Figure 3.

Cytosine demethylation marks the regions of chromatin transition and correlates with GR binding in a cell type-specific manner. DNA was extracted from untreated 3134 and AtT-20 cells and analysed by bisulphite sequencing. The following pre-programmed DHSs are shown: (A) Per1-R1, shared by 3134 and AtT-20; (B) Dusp1, 3134-specific; (C) Arhegf3, AtT-20-specific and (D) de novo, 3134-specific DHS site (Tsc22d3). Additional examples are shown in Supplementary Figures S3 and S5. The GR-binding profile (red track) is shown with reference to the gene location and position of CpG island (black boxes). Black frames indicate the GR-binding site analysed further by bisulphite sequencing. The distribution of CpGs (vertical lines), location of a GRE (green oval) and region covered by bisulphite sequencing (restricted by arrows) are shown below the GR-ChIP track. The CpG density is indicated (# of CpGs/100 bp) . The bisulphite sequencing results are described as follows: each line represents one clone with CpGs shown as open (unmethylated) or filled (methylated) dots and CpG number indicated on the bottom; the green bars show the position of GREs; the left side of each graph shows the DHS-seq and GR ChIP-seq tracks for the regions being queried. (D) CpG methylation pattern of Sgk regulatory elements reflects their tissue-specific utilization. The three upper tracks represent the treated (Dex, 1 h) and untreated DHS profiles and GR ChIP-seq profile detected in 3134 cells; the three lower tracks are specific for AtT-20 cells. The results of bisulphite sequencing of the three GR-binding regions (Sgk-R1: 3134 specific, de novo; Sgk-R2: AtT-20 specific, pre-programmed; Sgk-R3: common, de novo in 3134 and pre-programmed in AtT-20) are compared between 3134 and AtT-20 cells and presented as above. In (D, E), the differentially methylated CpGs, with at least 35% difference in methylation levels between the two cell lines are marked (^*).