CGCG elements are transcriptionally active in CpG islands and methylation abolishes its activity.

A) CGCG elements that occur in CGIs in K562 cells coincide with DNase-seq footprints and associate with divergent plus and minus GRO-Cap transcripts and RNA Pol II occupancy. ENCODE WGBS methylation data show increased methylation of CGCG elements in non-CGIs compared to those in CGIs. B) Methylation of CGCG elements suppresses their promoter activity. Methylation of a TCTCGCGAGA insert in a CpG-free Lucia reporter using M.SssI CpG methyltransferase and SAM. C) Methylation of TCTCGCGAGA in the construct assessed by agarose gel analyses after digestion with NheI plus BstUI (only cuts non-methyl CGCG), which produces a doublet band of 2.3Kb. D) Lucia vs SEAP (transfection control) activity driven by WT, mutant or methylated CGCG inserts in transfected HEK293T cells. Data represent the mean of three replicates +/- SD. E) Strand-specific CAGE-seq signals generated from raw CAGE-seq sequence reads described by Brocks et al. [32] from NCI-H1299 cells treated with either the DNMT inhibitor, DAC, or DMSO control.