VEZF1 Elements Mediate Protection from DNA Methylation (original) (raw)
Figure 2
Three footprinted sites in the HS4 barrier protect a promoter from DNA methylation.
(A) Schematic representation of the IL-2R transgene drawn to scale with the distribution of CpG dinucleotides shown below. Promoter region CpG dinucleotides subject to bisulfite genomic sequencing (BSEQ) analysis are indicated by the gray bar. (B) CpG methylation of transgene promoters flanked by wild-type or mutant HS4 insulators after 30 (left) or 90 (right) days of culture. The percentage of methylation for each CpG from 10 clones is plotted. The average methylation for all CpGs is indicated to the right of each plot. Scoring from individual clones can be found in Table S1. Numbers below each histogram refer to CpG numbering from [16], where CpG 4–11 and 12–18 reside in the promoter and coding sequence, respectively. Data are representative of two independent transgenic lines, with an average methylation variation of 7% or less. (C) Transgenic IL-2R expression for each line in (B) as monitored by flow cytometry. WT and ΔII lines retain IL-2R expression, whereas ΔI, ΔIII, ΔIV and ΔV all succumb to silencing following 20–40 days of culture.