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. 2001 Jul 1;15(13):1613–1618. doi: 10.1101/gad.198501

Figure 3.

Figure 3

Kaiso represses transcription in vivo in a methyl–CpG-dependent manner, and is a component of the MeCP1 doublet complex. (A) Methyl–CpG-dependent repression by Kaiso and its truncated derivatives (diagram, top) in a transient transfection assay. Kaiso expression constructs were cotransfected with an SV40-luciferase reporter into mouse cells that are compromised in methyl–CpG-dependent transcriptional repression (Mbd2−/−; shaded bars) or into their normal counterparts (Mbd2+/−, unshaded bars). Relative reporter activity is presented as a percentage (methylated reporter expression/nonmethylated reporter expression × 100). Results are the average of at least three experiments. (B) Kaiso is responsible for the faster migrating component of the MeCP1 complex that is seen in many cell types. Complexes between HeLa and NIH3T3 nuclear extracts and the methylated (M+CG11) or nonmethylated (CG11) probe were detected by EMSA using agarose gels. Increasing amounts (up to 1 μL) of ZFH6 anti-Kaiso antibodies (αKaiso) or preimmune serum (P.I.) were added to determine which complexes were supershifted. The KGB complex is arrowed. (C) Kaiso is responsible for formation of a methyl–CpG-specific complex on a fragment of the E-cadherin promoter. Nuclear extracts from NIH3T3 cells and either methylated (M+Ecad) or nonmethylated Ecad probe were used in EMSA using agarose gels. Increasing amounts (up to 1 μL) of ZFH6 anti-Kaiso antibodies (αKaiso) or anti-MBD2 (αMBD2) antibodies were added to determine which complexes were supershifted. The KGB complex is arrowed. (D) Kaiso forms a methyl–CpG-specific complex on a fragment of the Pgk (−430; +74 bp from transcriptional start of the gene) promoter. Nuclear extracts from NIH3T3 cells and either methylated (M+Pgk) or nonmethylated (Pgk) probes were used in EMSA. The slight supershift on lane 5 is nonspecific due to the high concentration of rabbit serum in this lane. (E) Kaiso comigrates with a 700-kD protein complex on a Superose 6 column as assayed by EMSA in an agarose gel. Molecular weight markers are arrowed. The same fractions (pooled in pairs) were analyzed by Western blot hybridization with anti-Kaiso and anti-MBD2 antibodies. Input lanes represent mouse liver nuclear extract. The anti-Kaiso cross-reacting band seen in fractions 25–31 has a different electrophoretic mobility from Kaiso.