XhoI fragment of ZNF200 encoding aa3–251, BamHI-XbaI fragment of ZNF200 encoding aa252–395 into the plasmid pM (Clonthech). For expression of a HA-tagged ZNF200, the fragment digested with BglII/SalI of pEGFP-C2-ZNF200 was cloned into BamHI/XhoI of the plasmid pcDNA3 (Invitrogen) to create the plasmid pcDNA3-HA-ZNF200.

Antibodies

GST-ZNF2003–393 fusion protein was expressed in E. coli BL21 (DE3). Lysates of cells were prepared by sonication in PBS containing 0.1% Triton X-100 and a mixture of protease inhibitors (Roche). The lysates were applied to GSTrap HP to purify the GST-ZNF2003–393 fusion protein according to the manufacturer's instructions (GE Healthcare). Anti-ZNF200 antibody was produced in rabbits by injection of the purified GST-ZNF2003–393 fusion protein. Rabbit serum was applied to MabTrap Protein G HP using MabTrap kit (GE Healthcare) to purify total rabbit IgG.

Immunoprecipitation

HEK293 cells were lysed with lysis buffer (20 mM Tris [pH8.0], 0.4 M KCl, 1 mM EDTA, 10% glycerol, 5 mM MgCl2, 0.1% Tween 20) containing a mixture of protease inhibitors (Nacalai tesque). The cell extracts equivalent to 1.5 mg protein was then added to Protein A Sepharose beads (GE Healthcare) cross-linked with anti-ZNF200 antibody. Normal rabbit IgG (Sigma) was used to immunoprecipitation the same amount of cell extract as a negative control. Samples were rotated 16 h at 4 °C. Sepharose beads were washed 3 times with wash buffer (10 mM Tris [pH7.5], 150 mM NaCl, 0.1% Nonidet P-40). The protein complex was eluted in PBS containing 2% SDS and analyzed by Western blot using anti-ZNF200 antibody (1 : 250 dilution) and anti-mouse G9a antibody (MBL, 1 : 10 dilution). Visualization was carried out using Super Signal West femto Maximum Sensitivity Substrate (Pierce).

HEK293 cells were plated in 6 cm diameter dishes (5 x 105/dish), cultured for 20 h, and co-transfected with pEGFP-ZNF200 and pIRESneo-FLAGHA-hG9a. The total amount of expression plasmids transfected was adjusted to 4 µg. After 48 h incubation, cells were harvested and suspended in lysis buffer (20 mM Hepes, 0.42 M NaCl, 1.5 mM MgCl2, 0.1% NP40, 20% glycerol, 0.5 mM PMSF, 0.5 mM DTT, and a mixture of protease inhibitors containing 4-(2-aminoethyl) benzenesulfonyl fluoride, aprotinin, E-64, leupeptin, pepstain, pepstatin A). After centrifugation for 30 min at 15 000g, supernatants were incubated with agarose conjugated with anti-FLAG IgG1 (ANTI-FLAG M2 affinity gel, Sigma). After incubation for 16 h at 4 °C, the beads were washed 3 times with the same buffer, and the immune complexes were collected and subjected to Western blot analysis. Bound FLAG tagged hG9a and EGFP tagged ZNF200 were detected with anti-FLAG monoclonal antibodies (ANTI-FLAG M5, Sigma), anti-GFP monoclonal antibodies (Clontech) and an ECL detection kit (GE Healthcare).

GST pull-down assay

HEK293 cells were transfected with pIRESneo-FLAGHA-hG9a, then cells were harvested and subjected to GST pull-down assays. The amounts of proteins were determined by BCA Protein Assay Reagent Kit (Pierce). GST or GST-ZNF200 fusion proteins were produced in E. coli BL21 (DE3) and purified with glutathione-Sepharose beads equilibrated with PBS. The amounts of each GST fusion protein were estimated by SDS-PAGE. Beads loaded with 10 µg of GST fusion proteins were rocked at 4 °C with 400 µg of HEK293 whole-cell extracts. After incubation for 16 h at 4 °C, the beads were washed 3 times with the wash buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM Na3VO4, 0.1% NP40) and bound proteins were subjected to Western blot analysis. Bound hG9a was detected by immunoblotting with anti-FLAG monoclonal antibodies (ANTI-FLAG M5, Sigma) and an ECL detection kit.

Fluorescent microscope analysis

HEK293 cells grown on glass coverslips were co-tranfected with pIRESneo-FLAGHA-hG9a and pEGFP-ZNF200 by the method described above, fixed with 4% paraformaldehyde in PBS for 30 min, and permeabilized with 0.2% Triton X-100 in PBS for 5 min. After washing with PBS, samples were blocked with 1% skim milk in PBS for 10 min. After washing with PBS, the samples were then incubated for 1 h with anti-HA Rat monoclonal antibody (clone 3F10, 1 : 150 dilution, Roche), washed further 3 times with 0.1% NP40 in PBS, and blocked again with 1% skim milk in PBS for 10 min. The samples were then incubated for 30 min with Alexa Fluor 594-conjugated secondary antibody (Molecular Probes, 1 : 400 dilution). After washing 3 times with 0.1% NP40 in PBS, the samples were stained with 4', 6-diamino-2-phenylindole (DAPI) for 5 min and examined under a confocal laser scanning microscope (LSM510, Carl Zeis).


   Acknowledgements
 
We thank Dr Y. Hayashi for technical advice, Dr M. Moore for comments on the English language in the manuscript and Dr M. Sekimata for pGL3-G5polß plasmid. This study was partially supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.


   Footnotes
 
Communicated by: Fuyuki Ishikawa

aPresent address: Developmental Biology, Stanford School of Medicine, 279 Campus Dr. B371, Stanford, CA 94305, USA. Back

* Correspondence: E-mail: myamaguc{at}kit.ac.jp


   References
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 Introduction
 Results
 Discussion
 Experimental procedures
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Accepted: 16 April 2007




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