Genome-wide investigation of the dynamic changes of epigenome modifications after DNA methylation editing [MBD-seq]

The aim of this study was to investigate the stability of DNA methylation, the dynamics of different histone modifications and the changes in expression after genome-wide introduction of DNA methylation in promotor CpG islands. Therefore, the catalytic domain of DNMT3A was fused to a zinc finger protein and expressed for three days to globally introduce de novo DNA methylation and subsequently, the changes in the epigenome were monitored for up to eleven days. A stable HEK293 cell line was constructed with the catalytic domain of DNMT3A fused to a zinc finger (ZnF-3AC) which is under the control of a doxycyclin (dox)-inducible promoter. GFP is co-expressed via an IRES and serves as a reporter. Cells were either harvested before dox treatment ("no dox") or grown under dox conditions for three days ("3d dox"). GFP-positive cells were enriched by FACS after 3d dox and a fraction of these cells was re-seeded on a 6-Well-plate without supplementation of dox. Five and eight days after dox removal ("5d / 8d / 11d off"), cells were harvested and a fraction of cells was re-seeded again. After eleven days of dox removal ("11d off"), cells were harvested and the experiment was stopped.