FIG. 6.

Repression of a methylated reporter gene by MBD1 depends on the TRD and methyl-CpG binding domains and is sensitive to TSA. (A) Mouse L929 cells were transfected with an SV40-luciferase reporter (2 μg) that was nonmethylated (M−) or methylated at 25 HhaI (GCGC) sites (M+). Levels of M+ and M− reporter expression (luciferase activity) were normalised to the expression level of cotransfected CMV β-galactosidase control reporter (1 μg). Transcription levels were expressed as the ratio of M+ to M− normalised expression levels. The low density of methylation in the pGL2 control reporter had a negligible effect on transcription in the absence of cotransfected MBD1 but caused repression relative to the nonmethylated reporter in the presence of 0.5 μg of intact MBD1 fused to a Myc epitope tag (5MT-MBD1). N-terminal or C-terminal deletions prevented repression. (B) Diagram of the pGL2 SV40-luciferase reporter, showing the locations of methylated HhaI sites. (C) Diagram of the 5MT-MBD1 proteins used to obtain data shown in panel A. (D) Western blots of proteins extracted from cells transfected by each of the 5MT-MBD1 constructs showing equivalent expression. The blots were probed with anti-Myc monoclonal antibody 9e10. (E) TSA (100 ng/ml) overcomes repression by MBD1 of a methylated reporter gene. The results shown are based on three independent transfections.