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First published online 17 November 2004
doi: 10.1242/dev.01549

Development 131, 6185-6194 (2004)
Published by The Company of Biologists 2004
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Kaiso is a genome-wide repressor of transcription that is essential for amphibian development

Alexey Ruzov1,2,3,*, Donncha S. Dunican1,3,*, Anna Prokhortchouk2, Sari Pennings1, Irina Stancheva1, Egor Prokhortchouk2 and Richard R. Meehan1,3,{dagger}

1 Department of Biomedical Sciences, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK
2 Institute of Gene Biology, Russian Academy of Sciences, Vavilova 34/5, Moscow, 119334, Russian Federation
3 Human Genetics Unit, MRC, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK



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  		Fig. 1. xKaiso is a methyl-CpG dependent transcriptional repressor. (A) Schematic of xKaiso illustrating the BTB-POZ and zinc-finger domains and identity and similarity (in %) with human and zebrafish protein sequences. The blue box indicates a region of high homology of unknown function. The bar indicates the region used in EMSA assays. (B) EMSA experiment using recombinant ZF domain of xKaiso (KaisoZFs) (1, 2, 4, 10, 20 and 40 ng of protein) with Sm (methylated and non-methylated) or human matrilysin (Hmat) probes. Arrow indicates the xKaiso ZF specific band shift in the reaction with methylated Sm, but not with non-methylated Sm or Hmat probe. (C) EMSA experiment with xKaiso ZF, methylated (Sm) probe and non-labelled competitors: either methylated or non-methylated Sm, or Hmat, at 5x, 10x, 100x, 1000x molar excess. No competitor is added to the reaction in the first lane. xKaiso ZF specific band (arrow) completely disappears at 1000 x molar excess of methylated Sm. Non-methylated Sm oligo shows virtually no competition with the methylated Sm probe. The xKaiso ZF band in the presence of 1000 x molar excess of Hmat competitor is stronger than in the presence of 100 x molar excess of methylated non-labelled Sm oligo. (D) Methyl-CpG-dependent repression by xKaiso in a transient transfection assay. Kaiso expression constructs were co-transfected with an SV40-luciferase reporter into mouse cells that are compromised in methyl-CpG-dependent transcriptional repression (Mbd2–/–). Relative percentage (methylated reporter expression/nonmethylated reporter expression) is the average of at least three experiments. Human kaiso (hKaiso) and human MeCP2 (MeCP2) expression constructs were used as positive controls for methyl-CpG dependent transcriptional repression.

 


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  		Fig. 3. The methyl-CpG binding function of kaiso is required to rescue the xKaiso knockdown phenotype. The percentages of presented phenotypes are indicated. (A) Embryos injected with 10 ng of CMO develop normally by tadpole stage. (B) Over-expression of 750 pg wild-type human kaiso RNA does not affect normal development of Xenopus embryos. (C) Injection of 750 pg of wild-type human kaiso RNA together with 5 ng KMO leads to complete rescue of 26% of the embryos. (D) Apoptotic phenotype produced by injection of 5 ng KMO. (E) Co-injection of 750 pg of C522R human kaiso mutant RNA with 5 ng KMO cannot rescue the phenotype of xKaiso depletion. (F) Location of the kaiso mutant C522R amino acid substitution (red) in the third zinc finger, leading to loss of the ability to bind methylated DNA. (G) Protein gel of recombinant wild-type human kaiso and C522R (C>R) mutant proteins (arrow). (H) Pull-down experiment showing p120ctn (arrow) binds both wild-type human kaiso and C522R (C>R) mutant proteins in vitro, but not human kaiso protein lacking the ZF domain ({Delta}ZF). (I) EMSA experiment using recombinant C522R (C>R) mutant and wild-type human kaiso proteins with methylated (lanes 1, 4), non-methylated Sm oligos (lanes 2, 5) and human matrilysin (Hmat) oligo (lanes 3, 6) as probes. The kaiso-specific band shift is arrowed. The C522R mutant shows no DNA-binding activity.

 


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  		Fig. 2. The phenotypes of xKaiso-depleted embryos. (A) Embryos injected at two-cell stage with 10 ng of a control morpholino (CMO) show normal neurulation (stage 15). (B) Injection of 5 ng of the xKaiso morpholino (KMO) leads to a failure of blastopore closure and developmental arrest of neurulation at stage 15. Arrows indicate the appearance of white apoptotic cells from the borders of the blastopore. (C) Apoptotic cells (arrowed) cover almost the entire surface of embryos at stage 21 and cell shedding is present. (D) Injection of low dose (0.5 ng) of KMO causes defects of neurulation and delay of blastopore closure. Apoptotic cells are arrowed. (E) The range of phenotypes produced at low dose (0.5 ng) of KMO: 44% of embryos look normal by stage 38 (upper embryo), 29% exhibit failure to develop normal dorsal structures (spina bifida, lower embryo). Other phenotypes are intermediate. (F) Embryos injected with 10 ng of xDnmt1 morpholino (DMO) show apoptotic phenotype virtually identical to that of 5 ng KMO. (G) Western blot using anti-xKaiso antibody and whole embryonic extracts derived from wild type (WT) and 5 ng KMO-injected embryos (KMO). Stages of development are indicated above the lanes. The kaiso-specific band in KMO-injected embryos disappears by stage 10.

 


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  		Fig. 4. The loss of xKaiso induces an apoptotic response in Xenopus embryos. Wild-type (A-C) and 5 ng KMO-injected (D-F) Xenopus embryos were assayed by TUNEL for the appearance of apoptotic cells. TUNEL-positive cells were not detectable in normal late blastula (A) and gastrula (B) embryos. Small numbers of apoptotic cells (arrowhead) appeared in 1-2% of wild-type embryos at late neurula stage (C). TUNEL-positive cells were detected in 11% and 15% of KMO-injected embryos at late blastula (D) and gastrula stages (E), respectively. More than 90% of KMO-injected embryos exhibited a general pattern of apoptosis at a stage corresponding to late neurula of normally developing embryos (F). Abbreviations: bp, blastopore; ant, anterior; post, posterior.

 


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  		Fig. 5. xKaiso depletion leads to premature activation of transcription of a subset of genes regulated by xDnmt1. (A) [{alpha}-35S]UTP incorporation detecting the activation of gene expression in wild-type (WT) and 0.5 ng KMO-injected embryos. WT and KMO-injected two-cell embryos were treated with 50 nCi [{alpha}-35S]UTP, cultured in parallel and collected at the same developmental stage. (B) RNA derived from wild type, 5 ng KMO and 5 ng DMO (xDnmt1 morpholino) injected embryos at stage 8 were assayed by RT-PCR for the presence of transcripts of genes (xOct-25, xBef, xDrak1) regulated by xKaiso and xDnmt1, respectively, with xODC as a loading control.

 


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  		Fig. 6. Depletion of xKaiso relieves transcriptional silence but can be rescued by overexpression of human kaiso mRNA. (A) Two array experiment comparisons were performed: WT8 versus KMO8 and WT8 versus KMO8rescue. The x-axis shows 57 genes that were differentially expressed over a minimum 1.5-fold threshold, and the y-axis shows the fold expression changes. Genes that are elevated in KMO8 or KMO8rescue relative to wild-type expression are shown in blue, and downregulated genes in red. (B) A representative array region showing the expression changes of five genes [Histone H1 (H1), Geminin H (GemH), cyclin A1 (CycA1), cyclin B2 (CycB2) and MEK1 (MEK1)], which are also indicated on the graph of genes shown in A. H1 expression was not rescued in KMO8rescue embryos, whereas CycB2 is, and GemH, MEK1 and CycA1 have reduced expression levels.

 


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  		Fig. 7. xKaiso-repressed genes: functional categories. (A) The 429 input (Total) genes and 55 genes upregulated relative to WT in Kaiso-depleted embryos (KMO/WT) were categorized as described (http://www.viagenx.ca/) and their distributions shown in separate pie charts. The numbers refer to the percentages of genes in each functional category in the left/right chart respectively. (B) CpG island analysis of the input (Total) and KMO/WT upregulated genes is shown in separate pie charts. Colour coding for the different groupings and their percentages of the total in each chart are shown on the right.

 

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© The Company of Biologists Ltd 2004