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J Biol Chem
2002 Mar 08;27710:8202-8. doi: 10.1074/jbc.M109508200.
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Isolation and characterization of XKaiso, a transcriptional repressor that associates with the catenin Xp120(ctn) in Xenopus laevis.
Kim SW
,
Fang X
,
Ji H
,
Paulson AF
,
Daniel JM
,
Ciesiolka M
,
van Roy F
,
McCrea PD
.
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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.
Figure 1
XKaiso sequence. The deduced amino acid sequence of XKaiso is aligned with that of mouse Kaiso. Sequences inshaded 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 12â117) are indicated by a filled line above thesequence, 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 numberAF420316.
Figure 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.
Figure 3
Whole-mount in situhybridization analysis of XKaiso expression in earlyXenopus development. Spatial expression pattern of XKaiso was evaluated via whole mount in situhybridization 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.
Figure 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 fromXenopus whole embryo lysates blotted with anti-myc monoclonal antibody. B, embryos were coinjected with mRNA coding for XKaiso tagged with mycepitopes (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.
Figure 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 Xenopuswhole embryo lysates blotted with anti-HA monoclonal antibody, indicating the existence of XKaiso homodimers or higher order oligomers.
Figure 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 âExperimental Proceduresâ) or nucleus-localized β-galactosidase (nbgal) mRNA were respectively coinjected as mRNA or protein expression controls. Exogenous expression of XKaiso consistently generated 10â20-fold repressions of the luciferase reporter, while significantly lesser effects arose from expression of Xp120ctn or Xarvcf.
