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Division of Gene Function in Animals, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan
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Abstract |
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Introduction |
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The C2H2-type zinc fingers are one of the commonest types of DNA-binding motifs. Approximately 5–10% of zinc finger proteins are estimated to contain the BTB (broad complex, tramtrack and bric-a-brac) domain, also known as the POZ (poxvirus and zinc fingers) domain, which serves as a protein-protein interaction interface (Bardwell & Treisman 1994; Zollman et al. 1994; Collins et al. 2001). Many of the BTB-zinc finger proteins, including oncogenic or tumor suppressor proteins such as PLZF, BCL-6 and HIC-1, are known to function as transcriptional repressors by altering chromatin structure through various mechanisms. For example, the BTB domain of PLZF and BCL-6 interacts with SMRT/NCoR and mSin3A corepressors and with HDAC1 (David et al. 1998; Dhordain et al. 1998; Huynh & Bardwell 1998; Lin et al. 1998), while the central region of PLZF and the zinc fingers of BCL-6 associate with ETO corepressor and Class II HDACs, respectively (Melnick et al. 2000; Lemercier et al. 2002). Moreover, the central region of HIC-1 binds to CtBP1 via a CtBP binding motif, but the BTB domain of HIC-1 fails to interact with SMRT/NCoR/HDAC complexes (Deltour et al. 1999, 2002).
CtBP1 was originally identified as a cellular protein that binds to the C-terminal region of adenovirus E1A oncoprotein and negatively regulates oncogenic transformation (Boyd et al. 1993). CtBP1 and the closely related protein CtBP2 have been reported to act as corepressors for diverse transcriptional repressors by binding to the PXDLS or related motifs (Chinnadurai 2002). Genetic and biochemical studies have shown that CtBPs play important roles in many biological processes in vertebrates and invertebrates. In Drosophila, dCtBP plays critical roles in embryo patterning during early development by interacting with the short-range transcriptional repressors Snail, Kruppel and Knirps (Nibu et al. 1998). In mammals, CtBPs bind to the Ras-regulated transcription factor Net, the cellular oncogene Evi-1, the Wnt signaling effector Tcf-4, and to many other transcription factors and cofactors, to strictly regulate cell differentiation or proliferation (Chinnadurai 2002). Furthermore, analysis of CtBP1- and CtBP2-deficient mice demonstrated that CtBPs have fundamental roles in mammalian development (Hildebrand & Soriano 2002). Although CtBPs act mainly as transcriptional corepressors in these biological processes, the detailed molecular mechanisms by which they mediate transcriptional repression remain unclear. CtBPs appear to repress transcription in an HDAC-dependent and -independent manner, depending on the cell type or promoter context (Koipally & Georgopoulos 2000; Sundqvist et al. 2001; Zhang et al. 2001). However, little is known about the correlation between subnuclear localization of CtBP and its corepressor activity.
We describe here the cloning and characterization of a novel BTB-zinc finger protein, CIBZ. CIBZ represses transcription via two independent domains, the BTB domain and a proximal domain referred to as RD2, in an HDAC-independent and -dependent manner, respectively. CIBZ specifically interacts with CtBPs via RD2 and relocalizes CtBPs to pericentromeric foci, suggesting that CIBZ exerts its repression activity via recruitment of the CtBP complex.
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Results |
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We performed gene trap mutagenesis in mouse ES cells and established the NAISTrap database, in which the nucleotide sequences of trapped genes are described <http://bsw3.naist.jp/kawaichi/naistrap.html> (Ishida & Leder 1999; Matsuda et al. 2004). Among the gene-trapped clones in the database, we focused on #2 V-43 that contains a nucleotide sequence encoding a novel BTB domain, because many BTB-zinc finger proteins identified so far are known to be involved in development or oncogenesis. To study the function of this gene, termed CIBZ, we obtained the entire cDNA of the coding sequence of CIBZ by 3'RACE and RT-PCR analysis based on ESTs in public databases. CIBZ consists of a 3594-bp open reading frame and encodes a deduced protein of 1197 amino acids with a calculated molecular weight of 134 kDa (Supplementary Figure S1A). A BLAST search against genome databases revealed that CIBZ was located at mouse chromosome 9E3.3.
In addition to the BTB domain that is typically located at the N terminus, CIBZ contains 10 zinc finger motifs separated into central (four C2H2 type and one C2HC type) and C-terminal (five C2H2 type) clusters; the middle portion, located between two zinc finger clusters, includes two potential bipartite nuclear localization signals (NLSs) (Supplementary Figure S1A,B). Thus, CIBZ belongs to the BTB-zinc finger family of transcription factors. Amino acid alignment of BTB domains (Supplementary Figure S1C) shows that CIBZ is most similar to Kaiso, which was originally identified as a protein that binds to p120 catenin by a yeast two-hybrid screen (Daniel & Reynolds 1999). A BLAST search of the GenBank‘ database identified human gene (XP_172341 [GenBank] ) and rat (AY623002 [GenBank] ) gene (ZENON) sharing high similarity in the BTB domain and zinc fingers (Supplementary Figure S1B). ZENON was recently identified as a protein that binds to the rat tyrosine hydroxylase promoter by yeast one-hybrid screening (Kiefer et al. 2005). We conclude that CIBZ is a mouse counterpart of ZENON because CIBZ locus (mouse 9E3.3) corresponds to rat 8p31 where ZENON is located.
CIBZ is expressed ubiquitously in adult mouse tissues
The expression pattern of CIBZ in adult mouse tissues was examined by Northern blot analysis using a cDNA fragment of CIBZ as a probe. A single CIBZ transcript of approximately 7–8 kb was detected in all tissues examined, but was relatively abundant in the kidney and thymus (Fig. 1A). The rat ortholog, ZENON, was shown to be expressed specifically in neural tissues and in kidney by high stringent Northern blotting (Kiefer et al. 2005), suggesting that CIBZ/ZENON is ubiquitously expressed but is relatively abundant in neurons and in kidney.
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314–580), was detected with anti-CIBZ antibody (Fig. 1C, right), whereas both proteins were detected with anti-Myc antibody (Fig. 1C, left), confirming the specificity of this antibody. CIBZ localizes to pericentromeric heterochromatin
To investigate the subcellular localization of CIBZ, expression plasmids for C-terminal Myc-tagged CIBZ were transiently transfected into NIH3T3 cells, and confocal immunofluorescence analysis was performed. In approximately 70% of transfected cells, CIBZ exhibited both a unique dot-like structure in the nuclei and a diffuse distribution in the cytoplasm (Fig. 1D,a). The remaining 30% of cells displayed diffuse cytoplasmic and/or nuclear localization (data not shown). In addition, similar dot-like structure was observed when CIBZ was tagged with FLAG or Myc epitope at the N terminus (data not shown). In mouse cells, major satellite repeats are concentrated in the pericentromeric region, considered to be typical constitutive heterochromatin, and this pericentromeric heterochromatin is stained with DAPI as bright dots (Maison & Almouzni 2004) (Fig. 1D,b). When CIBZ and DAPI images were merged, the nuclear dot-like structure of CIBZ almost co-localized with DAPI-stained dots, suggesting that CIBZ localizes to pericentromeric heterochromatin (Fig. 1D,c). These nuclear dots of CIBZ also co-localized with tri-methylated Lys9 of histone H3, a marker of pericentromeric heterochromatin (Fig. 1D,d–f) (Peters et al. 2003), confirming that exogenously expressed CIBZ localizes to pericentromeric heterochromatin.
CIBZ represses transcription via two independent repression domains, BTB and RD2
Since CIBZ localizes at transcriptionally inactive heterochromatin region (Fig. 1), we considered that it might function as a transcriptional repressor. To address this possibility, we performed a luciferase reporter assay using full-length CIBZ fused to the GAL4 DNA-binding domain (GAL4-DBD). As shown in Fig. 2A, GAL4-CIBZ repressed transcription of a reporter gene (pGL3-G5SV) containing five GAL4-binding sites in a dose-dependent manner, but did not affect transcription of a reporter gene lacking GAL4-binding sites (pGL3-SV). Similar repression activity was also observed in HEK293 cells (data not shown). These results indicate that CIBZ possesses transcriptional repressor activity when heterologously targeted to DNA.
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CIBZ-mediated repression is associated with HDACs
To investigate whether HDACs are involved in CIBZ-mediated transcriptional repression, we tested the effect of TSA, a specific inhibitor of HDACs, by luciferase assay. BTB-mediated repression was not affected by TSA (Fig. 3A), suggesting that HDACs are not responsible for BTB-mediated repression. In contrast, RD2-mediated repression activity was markedly reduced by TSA treatment (Fig. 3A), implying that RD2 uses HDACs to repress transcription. The repression by BTB-RD2 was also relieved by TSA. These results suggest that CIBZ uses both HDAC-dependent and -independent repression mechanisms.
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CIBZ interacts specifically with CtBP corepressor in yeast and mammalian cells
To further elucidate the mechanisms underlying CIBZ-mediated transcriptional repression, we searched for CIBZ-binding proteins by yeast two-hybrid screening, using the N-terminal region of CIBZ (1–339) encompassing two repression domains (BTB and RD2) as bait. Of approximately 4.5 x 106 clones screened in a mouse 17-day embryonic cDNA library, three were found to encode CtBP1. To confirm the interaction of CIBZ with CtBP1 and further map the CtBP interaction domain of CIBZ, deletion mutants of CIBZ and full-length CtBP1 were prepared and used in yeast two-hybrid assays. As shown in Fig. 4A, full-length CIBZ (1–1197) and CIBZ lacking the BTB domain (158–1197) interacted with CtBP1, but not with the GAL4 activation domain alone, whereas CIBZ lacking both the BTB domain and RD2 (335–1197) failed to interact with CtBP1, suggesting that RD2, but not the BTB domain, is responsible for CtBP binding. Furthermore, the BTB domain (1–158) did not interact with CtBP1, whereas RD2 (158–339) as well as the N-terminal region (1–339) interacted strongly with CtBP1, supporting the result that RD2 is sufficient for interaction with CtBP1. Similar results were obtained in an interaction assay between CIBZ and CtBP2 (Fig. 4A). Importantly, these data are compatible with the result that RD2-mediated repression is dependent on HDACs (Fig. 3), because CtBP is known to interact with HDACs in vivo and in vitro (Zhang et al. 2001; Sundqvist et al. 2001). Taken together, these data indicate that CIBZ interacts with both CtBP1 and CtBP2 (CtBP1/2) via RD2 in yeast.
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CIBZ interacts with CtBP1 through a PLDLR motif
Sequence analysis of CIBZ revealed that RD2 contains two potential CtBP-binding motifs, PLDLR (182–186) and PLSLV (281–285), which are similar to the consensus sequence PXDLS (Chinnadurai 2002) (Fig. 5A). The PLDLR and PLSLV motifs are identical to those of MITR and Hairy, respectively (Fig. 5A). To examine whether these motifs are responsible for the interaction between CIBZ and CtBP1, we performed an immunoprecipitation assay. PLDLR and PLSLV were mutated to PLASR and PLASV, respectively, and three types of Myc-tagged CIBZ mutants, CIBZ (DL/AS), CIBZ (SL/AS) and CIBZ (DL/AS, SL/AS), were constructed and co-transfected into 293T cells together with FLAG-CtBP1. Myc-tagged wild-type (WT) CIBZ and CIBZ (SL/AS) were detected in FLAG-CtBP1 immunoprecipitates (Fig. 5B, lanes 2, 4), whereas CIBZ (DL/AS) and CIBZ (DL/AS, SL/AS) were almost or completely undetectable in the FLAG-CtBP1 immunoprecipitates (Fig. 5B, lanes 3, 5). These results suggest that only the PLDLR motif is responsible for the interaction of CIBZ with CtBP1.
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Having established that CIBZ physically interacts with CtBPs via the PLDLR motif, we next examined, by confocal immunofluorescence, whether CIBZ and CtBPs co-localize in NIH3T3 cells. We found that in nearly 70% of transfected cells, CIBZ (DL/AS)-Myc also localized to pericentromeric heterochromatin in the interphase nuclei (Fig. 5C,a–c), as did CIBZ (WT)-Myc (Fig. 1D). In contrast, FLAG-tagged CtBP1 was distributed diffusely in the nucleus and cytoplasm (Fig. 5C,d–f). When CIBZ (WT)-Myc was co-expressed with FLAG-CtBP1, CtBP1 was concentrated in pericentromeric foci (Fig. 5C,g–j). In almost 99% of foci-positive and CtBP1-expressing cells, CtBP1 also displayed pericentromeric localization (data not shown). In contrast, when CIBZ (DL/AS)-Myc and FLAG-CtBP1 were co-expressed (Fig. 5C,k–n), the diffuse nucleocytoplasmic distribution of CtBP1 was unchanged, although CIBZ (DL/AS)-Myc was localized to pericentromeric foci itself in approximately 70% of transfected cells.
We then tested whether endogenous CtBPs also co-localize with over-expressed CIBZ. Endogenous CtBPs displayed diffuse localization, predominantly in the nuclei of non-transfected NIH3T3 cells (Fig. 5D,a–c). When CIBZ (WT)-Myc was expressed, endogenous CtBPs in all CIBZ foci-positive cells became concentrated in pericentromeric foci that co-localize with tri-methylated K9 of histone H3 (Fig. 5D,d–f). In contrast, CIBZ (DL/AS)-Myc failed to relocalize CtBPs to pericentromeric foci (Fig. 5D,g–i). Taken together, these results suggest that CIBZ can recruit CtBPs to pericentromeric heterochromatin through the PLDLR motif.
CtBP binding correlates with repressor activity of RD2
To investigate whether RD2-mediated repression activity depends on CtBP binding, we first tested the interaction of RD2 with CtBP1 by a mammalian two-hybrid assay. RD2 or RD2 mutants fused to GAL4-DBD (Fig. 6A,a–e) were co-transfected with CtBP1 fused to VP16, or with VP16 alone, into NIH3T3 cells. As shown in Fig. 6B, co-expression of GAL4-RD2 (WT) with VP16-CtBP1 strongly activated the reporter gene, whereas co-expression with VP16 did not, suggesting that RD2 functionally interacts with CtBP1 in mammalian cells. In addition, requirement of the PLDLR motif for CtBP interaction was confirmed in this assay because GAL4-RD2 (DL/AS) and -RD2 (DL/AS, SL/AS), but not GAL4-RD2 (SL/AS), failed to activate transcription when co-transfected with VP16-CtBP1. These results suggest that RD2 interacts specifically with CtBP1 via the PLDLR motif.
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Although RD2 represses transcription via recruitment of CtBP corepressor, BTB-RD2 (DL/AS) retained the ability to repress transcription (Fig. 6C). These data are consistent with the result that the BTB domain alone showed transcriptional repressor activity (Fig. 2B). Therefore, these findings suggest that the BTB domain of CIBZ has additional repression mechanism(s) independent of CtBP binding.
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Discussion |
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The pericentromeric localization of CIBZ is clearly distinct from that of several other BTB transcription factors such as PLZF and Bach2, which form nuclear foci associated with PML nuclear bodies (Ruthardt et al. 1998; Tashiro et al. 2004). However, the following evidence suggests that pericentromeric foci of CIBZ may be significant. First, pericentromeric foci of exogenous CIBZ were resistant to extraction with Triton X-100 (data not shown), suggesting that CIBZ is tightly associated with heterochromatin. Second, exogenous CIBZ could redistribute both over-expressed and endogenous CtBPs to pericentromeric heterochromatin via the PLDLR motif, whereas the PLDLR mutant of CIBZ failed to do so. These results support the idea that CIBZ is specifically associated with pericentromeric heterochromatin. Further immunostaining experiments are required to confirm whether endogenous CIBZ also localizes to pericentromeric heterochromatin.
The mechanism by which CIBZ localizes to pericentromeric foci remains unknown. It is possible that CIBZ binds directly to repetitive DNA sequences such as 
-satellite DNA via zinc fingers in a manner similar to Ikaros and YY1 (Cobb et al. 2000; Shestakova et al. 2004). CIBZ may also bind to methylated CpG dinucleotides, which are concentrated in satellite repeats of pericentromeric heterochromatin, because several of the zinc fingers of CIBZ share high sequence similarity with those of Kaiso, which recognize methylated DNA (Prokhortchouk et al. 2001; Yoon et al. 2003). Another possibility is that CIBZ is recruited to pericentromeric foci through interaction with heterochromatin-associated proteins, such as HP1 (Maison & Almouzni 2004). To elucidate the mechanism of pericentromeric targeting of CIBZ, it will be important to determine the region responsible for pericentromeric targeting and to identify the CIBZ-interacting proteins.
CtBPs are well characterized as corepressors for many types of DNA-binding transcriptional repressors, and corepressor activity is regulated by nuclear-to-cytoplasmic translocation through interaction with the PDZ protein nNOS (Riefler & Firestein 2001). However, the correlation between subnuclear localization of CtBPs and their corepressor activity is poorly understood. Although Net and HIC-1 both recruit CtBPs to the nucleus from the cytoplasm, little is known about the significance of nuclear targeting of CtBP (Criqui-Filipe et al. 1999; Deltour et al. 2002). Our finding that CtBPs are targeted to pericentromeric foci may provide insights into a novel transcriptional repression mechanism of CtBPs as a heterochromatin-associated corepressor. CIBZ may target CtBPs to pericentromeric foci and form the ternary complex comprising CtBP, HDAC and CIBZ, resulting in the silencing of target genes. Interestingly, the CtBP complex purified from Hela cells contains two histone methyltransferases (G9a and EuHMT) and the polycomb protein HPC2, as well as HDACs (Shi et al. 2003). Further experiments are required to determine whether these proteins are also included in the CIBZ/CtBP complex.
We showed that RD2 is necessary and sufficient for CIBZ to interact with CtBPs. Although RD2 of CIBZ contains two potential CtBP-binding motifs similar to the consensus PXDLS motif (Chinnadurai 2002), only the PLDLR motif was responsible for CtBP binding. This result further supports the notion that the five consensus amino acid residues are not the determinant for CtBP binding (Zhang et al. 2001; Hickabottom et al. 2002), and thus suggests that flanking residues of CtBP-binding motifs or protein folding may play critical roles in binding to CtBPs.
Several lines of evidence suggest that the BTB domain has other repression mechanism(s) different from that of RD2. First, although the BTB domain could not interact with CtBPs, it repressed transcription significantly when heterologously targeted to DNA. It is noteworthy that we could not observe synergistic transcriptional repression between the BTB domain and RD2 (Fig. 2B). This implies that the BTB domain and RD2 function independently through recruitment of different repressor complexes on target promoters depending on the situation. Second, BTB-RD2 (DL/AS) and full-length CIBZ (DL/AS), which failed to interact with CtBPs, efficiently repressed transcription (Fig. 6C and data not shown), supporting the idea that CtBP recruitment is not the only mechanism used by CIBZ. Several BTB transcription factors are known to associate with SMRT/NCoR, mSin3A corepressors and HDAC1 via the BTB domain. However, it is important to note that repression by the BTB domain of CIBZ was insensitive to the HDAC inhibitor TSA, and that recruitment of corepressor/HDAC complex by the BTB domain is not a general property of BTB-transcription factors (Deltour et al. 1999). Further studies are required to clarify the transcriptional silencing mechanism mediated by the BTB domain of CIBZ.
Although many BTB-zinc finger proteins identified so far act as transcriptional repressors (Deltour et al. 2002; Yoon et al. 2003), some function as transcriptional activators (Kobayashi et al. 2000) or as both activators and repressors (Kaplan & Calame 1997). We found that CIBZ possesses weak transcriptional activation activity in its middle region, located between two zinc finger clusters (Fig. 2B). In addition, rat ZENON seems to activate transcription via binding to the E-box sequence of the TH promoter (Kiefer et al. 2005). This suggests that CIBZ may also function as a transcriptional activator depending on the context, such as promoter or cell type.
Our data provide the first evidence that CtBPs are recruited to pericentromeric heterochromatin via binding to a BTB-zinc finger protein. Further studies are required to clarify the biological significance of the CIBZ/CtBP interaction and the physiological functions of CIBZ. Identification of CIBZ target genes and analysis of CIBZ-deficient mice will lead to the elucidation of CIBZ function.
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Experimental procedures |
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Details of gene trapping and 3' rapid amplification of cDNA ends (3' RACE) have been previously described (Matsuda et al. 2004). Full-length CIBZ cDNA was obtained from total RNA of mouse kidney using-3' RACE and reverse transcription-PCR (RT-PCR) analysis based on expressed sequence tags (ESTs), and subcloned into pBluescript II KS(+) (Stratagene). DNA sequences were verified using the BigDye terminator and an automated sequencer (ABI PRISM310).
Plasmids
For the C-terminal Myc-tagged CIBZ construct, full-length CIBZ cDNA including the Kozak consensus sequence at the 5' end was ligated into the BamHI and XhoI sites of pcDNA3 vector (Invitrogen) in-frame with six repeats of the c-Myc epitope. For the GAL4-CIBZ (amino acids 1–1197) construct, full-length CIBZ cDNA was inserted into the HindIII and XhoI sites of pcDNA3 along with cDNA encoding the GAL4 DNA-binding domain (GAL4-DBD) (1–147). For GAL4-CIBZ (1–158), -CIBZ (158–339), -CIBZ (335–538), -CIBZ (967–1197) and -CIBZ (1–339) constructs, each CIBZ fragment was generated by PCR and inserted into pcDNA3 in-frame with GAL4-DBD. GAL4-CIBZ (158–1197), -CIBZ (335–1197), -CIBZ (539–1197), -CIBZ (539–1012) and -CIBZ (1–538) constructs were obtained by PCR and/or exchange of restriction fragments from the relevant chimeras of CIBZ. For yeast two-hybrid constructs, deletion mutants of CIBZ were digested from GAL4-fusion constructs and inserted into the BamHI and SalI sites of pGBT9 (Clontech).
CtBP1, CtBP2 and HDACs were generated by RT-PCR and subcloned into pGAD424 (Clontech) or a modified pcDNA3 vector containing a FLAG epitope coding sequence. GAL4-RD2 (DL/AS), -RD2 (SL/AS) and -RD2 (DL/AS, SL/AS) mutant constructs were generated by a two-step PCR method. Myc-tagged CIBZ (DL/AS), CIBZ (SL/AS) and CIBZ (DL/AS, SL/AS) constructs were generated by site-directed mutagenesis (Stratagene). VP16-CtBP1 was generated by inserting the EcoRI/SalI fragment of CtBP1 into the pCMX-VP16 vector (Agata et al. 1999). The luciferase reporters (pGL3-G5SV, pGL3-SV and pGL3-G5B) and internal control reporter (pRL-SV) for repression and mammalian two-hybrid assays have been previously described (Agata et al. 1999).
Northern blot analysis
Total RNA was extracted from various adult mouse tissues using ISOGEN, according to the manufacturer's instructions (Nippongene). Approximately 20 µg total RNA was separated on a 1.0% agarose-formaldehyde gel, transferred on to GeneScreen Plus membrane (NEN Research Products) and UV cross-linked. After prehybridization for 2 h at 42 °C, a 32P-labeled CIBZ cDNA probe (nucleotides 436–1022) was added and hybridized overnight at 42 °C. The membrane was washed once with 2 x SSPE/0.1% SDS for 15 min at room temperature, twice with 1 x SSPE/0.1% SDS for 15 min at 55 °C, and exposed to X-ray film.
Yeast two-hybrid screening and interaction assay
Yeast two-hybrid screening was performed according to the manufacturer's protocol (Clontech) using yeast strain HF7c and the N-terminal region of CIBZ (1–339) as bait. Approximately 4.5 x 106 clones were screened from a mouse 17-day embryonic cDNA library (Clontech) in SD minimal medium (-His/-Leu/-Trp), and positive clones were further tested for ß-galactosidase activity in yeast strain SFY526. Positive clones were sequenced.
For interaction assays, a series of CIBZ deletion mutants inserted into the pGBT9 vector, and CtBP1 or CtBP2 inserted into the pGAD424 vector, were co-transformed into yeast strain SFY526. Three independent transformants were liquid-cultured and ß-galactosidase activity was measured according to the manufacturer's protocol (Clontech).
Cell culture and luciferase assay
NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque) supplemented with 10% calf serum. HEK293 and 293T cells were cultured in DMEM containing 10% fetal calf serum. Reporter and mammalian two-hybrid assays were performed as previously described (Agata et al. 1999; Matsuda et al. 2001). For reporter assays, 0–250 ng of expression plasmids for GAL4-fusion proteins, and 100 ng of the firefly luciferase reporter genes pGL3-G5SV or pGL3-SV, were used. Mammalian two-hybrid assays used 200 ng of plasmids for GAL4-fusion protein, 50 ng of plasmids for VP16 or VP16 fusion proteins, and 100 ng of the firefly luciferase reporter gene pGL3-G5B. Four nanograms of pRL-SV reporter (Promega) was used as an internal control. The total amounts of expression plasmids were equalized by adding the empty plasmid, pcDNA3. For TSA treatment, 24 h after transfection, the cells were either mock-treated (DMSO) or treated with 100 ng/mL TSA for a further 24 h before harvesting. Firefly luciferase activity was normalized for transfection efficiency as determined by internal control luciferase activity. All experiments were performed in duplicate; the data shown represent at least three independent experiments, with standard deviation.
Immunoprecipitation and Western blotting
293T cells were transfected with the indicated plasmid using the CellPhect transfection kit (Amersham Biosciences) as previously described (Agata et al. 1999). At 40 h after transfection, the cells were lyzed in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Nonidet-P40) containing protease inhibitor cocktail (Roche Applied Science) for 30 min on ice. For extraction of protein from mouse tissues, tissues dissected individually from adult mice were pulverized in a liquid nitrogen-immersed mortar and pestle, suspended in ice-cold lysis buffer (50 mM HEPES, pH 7.4, 250 mM NaCl, 0.1% NP-40, 1 mM EDTA, 1 mM dithiothreitol) containing a protease inhibitor cocktail, and then homogenized. Cell debris was removed by centrifugation and soluble proteins were immunoprecipitated with FLAG M2 agarose (Sigma) or 2 µg of anti-CtBP monoclonal antibody followed by incubation with protein G-Sepharose beads (Amersham Biosciences) for 4 h at 4 °C. After four washes with 1 mL of lysis buffer, precipitated proteins were released by boiling in SDS-sample buffer, resolved by 6 or 8% SDS-PAGE, and subjected to Western blotting as described (Agata et al. 1999).
Antibodies
Anti-CIBZ, a rabbit polyclonal antibody (Ab) provided by TransGenic Inc., was raised against KLH-conjugated CIBZ peptide corresponding to amino acids 310–322 (NEGDIHFPREDEN). For Western blotting, anti-Myc Ab (9E10 ascites), anti-FLAG Ab (M2; Sigma), anti-CtBP Ab (E-12; Santa Cruz Biotechnology) and anti-tubulin Ab were used. HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Amersham Biosciences) were used as secondary antibodies for Western blotting. For immunofluorescence staining, Cy3-conjugated anti-Myc Ab (Matsuda et al. 2001), biotinylated anti-FLAG Ab (Sigma), anti-CtBP Ab (E-12) and anti-tri-methylated K9 of histone H3 Ab (ab8898; Abcam) were used. The following were used as secondary antibodies: FITC-labeled antibiotin Ab (F6762), Texas Red-labeled anti-rabbit IgG and FITC-labeled anti-mouse IgG (Jackson ImmunoResearch Laboratories).
Immunofluorescence microscopy
Immunofluorescence analyses were performed as previously described (Matsuda et al. 2001). Briefly, NIH3T3 cells were plated on 4- or 8-chamber slides precoated with 0.1% gelatin and transfected using Lipofectamine 2000 (Invitrogen). At 24 h after transfection, cells were fixed in 3% paraformaldehyde/PBS for 10 min at room temperature and permeabilized with 0.2% Triton-X100/PBS for 5 min. Primary and secondary antibodies were used at 1 : 50–1 : 400 dilutions in blocking solution. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma). The slides were mounted with Perma Fluor‘ (Shandon/Lipshaw). Confocal microscopy analysis was performed using a Zeiss LSM510 confocal microscope. Images were captured with Zeiss LSM510 v3.0 software and were processed using Adobe Photoshop 5.0. All staining experiments were repeated more than three times.
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Acknowledgements |
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Footnotes |
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These authors contributed equally to this work. 
* Correspondence: E-mail: ematsuda{at}bs.naist.jp |
References |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Bardwell, V.J. & Treisman, R. (1994) The POZ domain: a conserved protein–protein interaction motif. Genes Dev.
8, 1664–1677.
Boyd, J.M., Subramanian, T., Schaeper, U., La Regina, M., Bayley, S. & Chinnadurai, G. (1993) A region in the C-terminus of adenovirus 2/5 E1a protein is required for association with a cellular phosphoprotein and important for the negative modulation of T24-ras mediated transformation, tumorigenesis and metastasis. EMBO J. 12, 469–478.[Medline]
Chinnadurai, G. (2002) CtBP, an unconventional transcriptional corepressor in development and oncogenesis. Mol. Cell 9, 213–224.[CrossRef][Medline]
Cobb, B.S., Morales-Alcelay, S., Kleiger, G., Brown, K.E., Fisher, A.G. & Smale, S.T. (2000) Targeting of Ikaros to pericentromeric heterochromatin by direct DNA binding. Genes Dev.
14, 2146–2160.
Collins, T., Stone, J.R. & Williams, A.J. (2001) All in the family: the BTB/POZ, KRAB, and SCAN domains. Mol. Cell. Biol.
21, 3609–3615.
Criqui-Filipe, P., Ducret, C., Maira, S.M. & Wasylyk, B. (1999) Net, a negative Ras-switchable TCF, contains a second inhibition domain, the CID, that mediates repression through interactions with CtBP and de-acetylation. EMBO J. 18, 3392–3403.[CrossRef][Medline]
Daniel, J.M. & Reynolds, A.B. (1999) The catenin p120ctn interacts with Kaiso, a novel BTB/POZ domain zinc finger transcription factor. Mol. Cell. Biol.
19, 3614–3623.
David, G., Alland, L., Hong, S.H., Wong, C.W., DePinho, R.A. & Dejean, A. (1998) Histone deacetylase associated with mSin3A mediates repression by the acute promyelocytic leukemia-associated PLZF protein. Oncogene 16, 2549–2556.[CrossRef][Medline]
Deltour, S., Guerardel, C. & Leprince, D. (1999) Recruitment of SMRT/N-CoR-mSin3A-HDAC-repressing complexes is not a general mechanism for BTB/POZ transcriptional repressors: the case of HIC-1 and FBP-B. Proc. Natl. Acad. Sci. USA
96, 14831–14836.
Deltour, S., Pinte, S., Guerardel, C., Wasylyk, B. & Leprince, D. (2002) The human candidate tumor suppressor gene HIC1 recruits CtBP through a degenerate GLDLSKK motif. Mol. Cell. Biol.
22, 4890–4901.
Dhordain, P., Lin, R.J., Quief, S., et al. (1998) The LAZ3 (BCL-6) oncoprotein recruits a SMRT/mSIN3A/histone deacetylase containing complex to mediate transcriptional repression. Nucleic Acids Res.
26, 4645–4651.
Hickabottom, M., Parker, G.A., Freemont, P., Crook, T. & Allday, M.J. (2002) Two nonconsensus sites in the Epstein-Barr virus oncoprotein EBNA3A cooperate to bind the co-repressor carboxyl-terminal-binding protein (CtBP). J. Biol. Chem.
277, 47197–47204.
Hildebrand, J.D. & Soriano, P. (2002) Overlapping and unique roles for C-terminal binding protein 1 (CtBP1) and CtBP2 during mouse development. Mol. Cell. Biol.
22, 5296–5307.
Huynh, K.D. & Bardwell, V.J. (1998) The BCL-6 POZ domain and other POZ domains interact with the co-repressors N-CoR and SMRT. Oncogene 17, 2473–2784.[CrossRef][Medline]
Ishida, Y. & Leder, P. (1999) RET: a poly A-trap retrovirus vector for reversible disruption and expression monitoring of genes in living cells. Nucleic Acids Res.
27, e35.
Jepsen, K. & Rosenfeld, M.G. (2002) Biological roles and mechanistic actions of co-repressor complexes. J. Cell Sci.
115, 689–698.
Kaplan, J. & Calame, K. (1997) The ZiN/POZ domain of ZF5 is required for both transcriptional activation and repression. Nucleic. Acids Res.
25, 1108–1116.
Kiefer, H., Chatail-Hermitte, F., Ravassard, P., Bayard, E., Brunet, I. & Mallet, J. (2005) ZENON, a novel POZ Kruppel-Like DNA binding protein associated with differentiation and/or survival of late postmitotic neurons. Mol. Cell. Biol.
25, 1713–1729.
Kobayashi, A., Yamagiwa, H., Hoshino, H., et al. (2000) A combinatorial code for gene expression generated by transcription factor Bach2 and MAZR (MAZ-related factor) through the BTB/POZ domain. Mol. Cell. Biol.
20, 1733–1746.
Koipally, J. & Georgopoulos, K. (2000) Ikaros interactions with CtBP reveal a repression mechanism that is independent of histone deacetylase activity. J. Biol. Chem.
275, 19594–19602.
Lemercier, C., Brocard, M.P., Puvion-Dutilleul, F., Kao, H.Y., Albagli, O. & Khochbin, S. (2002) Class II histone deacetylases are directly recruited by BCL6 transcriptional repressor. J. Biol. Chem.
277, 22045–22052.
Lin, R.J., Nagy, L., Inoue, S., Shao, W., Miller, W.H. Jr & Evans, R.M. (1998) Role of the histone deacetylase complex in acute promyelocytic leukaemia. Nature 391, 811–814.[CrossRef][Medline]
Maison, C. & Almouzni, G. (2004) HP1 and the dynamics of heterochromatin maintenance. Nat. Rev. Mol. Cell Biol. 5, 296–304.[CrossRef][Medline]
Matsuda, E., Agata, Y., Sugai, M., Katakai, T., Gonda, H. & Shimizu, A. (2001) Targeting of Kruppel-associated box-containing zinc finger proteins to centromeric heterochromatin. Implication for the gene silencing mechanisms. J. Biol. Chem.
276, 14222–14229.
Matsuda, E., Shigeoka, T., Iida, R., Yamanaka, S., Kawaichi, M. & Ishida, Y. (2004) Expression profiling with arrays of randomly disrupted genes in mouse embryonic stem cells leads to in vivo functional analysis. Proc. Natl. Acad. Sci. USA
101, 4170–4174.
Melnick, A.M., Westendorf, J.J., Polinger, A., et al. (2000) The ETO protein disrupted in t(8;21)-associated acute myeloid leukemia is a corepressor for the promyelocytic leukemia zinc finger protein. Mol. Cell. Biol.
20, 2075–2086.
Nibu, Y., Zhang, H., Bajor, E., Barolo, S., Small, S. & Levine, M. (1998) dCtBP mediates transcriptional repression by Knirps, Kruppel and Snail in the Drosophila embryo. EMBO J. 17, 7009–7020.[CrossRef][Medline]
Peters, A.H., Kubicek, S., Mechtler, K., et al. (2003) Partitioning and plasticity of repressive histone methylation states in mammalian chromatin. Mol. Cell 12, 1577–1589.[CrossRef][Medline]
Prokhortchouk, A., Hendrich, B., Jorgensen, H., et al. (2001) The p120 catenin partner Kaiso is a DNA methylation-dependent transcriptional repressor. Genes Dev.
15, 1613–1618.
Richards, E.J. & Elgin, S.C. (2002) Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell 108, 489–500.[CrossRef][Medline]
Riefler, G.M. & Firestein, B.L. (2001) Binding of neuronal nitric-oxide synthase (nNOS) to carboxyl-terminal-binding protein (CtBP) changes the localization of CtBP from the nucleus to the cytosol: a novel function for targeting by the PDZ domain of nNOS. J. Biol. Chem.
276, 48262–48268.
Ruthardt, M., Orleth, A., Tomassoni, L., et al. (1998) The acute promyelocytic leukaemia specific PML and PLZF proteins localize to adjacent and functionally distinct nuclear bodies. Oncogene 16, 1945–1953.[CrossRef][Medline]
Shestakova, E.A., Mansuroglu, Z., Mokrani, H., Ghinea, N. & Bonnefoy, E. (2004) Transcription factor YY1 associates with pericentromeric gamma-satellite DNA in cycling but not in quiescent (G0) cells. Nucleic Acids Res.
32, 4390–4399.
Shi, Y., Sawada, J., Sui, G., et al. (2003) Coordinated histone modifications mediated by a CtBP co-repressor complex. Nature 422, 735–738.[CrossRef][Medline]
Sundqvist, A., Bajak, E., Kurup, S.D., Sollerbrant, K. & Svensson, C. (2001) Functional knockout of the corepressor CtBP by the second exon of adenovirus E1a relieves repression of transcription. Exp. Cell Res. 268, 284–293.[CrossRef][Medline]
Tashiro, S., Muto, A., Tanimoto, K., et al. (2004) Repression of PML nuclear body-associated transcription by oxidative stress-activated Bach2. Mol. Cell. Biol.
24, 3473–3484.
Yoon, H.G., Chan, D.W., Reynolds, A.B., Qin, J. & Wong, J. (2003) N-CoR mediates DNA methylation-dependent repression through a methyl CpG binding protein Kaiso. Mol. Cell 12, 723–734.[CrossRef][Medline]
Zhang, C.L., McKinsey, T.A., Lu, J.R. & Olson, E.N. (2001) Association of COOH-terminal-binding protein (CtBP) and MEF2-interacting transcription repressor (MITR) contributes to transcriptional repression of the MEF2 transcription factor. J. Biol. Chem.
276, 35–39.
Zollman, S., Godt, D., Prive, G.G., Couderc, J.L. & Laski, F.A. (1994) The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc. Natl. Acad. Sci. USA
91, 10717–10721.
Received: 20 April 2005
Accepted: 29 May 2005
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