Figure 4. Induction of Hypermethylation and Silencing of the RIL Short Allele in Vitro.

(A) Summary of transient allele-specific RIL/reporter gene expression in 4 cell lines. ∼0.2 KB (A), ∼0.6 KB (B) and ∼1.1 KB (C) RIL L and S promoter fragments were cloned upstream of pGL3 luciferase and transiently cotransfected with renilla thymidine kinase as a transfection control into the indicated cell lines in 6-well plates. Cells were harvested at 48 hrs and luciferase activities were analyzed. Luciferase/renilla ratios were plotted on the Y-axis. Transfections were performed in triplicates, and standard error bars for each experiment are shown on the graph. (B) Expression of stably transfected HpaII-seeded constructs. Long and short RIL allele-specific constructs (construct B, ∼0.6 KB) were seeded with HpaII methylase, stably cotransfected with pcDNA3.1 into NIH3T3 cells, selected in neomycin and pooled clones monitored for the indicated time period. Notice initial equal levels of expression, followed by maintenance of the long allele construct expression and rapid decline in expression of the short allele construct. (C) Bisulfite sequencing results for allele-specific seeded constructs. The methylation status of the transfected allele-specific constructs was analyzed by bisulfite sequencing at indicated time points, using plasmid-specific primers. Note equal methylation levels and presence of initial “seeds” of HpaII methylation (arrowheads) on day 19 and increased methylation spreading for the short allele construct at day 57. Percent methylation for each time point is indicated. (D) Pyrosequencing results for allele-specific seeded constructs. The methylation status of the transfected allele-specific constructs was analyzed by pyrosequencing at indicated time points, using plasmid-specific primers. One site was analyzed by the assay, which corresponds to CpG site # 9 in bisulfite sequencing figure. The pyrosequencing results shown agree with expression and bisulfite sequencing data.