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Development
2004 Dec 01;13124:6185-94. doi: 10.1242/dev.01549.
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Kaiso is a genome-wide repressor of transcription that is essential for amphibian development.
Ruzov A
,
Dunican DS
,
Prokhortchouk A
,
Pennings S
,
Stancheva I
,
Prokhortchouk E
,
Meehan RR
.
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DNA methylation in animals is thought to repress transcription via methyl-CpG specific binding proteins, which recruit enzymatic machinery promoting the formation of inactive chromatin at targeted loci. Loss of DNA methylation can result in the activation of normally silent genes during mouse and amphibian development. Paradoxically, global changes in gene expression have not been observed in mice that are null for the methyl-CpG specific repressors MeCP2, MBD1 or MBD2. Here, we demonstrate that xKaiso, a novel methyl-CpG specific repressor protein, is required to maintain transcription silencing during early Xenopus laevis development. In the absence of xKaiso function, premature zygotic gene expression occurs before the mid-blastula transition (MBT). Subsequent phenotypes (developmental arrest and apoptosis) strongly resemble those observed for hypomethylated embryos. Injection of wild-type human kaiso mRNA can rescue the phenotype and associated gene expression changes of xKaiso-depleted embryos. Our results, including gene expression profiling, are consistent with an essential role for xKaiso as a global repressor of methylated genes during early vertebrate development.
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 (δZF). (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.
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.
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.
