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.