FIG. 2.

Methylation is associated with silencing of a cyclin A1 promoter-EGFP transgene in MG63 cells, and MeCP2 represses the methylated cyclin A1 promoter by an HDAC-dependent mechanism. (a) MG63 osteosarcoma cells were stably transfected with a cyclin A1 promoter-EGFP construct. After 2 months of continuous culture, a fraction of neomycin-resistant cells had lost expression of EGFP. Both populations were sorted by flow cytometry and analyzed for CpG methylation of the cyclin A1 promoter transgene. Specific primers were designed for the promoter-EGFP construct to avoid analysis of the endogenous cyclin A1 locus. Strong methylation was observed in nonexpressing cells, whereas negligible or very low levels of methylation were observed in expressing cells. (b) Methylated (+) or mock-methylated (−) reporter constructs were transfected into S2 Drosophila cells along with an Sp1 expression plasmid and either an empty vector (−) or an MeCP2 expression vector (+). Fold repression was calculated as 1/relative activity, with the activity of the control set as 1. The results are shown as the mean + SE of three independent experiments. (c) TSA relieves MeCP2-mediated repression of the methylated cyclin A1. Twenty-four hours after transfection of the methylated construct and the MeCP2 expression vector, TSA was added in different concentrations as indicated. The next day, luciferase activities were analyzed.