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J. Biol. Chem., Vol. 277, Issue 10, 8202-8208, March 8, 2002

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Isolation and Characterization of XKaiso, a Transcriptional Repressor That Associates with the Catenin Xp120^ctn in Xenopus laevis^*

Si Wan Kim, Xiang Fang, Hong Ji, Alicia F. Paulson, Juliet M. Daniel^§, Malgorzata Ciesiolka^¶, Frans van Roy^¶, and Pierre D. McCrea^**

From the  Department of Biochemistry and Molecular Biology,  Program in Genes and Development, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, the ^§ Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada, and the ^¶ Department of Molecular Biology, VIB-University of Gent, Ledeganckstraat 35, B-9000 Gent, Belgium

Received for publication, October 2, 2001, and in revised form, December 6, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Armadillo family of catenin proteins function in multiple capacities including cadherin-mediated cell-cell adhesion and nuclear signaling. The newest catenin, p120^ctn, differs from the classical catenins and binds to the membrane-proximal domain of cadherins. Recently, a novel transcription factor Kaiso was found to interact with p120^ctn, suggesting that p120^ctn also possesses a nuclear function. We isolated the Xenopus homolog of Kaiso, XKaiso, from a Xenopus stage 17 cDNA library. XKaiso contains an amino-terminal BTB/POZ domain and three carboxyl-terminal zinc fingers. The XKaiso transcript was present maternally and expressed throughout early embryonic development. XKaiso's spatial expression was defined via in situ hybridization and was found localized to the brain, eye, ear, branchial arches, and spinal cord. Co-immunoprecipitation of Xenopus p120^ctn and XKaiso demonstrated their mutual association, whereas related experiments employing differentially epitope-tagged XKaiso constructs suggest that XKaiso additionally self-associates. Finally, reporter assays employing a chimera of XKaiso fused to the GAL4 DNA binding domain indicate that XKaiso is a transcriptional repressor. These data suggest that XKaiso functions throughout development and that its repressor functions may be most apparent in the context of neural tissues. The significance of the XKaiso-p120^ctn interaction has yet to be determined, but it may include transducing information from cadherin-mediated cell-cell contacts to transcriptional processes within the nucleus.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intercellular adherens junctions composed of cadherin-catenin complexes have roles not only in embryonic development but also in maintaining adult tissue integrity and differentiated cellular identity (1-3). The Armadillo family of catenins are a group of proteins characterized by the presence of 10-13 Armadillo repeats, which bind to cadherin intracellular domains (4-7). In the case of the classical catenins -catenin and plakoglobin, they also bind to the cortical actin cytoskeleton via proteins including -catenin and -actinin (8, 9). In addition to the modulation of cadherin-mediated cell-cell contacts at the plasma membrane (10, 11), the catenins have nuclear functions. Best characterized is -catenin, which, in the context of the Wnt signaling pathway (12-14), translocates from the cytosol into the nucleus to selectively regulate gene transcription in conjunction with members of the LEF/TCF (for lymphoid enhancer factor/T cell factor)¹ family of transcription factors (15-18).

Another member of the Armadillo protein family is the catenin p120^ctn, a substrate of Src kinase and receptor-tyrosine kinases (19-22) and protein-tyrosine phosphatases (23, 24). Accumulating evidence indicates that p120^ctn regulates both cadherin-mediated cell adhesion (25-31) and the activity of small G-proteins controlling actin filament-dependent cell adhesion and motility (32-34). p120^ctn is also present in the nucleus, a localization believed to be influenced by the extent of its nuclear export and by cadherin expression levels (35, 36). Recently, a nuclear transcription factor interacting with p120^ctn was isolated through yeast two-hybrid screening of a mouse cDNA library (37). This protein, named Kaiso, was found to be a BTB/POZ (for Broad-Complex, Tramtrack, and Bric à brac/poxviruses and zinc finger) protein (38-40) containing a BTB/POZ protein-protein interaction domain at the amino terminus and three Cys-2-His-2 type DNA-binding zinc fingers at the carboxyl terminus. Immunofluorescence analysis of Kaiso showed it localized to the nucleus (37). More recently, Kaiso was shown to specifically bind nonmethylated DNA sequences and to bind other distinct DNA sequences in a methylation-dependent manner in mammalian cells (41).² This work also indicated that Kaiso's binding to DNA targets occurs via its zinc finger domain and suggested the further association of co-repressors such as the histone deacetylase complex and/or components of methyl-CpG binding complexes in effecting transcriptional repression and/or gene silencing. While Kaiso interacts with p120^ctn, Kaiso does not bind the related Armadillo domain-containing protein -catenin (37), indicating that Kaiso in complex with p120^ctn has a nuclear function distinct from that of LEF/TCF when associated with -catenin upon Wnt signaling, a view further supported by exogenous expression studies of p120^ctn carried out in developing Xenopus embryos (26, 30).

The BTB/POZ proteins are generally divided into two groups, actin-binding proteins and DNA-binding proteins (39). Nuclear BTB/POZ members have been characterized in mammalian cells and invertebrates such as Drosophila. Some mammalian BTB/POZ proteins have been found to be oncogenic. For example, the BCL6 gene is rearranged in human non-Hodgkin's lymphomas (43, 44), and the promyelocytic leukemia zinc finger (PLZF) protein is fused to the retinoic acid receptor  in translocations associated with acute promyelocytic leukemia (45). Drosophila BTB/POZ proteins have been found in turn to be important in development. For example, Tramtrack is a repressor of pair-rule segmentation genes such as ftz, eve, runt, and odd (46) and is required for cell fate determination in the Drosophila eye (47). Another BTB/POZ protein, Broad-Complex, is involved in Drosophila metamorphosis (48, 49). A Xenopus BTB/POZ zinc finger protein, Champignon, has been recently isolated and shown to interfere with gastrulation movements when exogenously expressed during embryonic development (50).

To better understand the nuclear function of p120^ctn during development, we isolated the Xenopus homolog of Kaiso, XKaiso, from a neurula stage Xenopus cDNA library (51). XKaiso was found to be highly homologous with Kaiso especially within the BTB/POZ and zinc finger domains. The temporal and spatial expression pattern of the XKaiso transcript was determined via RT-PCR analysis and in situ hybridization, respectively, revealing localized expression in neuronal tissues. XKaiso specifically associated with Xenopus p120^ctn (Xp120^ctn),³ whereas co-immunoprecipitation experiments employing differentially epitope-tagged XKaiso constructs additionally suggested the existence of XKaiso homodimers and/or higher order oligomers. XKaiso behaved as a transcriptional repressor when assayed in the context of a chimeric fusion with the GAL4 DNA binding domain (GAL4BD), a characteristic shared with other members of the BTB/POZ zinc finger family. This study represents the first characterization of XKaiso, a direct binding partner of Xp120^ctn, in a developmental context. Experiments are under way to determine the function of XKaiso and the XKaiso-Xp120^ctn complex.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNA Cloning-- We employed standard hybridization screening methods to obtain the full-length XKaiso cDNA from a Xenopus stage 17 embryo cDNA library constructed in the gt10 vector (51). The random primed probe (Random Primers DNA Labeling System; Invitrogen) was generated by PCR amplification (50 °C and 25 cycles) using the Xenopus library as template and a mouse Kaiso primer set (5'-GCTACAGACATTCAGTAC-3', 5'-TTGTTCTGAGGAGGAGTA-3'), corresponding to coding regions within the BTB/POZ and zinc finger domains, respectively (37). The hybridization was carried out at 50 °C for 12-16 h according to published procedures (52). The membranes were then washed 3-5 times in 2× SSC (20× SSC: 17.5% NaCl, 8.8% sodium citrate, pH 7.0) and 0.1% SDS at room temperature for a total of 20 min and washed once in 2× SSC, 0.1% SDS at 50 °C for 15 min. Positive clones were further identified by PCR (55 °C and 30 cycles) with gt10-specific primers using the Expand High Fidelity PCR System (Roche Molecular Biochemicals). Clones were sequenced at the institutional core sequencing facility, and a full-length cDNA was generated from the cDNA library by PCR (55 °C and 30 cycles) with the Xenopus Kaiso-specific primers that were designed based on the above known coding sequence. The product was then gel-purified using a GeneClean kit (Bio 101) and sequenced. The XKaiso sequence is available from GenBank^TM under accession number AF420316.

Embryos-- Xenopus eggs were obtained and fertilized using standard methods (53). Embryos were dejellied by treating with 2% cysteine HCl (pH 8.0) for 5 min before the first cleavage stage and were rinsed and incubated in 0.1× MMR (10× MMR: 100 mM NaCl, 2 mM KCl, 1 mM MgSO₄, 2 mM CaCl₂, 5 mM HEPES, pH 7.4). Embryo stages were determined by observation under a standard binocular dissecting microscope (model SMZ-U; Nikon) based on the normal table of Xenopus laevis development (54).

Microinjection-- mRNA or plasmid DNA constructs were microinjected into the animal hemisphere of two blastomeres in 2-4 cell stage embryos incubated in 5% Ficoll, 0.3× MMR (pH 7.4). The Ficoll solution was replaced with 0.1× MMR (pH 7.4) 1 h after the injection. Embryos were injected using borosilicate glass capillary tubes (0.75 mm in diameter; Sutter) drawn out using a P-30 pulling instrument (Sutter) and bevelled by a K. T. Brown Type Micropipette beveller (Sutter). The total volume of DNA or RNA injected ranged from 20 to 40 nl (total doses from 250 to 500 pg) as manipulated by the NA-1 oil-driven microinjector (Sutter).

cDNA Constructs-- XKaiso was differentially tagged on its amino terminus with three hemagglutinin (HA) or six myc epitopes. A XKaiso full-length cDNA was amplified by PCR using the following primers: for HA tag, 5'-GCTCTAGAAATGGAGACAAAAAAGCTG-3' and 5'-GCTCTAGACTAGTACGATTCTGGTAT-3'; for myc tag, 5'-GCTCTAGAATGGAGACAAAAAAGCTG and 5'-GCTCTAGACTAGTACGATTCTGGTAT-3'. The resulting products were cloned in frame within the vectors pCS2+3HA or pCS2+MT (55, 56) to generate XKaiso/pCS2 + 3HA or XKaiso/pCS2+MT, respectively. A XKaiso construct with carboxyl-terminal triplet HA epitopes (XKaiso/pCS2+C-3HA) was generated by PCR (using primers 5'-AAGGCCTATGGAGACAAAAAAGCTG-3' and 5'-AAGGCCTGTACGATTCTGGTATTAC-3') and subcloned in frame into pCS2+C-3HA at the StuI site. A XKaiso frameshifted construct (XKaisoFS) was engineered by SacI restriction digestion of the XKaiso cDNA coding sequence at base pair 131, T4 DNA polymerase (Roche Molecular Biochemicals) removal of the 3'-overhangs, and T4 DNA ligase (Roche Molecular Biochemicals) blunt end religation of the preexisting insert within the pCS2+3HA vector.

A cDNA encoding a carboxyl-terminal fragment of a Xenopus p120^ctn-like protein was cloned by screening a yeast two-hybrid library of X. laevis oocytes using a membrane-proximal cytoplasmic domain of Xenopus N-cadherin as a bait.³ This p120^ctn-like cDNA was completed by 5'-rapid amplification of cDNA ends (GenBank^TM accession number AF150744) and turned out to be a true Xenopus ortholog of human p120^ctn isoform 1AD (57, 58), showing highest homology in the central Armadillo domain. Because it is the longer of two Xenopus isoforms isolated, it was designated Xp120^ctniso1. A derivative construct having six carboxyl-terminal myc epitopes (Xp120iso1/pCS2+C-MT) was generated by PCR (using primers 5'-GAATTCGATGGATGAGCCAGAG-3' and 5'-GAATTCACACGCTGATCTTC-3') and subcloned in frame into pCS2+C-MT at the EcoRI site. A Xenopus ARVCF (for Armadillo gene deleted in velocardiofacial syndrome) isoform 1B (Xarvcf1B) (59) construct having three amino-terminal HA epitopes (Xarvcf1B/pCS2+3HA) was generated by PCR (using primers 5'-GCTCTAGAATGATGCAGGAACC-3' and 5'-GCTCTAGACCCAAAAAGGGTCACTGC-3') and subcloned in frame into pCS2+3HA at the XbaI site.

To generate a template construct for labeling of in situ hybridization probe of XKaiso, XKaiso full-length cDNA was amplified by PCR using the following primers: 5'-CCATCGATATGGAGACAAAAAAGCTGA-3' and 5'-CCGCTCGAGCGTACGATTCTGGTATTACAAAC-3'. The resulting products were cloned into the TA cloning vector pCRII (Invitrogen) to generate XKaiso/pCRII.

To generate fusion constructs with the GAL4BD, XKaiso, Xp120^ctniso1, and Xenopus ARVCF isoform 1A (Xarvcf1A) (59) were cloned into the plasmid pGBT9 (CLONTECH) at the SmaI, EcoRI, and EcoRI sites, respectively. The following oligonucleotides were used for PCR: for XKaiso, 5'-TCCCCCGGGGATGGAGACAAAAAAGCTG-3' and 5'-TCCCCCGGGCTAGTACGATTCTGGTATTAC-3'; for Xp120^ctniso1, 5'-GGAATTCATGGATGAGCCAGAGTCT-3' and 5'-GGAATTCTTAGACACGCTGATCTTCAG-3'; for Xarvcf1A, 5'-GGAATTCATGCCTGCCGAACTCCAA-3' and 5'-GGAATTCTTAGACCCAGGAGTCAAC-3'. The GAL4BD-fused XKaiso, Xp120^ctniso1, and Xarvcf1A were then PCR-amplified and subcloned into pCS2+ (55, 56) to generate GAL4XKaiso/pCS2+, GAL4Xp120iso1/pCS2+, and GAL4Xarvcf1A/pCS2+, respectively. The PCR primers used were as follows: for GAL4BDXKaiso, 5'-AAGGCCTATGAAGCTACTGTCTTCT-3' and 5'-AAGGCCTCCCGGGCTAGTACGATT-3'; for GAL4BDXp120^ctniso1, 5'-AAGGCCTATGAAGCTACTGTCTTCT-3' and 5'-AAGGCCTGAATTCTTAGACACGCTG-3'; for GAL4BDXarvcf1A, 5'-AAGGCCTATGAAGCTACTGTCTTCT-3' and 5'-AAGGCCTGAATTCTTAGACCCAGGA-3'.

PCR was conducted using the Expand High Fidelity PCR system (Roche Molecular Biochemicals), and the insert sequence of each construct was verified by sequencing at the institutional core sequencing facility.

In Vitro Transcription-- All DNA constructs were transcribed in vitro into capped mRNA using the SP6 mMessage mMachine kit according to the manufacturer's protocol (Ambion). Unincorporated nucleotides were removed by filtration through Quick Spin Columns, Sephadex G-50 (Roche Molecular Biochemicals). The concentration and integrity of the mRNA were determined by measuring the optical density (OD_260/280) and mobility on standard RNA formaldehyde agarose gels (52).

Immnunoprecipitation and Western Blotting-- Embryos were coinjected with 500 pg each of mRNA of epitope-tagged XKaiso and Xp120^ctn, XKaiso, and Xarvcf1B or with differentially tagged XKaisos. The injected embryos were harvested at stage 9, and whole embryo lysates were prepared by pipetting with a prechilled TX buffer (10 mM HEPES, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.5% Triton X-100, pH 7.4) supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 4 µg/ml aprotinin, 1 µg/ml pepstatin A, 2 µg/ml leupeptin, 10 µg/ml antipain, 50 µg/ml benzamidine, 10 µg/ml soybean trypsin inhibitor, 100 µg/ml iodoacetamide, and 40 µg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone). Yolk proteins and cellular debris were cleared from lysates by centrifugation at 14,000 × g, 4 °C for 15 min. The lysates was incubated with anti-HA (12CA5) or anti-myc (9E10) monoclonal antibodies at 1:1,000 dilution by rotation at 4 °C for at least 1 h. Protein A- and G-Sepharose 4B beads (Sigma) were then added and incubated for an additional 1 h. The resulting immunocomplexes were precipitated at 14,000 × g, 4 °C for 10 s. The precipitates were washed in prechilled TX buffer and resuspended in an SDS sample buffer (125 mM Tris, 4% SDS, 20% glycerol, 2% -mercaptoethanol, 1% bromphenol blue, pH 6.8). The resuspension was boiled for 5 min in the presence of 20 mM dithiothreitol. The samples were then electrophoresed on 8% polyacrylamide gels and transferred to nitrocellulose membranes. The blots were probed with anti-HA (1:4,000 dilution) or anti-myc antibodies (1:4,000 dilution), followed by a second incubation with goat anti-mouse antibodies (1:3,000 dilution) conjugated to horseradish peroxidase (Bio-Rad). The signal was detected with enhanced chemiluminescence (Amersham Biosciences).

RT-PCR Analysis-- Total RNA was isolated from frog eggs and embryos using Trizol (Life Technologies, Inc.) according to the manufacturer's protocol. To detect transcripts by PCR, cDNA was generated from 1 µg of total RNA using Moloney murine leukemia virus reverse transcriptase (Roche Molecular Biochemicals), employing random hexamers (Roche Molecular Biochemicals). The RT-PCR assays were semiquantitative. The linear range for each primer set was determined by removing 5-µl aliquots from the PCR tube at five-cycle intervals and comparing band intensities of the products resolved on agarose gels. The PCR conditions and primers used were as follows: for XKaiso transcripts, 50 °C, 30 cycles, 5'-ATCTCCATCGAAACCTGGC-3', 5'-TTTTCCCAGGAATGGACG-3'; for ornithine decarboxylase, 55 °C, 25 cycles, 5'-GGAGCTGCAAGTTGGAGA-3', 5'-CTCAGTTGCCAGTGTGGTC-3'.

Whole-mount in Situ Hybridization-- Digoxigenin-labeled RNA probes were prepared using the DIG RNA labeling kit (Roche Molecular Biochemicals). XKaiso/pCRII was digested with NotI (sense linearization) or SpeI (antisense linearization) and then treated with SP6 and T7 polymerase to produce the sense and antisense probes, respectively. Eggs and embryos of different stages were collected from albino frogs and processed using the published protocol (53).

Luciferase Assay-- Embryos were coinjected with 250 pg of mRNA of different GAL4 fusion constructs and 250 pg of luciferase reporter plasmid, p17X4TKlucSV40pA (a generous gift from Drs. Zafar Nawaz and Ming Tsai, Baylor College of Medicine), containing a minimal thymidine kinase promoter under the control of four GAL4 binding sites. The injected embryos at the early gastrula stage (stages 10 and 11) were harvested into three separate groups of five for each assay. The collected embryos were lysed and assayed for luciferase activity using the Luciferase Assay System according to the manufacturer's protocol (Promega). Uninjected embryo lysates were used to measure base-line activity. All experiments were repeated a minimum of three times.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

XKaiso cDNA Isolation-- To isolate the Xenopus homolog of Kaiso, XKaiso, a screen was undertaken of a Xenopus stage 17 embryo cDNA library using the mouse Kaiso coding region to design primers for generating the hybridization probe (described under "Experimental Procedures"). A full-length cDNA was isolated that showed overall 53% identity and 66% amino acid similarity compared with murine Kaiso (Fig. 1). XKaiso contains an amino-terminal BTB/POZ domain, generally involved in protein-protein interactions, and a carboxyl-terminal zinc finger domain containing three Cys-2-His-2 zinc fingers, shown in murine Kaiso¹ and other family members to bind DNA (39). The BTB/POZ and zinc finger domains of XKaiso show 87 and 90% amino acid identity, respectively, to mouse Kaiso, suggesting that we have isolated the Xenopus ortholog.

View larger version (89K): [in this window] [in a new window]   Fig. 1.   XKaiso sequence. The deduced amino acid sequence of XKaiso is aligned with that of mouse Kaiso. Sequences in shaded and boldface type represent the presence of identical amino acid residues, and boxed sequences indicate the presence of identical and/or similar residues. The BTB/POZ domain residues (amino acids 12-117) are indicated by a filled line above the sequence, while the residues of three Cys-2-His-2 zinc fingers are indicated by gray lines above the sequence. The XKaiso sequence is available from GenBankTM under accession number AF420316.

Temporal Expression of XKaiso-- To determine the temporal expression patterns of XKaiso, semiquantitative RT-PCR analysis was performed on total RNAs isolated from Xenopus eggs and embryos of different developmental stages. As shown in Fig. 2, the XKaiso transcript was present maternally and expressed throughout early embryonic development. The XKaiso transcript was not detectable via Northern blotting using 20 µg of total RNA of varying Xenopus embryonic stages and a ³²P-labeled random primed probe (data not shown). The low expression levels of XKaiso were further suggested by our inability to detect the XKaiso protein using an anti-XKaiso polyclonal antibody that effectively resolved exogenously expressed XKaiso. In all events, XKaiso's RT-PCR temporal expression and whole-mount in situ patterns (Figs. 2 and 3) indicate that XKaiso mRNA is continually present from the egg to early tadpole stages of embryonic development.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   RT-PCR analysis of XKaiso expression at the indicated embryonic stages. Top panel, semiquantitative RT-PCR evaluation of total XKaiso mRNA transcript expression at the indicated developmental stages. Bottom panel, control RT-PCR of ornithine decarboxylase that was used as a reaction and loading control. The XKaiso transcript is present throughout development.

View larger version (77K): [in this window] [in a new window]   Fig. 3.   Whole-mount in situ hybridization analysis of XKaiso expression in early Xenopus development. Spatial expression pattern of XKaiso was evaluated via whole mount in situ hybridization using a dioxigenin-labeled antisense (A-K) and sense (B', D', K') XKaiso full-length RNA probe. Developmental stages: egg (A), cleavage (B) (stage 3); blastula (C) (stage 8); gastrula (D) (stage 11); neurula (E) (stage 17) (dorsal); neurula (F) (stage 17) (ventral); neurula (G) (stage 17) (anterior); neurula (H) (stage 17) (posterior); early tailbud (I) (stage 23) (dorsal); early tailbud (J) (stage 23) (lateral); tailbud (K) (stage 28); cleavage (B') (stage 3); gastrula (D') (stage 11); tailbud (K') (stage 28). The XKaiso transcript is principally present in neural cell precursors and neural tissues.

Spatial Expression of XKaiso-- The spatial expression pattern of XKaiso was examined by whole-mount in situ hybridization of Xenopus embryos using a dioxigenin-labeled antisense XKaiso full-length RNA probe (Fig. 3). In situ hybridization of sectioned embryos further confirmed the expression pattern (data not shown), while a series of negative control embryos hybridized and processed in parallel with the complimentary sense XKaiso RNA probe did not show any significant signal (Fig. 3 (B', D', and K') and data not shown). XKaiso was revealed to be expressed in the animal hemisphere of eggs (Fig. 3A) and cleavage stage embryos (stage 3, Fig. 3B). At blastula (stage 8, Fig. 3C) and gastrula stages (stage 11, Fig. 3D), XKaiso was expressed in the ectodermal region. At neurula stages, the anterior (stage 17, Fig. 3G) and dorsal regions (stage 17, Fig. 3E) displayed localized XKaiso signals. Most prominently, tailbud stage embryos showed a distinctive expression pattern in the brain, eye, ear, branchial arches, and spinal cord (stage 28, Fig. 3K), indicating that the XKaiso transcript was principally present within neuronal cell derivatives.

Interaction of XKaiso and Xp120^ctn-- Mouse Kaiso was first identified as a direct binding partner of p120^ctn following a yeast two-hybrid screen and subsequent co-immunoprecipitation analysis (37). To determine if XKaiso is likewise capable of associating with Xp120^ctn, co-immunoprecipitation experiments were performed after expressing differentially epitope-tagged XKaiso and Xp120^ctn constructs in Xenopus embryos. As shown in Fig. 4A, Xp120^ctniso1 tagged on its carboxyl terminus with myc epitopes co-immunoprecipitated with XKaiso tagged on its carboxyl terminus with HA epitopes (lane 3). Similar levels of the immunocomplex were precipitated in experiments using differentially amino-terminal tagged forms of Xp120^ctniso1 and XKaiso or Xp120^ctn isoform 2 and XKaiso (data not shown). Another member of the p120^ctn subfamily of Armadillo proteins (7, 60), ARVCF, was recently identified in Xenopus (59). To determine the specificity of the interaction of XKaiso with p120^ctn, we assessed if Xarvcf1B could be co-immunoprecipitated with XKaiso (Fig. 4B). Xarvcf1B was not able to co-immunoprecipitate with XKaiso (lane 3), lending further support to XKaiso's specific association with Xp120^ctn and to XKaiso being the Xenopus ortholog of mouse Kaiso.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Specific interaction of XKaiso with Xp120ctn. A, embryos were coinjected with mRNA coding for XKaiso tagged on the carboxyl terminus with HA epitopes (XKaisoHA) and Xp120ctniso1 tagged on the carboxyl terminus with myc epitopes (Xp120myc). Following immunoprecipitation of Xp120myc or XKaisoHA, the coimmunoprecipitation of Xp120myc was detected from Xenopus whole embryo lysates blotted with anti-myc monoclonal antibody. B, embryos were coinjected with mRNA coding for XKaiso tagged with myc epitopes (XKaisomyc) and Xarvcf1B tagged with HA epitopes (XarvcfHA) and were immunoblotted for XarvcfHA following immunoprecipitation of XKaisomyc or XarvcfHA. Xp120myc robustly coimmunoprecipitated with XKaisoHA in contrast to XarvcfHA.

Homomeric Interaction of XKaiso-- Previously identified BTB/POZ proteins, such as the ZID (for zinc finger with interaction domain) protein (38), PLZF, Tramtrack, and Bric à brac (61), have been found to homodimerize via their respective BTB/POZ domains (38, 62). In addition, murine Kaiso has been shown to homodimerize (37). The BTB/POZ domains also mediate heteromeric interactions, for example, Tramtrack and PLZF interact with Drosophila GAGA factor (63, 64) and BCL6 (38, 65), respectively. To determine whether XKaiso might homoassociate, we tested whether differentially epitope-tagged XKaiso constructs could be co-immunoprecipitated following the microinjection of their respective mRNAs in Xenopus embryos. As shown in Fig. 5, HA-tagged XKaiso co-immunoprecipitated with myc-tagged XKaiso (lane 1), suggesting that XKaiso homo-oligomerizes, probably as a homodimer in keeping with other BTB/POZ family members.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Homomeric interaction of XKaiso. Embryos were coinjected with mRNA coding for differentially tagged XKaiso constructs (XKaisomyc and XKaisoHA). Following immunoprecipitation of XKaisomyc or XKaisoHA, the coimmunoprecipitation of XKaisoHA was detected from Xenopus whole embryo lysates blotted with anti-HA monoclonal antibody, indicating the existence of XKaiso homodimers or higher order oligomers.

Transcriptional Repressor Activity of XKaiso-- A majority of BTB/POZ zinc finger proteins act as transcriptional repressors (66-72), interacting with transcriptional corepressors such as the silencing mediator of retinoid and thyroid hormone receptor, the nuclear receptor corepressor, and histone deacetylase (73-75). To assess the transregulatory activity of XKaiso, full-length XKaiso was fused to the GAL4 DNA binding domain (XKaiso-GAL4BD chimera), and its mRNA was co-injected into early Xenopus embryos with a GAL4-responsive luciferase reporter plasmid. As shown in Fig. 6, XKaiso-GAL4BD repressed the reporter's basal transcriptional activity by 10-20-fold compared with the expression of GAL4BD alone. Full-length Xp120^ctn-GAL4BD or Xarvcf1A-GAL4BD had significantly lesser effects on the reporter's luciferase activity. The reporter activity observed upon the co-expression of full-length Xp120^ctn and XKaiso-GAL4BD was similar to that obtained upon expressing XKaiso-GAL4BD alone, and likewise, the co-expression of full-length XKaiso and Xp120^ctn-GAL4BD resulted in reporter activities equivalent to those observed following the isolated expression of Xp120^ctn-GAL4BD (data not shown). Frameshifted XKaiso (XKaisoFS; see "Experimental Procedures"), and nucleus-localized -galactosidase (nbgal), were tested as respective mRNA and protein injection controls (55, 56). While neither control grossly altered the reporter's basal activity relative to XKaiso-GAL4BD, the reduction observed was consistent and might reflect the nonspecific titration of protein co-factors directly or indirectly employed in basal reporter transcription. As a positive control of the system (76, 77), a VP16-GAL4BD fusion construct was expressed, resulting in 5-7-fold reporter activation. These data suggest that in keeping with a number of other BTB/POZ zinc finger proteins (66-72), XKaiso functions as a transcriptional repressor, a view further supported by recent work in mammalian cell lines (41).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Repressive activity of XKaiso on the transcriptional reporter. A GAL4 responsive reporter plasmid, p17X4TKlucSV40pA, was coinjected with mRNA coding for chimeric fusions of GAL4BD with XKaiso, Xp120ctniso1, or Xarvcf1A (XKaiso-, Xp120-, or Xarvcf-GAL4BD), and luciferase reporter activities were measured. The VP16-GAL4BD fusion (VP16-GAL4BD) mRNA was coinjected with the reporter as an activation control. Frameshifted XKaiso mutant (XKaisoFS; see "Experimental Procedures") or nucleus-localized -galactosidase (nbgal) mRNA were respectively coinjected as mRNA or protein expression controls. Exogenous expression of XKaiso consistently generated 10-20-fold repressions of the luciferase reporter, while significantly lesser effects arose from expression of Xp120ctn or Xarvcf.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We report here the isolation and initial characterization of Xenopus Kaiso, XKaiso, from a hybridization screen of a Xenopus stage 17 embryo cDNA library. We show that XKaiso contains an amino-terminal BTB/POZ domain and a carboxyl-terminal zinc finger domain with an overall identity of 53% to its mouse ortholog (37). The XKaiso transcript was present maternally and expressed throughout early embryonic development. XKaiso's spatial expression was defined via in situ hybridization, which revealed early expression in the embryonic ectoderm and later localized expression in the brain, eye, ear, branchial arches, and spinal cord. Co-immunoprecipitation of Xp120^ctn and XKaiso demonstrated their specific mutual association, while related experiments employing differentially epitope-tagged constructs of XKaiso suggest that it homooligomerizes, consistent with the homodimerization reported for other BTB/POZ zinc finger family members (37, 38, 62). Luciferase reporter assays employing a chimeric GAL4BD fusion of XKaiso indicate that XKaiso is a transcriptional repressor.

The spatial expression pattern of the Drosophila BTB/POZ zinc finger proteins, Tramtrack, Bric à brac, and Broad-Complex, have been reported in developing embryos (42, 78, 79). Whole-mount in situ hybridization of Xenopus embryos revealed that XKaiso is expressed in the ectodermal region of blastula and gastrula stage embryos and at the anterior and dorsal regions of neurula stage embryos, patterns reflecting the positions of neural precursor cells. In tailbud stage embryos, a distinctive expression pattern was observed in the brain, eye, ear, branchial arches, and spinal cord, indicating that the XKaiso transcript is indeed most highly expressed within neuronal cell derivatives, perhaps reflecting greater functionality within these tissues.

Yeast two-hybrid analysis and co-immunoprecipitation showed that mouse Kaiso interacts with mouse p120^ctn (37). Regions including and adjoining Kaiso's zinc finger domain associate with p120^ctn, which in turn binds Kaiso via its central Armadillo domain (37). It is likely that corresponding regions of XKaiso and Xp120^ctn are responsible for their association in Xenopus embryos. In keeping with this, the Armadillo domain shows the highest homology between Xenopus and human p120^ctn proteins.³ Another Xenopus member of the p120^ctn subfamily, Xarvcf1B, did not interact with XKaiso, indicating that the interaction of XKaiso and Xp120^ctn is specific.

Other BTB/POZ zinc finger proteins such as mouse Kaiso, PLZF, and Tramtrack are known to form homodimers (37, 38, 62). Differentially tagged XKaiso constructs were found to co-immunoprecipitate with each other indicating that XKaiso is likewise likely to dimerize. In addition to homomeric interactions, some BTB/POZ zinc finger members engage in heteromeric interactions with other BTB/POZ zinc finger proteins, in particular BCL6 with PLZF (38, 65) and Tramtrack with GAGA factor (63, 64). BCL6 and PLZF have also been shown to interact with transcriptional repressors such as histone deacetylase, silencing mediator of retinoid and thyroid hormone receptor, and nuclear receptor corepressor (73-75). Subsequently, the identification of XKaiso binding partners in addition to Xp120^ctn will permit us to understand better the function of XKaiso at the molecular level.

While the gene targets or nuclear roles of XKaiso are unknown, we determined that a chimera of XKaiso, XKaiso-GAL4BD, repressed basal transcription from a GAL4 luciferase reporter. This result is further supported by recent reports that Kaiso acts as a transcriptional repressor in mammalian cells upon binding methylation-dependent (CpG) DNA sequences as well as having the capacity to associate with specific and distinct nonmethylated DNA consensus motifs (41).² This work further indicated that Kaiso's binding to DNA targets occurs via its zinc finger domain and that its association with components of methyl-CpG binding complexes and/or likely association with additional co-repressors such as the histone deacetylase complex assists in promoting transcriptional gene silencing and/or repression.

Ultimately, it will be important to resolve the developmental signaling pathways governing the expression and activity of XKaiso, which in turn are likely to be linked to those influencing Xp120^ctn. p120^ctn is found in the cytoplasm and nucleus but is most prominently localized to the plasma membrane in cells expressing cadherins (31, 36). Because p120^ctn binds cadherin membrane proximal domains and probably modulates their function (25-31) and that of small G-proteins (32-34), it is conjectured that XKaiso's nuclear functions are indirectly or directly linked to adhesive events taking place at the plasma membrane and/or actin-associated events contributing to cell motility. If this proves correct, XKaiso will represent a nuclear partner of Xp120^ctn facilitating the transcriptional integration of adhesion and motility information. Because -catenin bears homology to p120^ctn (both contain central Armadillo repeats) and -catenin is capable of relieving LEF/TCF-mediated repression of Wnt pathway gene targets, we were curious to test if Xp120^ctn might analogously relieve XKaiso mediated repression. While no such relief of repression was observed upon co-expressing Xp120^ctn with XKaiso (and conversely no additional repression was apparent), the GAL4 fusion system we employed is artificial in nature, leaving open the possibility that Xp120^ctn modulates XKaiso's transcriptional functions in an in vivo context. The physiological roles of XKaiso and Xp120^ctn will be more easily addressed following the identification of XKaiso's consensus binding site(s) and endogenous gene targets.

	ACKNOWLEDGEMENTS

We are grateful to Werner Montross, Dr. Agnes Chan, Byong Su Kim, Dr. Kwang Won Seo, and Charles Chung for providing technical advice, to Dr. Zafar Nawaz and Dr. Ming Tsai for providing needed reagents, and to Travis Vaught and Jon P. Lyons for reading the manuscript.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health RO1 Grant GM 52112, a Pharmacia/Monsanto Research Award, a Kleberg Foundation Award, and Cancer Center Support Grant Funds CCSG-CA 16672 (to P. D. M.) and by the Fund for Scientific Research-Flanders and Fortis Verzekeringen (Belgium) (to F. V. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF420316.

^** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Box 117, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-8979; Fax: 713-791-9478; E-mail: pmccrea@odin.mdacc.tmc.edu.

Published, JBC Papers in Press, December 19, 2001, DOI 10.1074/jbc.M109508200

³ M. Ciesiolka, A. Vanlandschoot, K. Staes, K. Vlemìnckx, and F. van Roy, manuscript in preparation.

² J. M. Daniel, C. M. Spring, H. C. Crawford, A. B. Reynolds, and A. Baig, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: LEF/TCF, lymphoid enhancer factor/T cell factor; PLZF, promyelocytic leukemia zinc finger; Xp120^ctniso1, Xenopus p120^ctn isoform 1; GAL4BD, GAL4 DNA binding domain; HA, hemagglutinin; ARVCF, Armadillo gene deleted in velocardiofacial syndrome; Xarvcf1A or 1B, Xenopus ARVCF isoform 1A or 1B; RT, reverse transcription.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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