Persistent epigenetic memory impedes rescue of the telomeric phenotype in human ICF iPSCs following DNMT3B correction.

DNA methyltransferase 3B (DNMT3B) is the major DNMT that methylates mammalian genomes during early development. Mutations in human DNMT3B disrupt genome-wide DNA methylation patterns and result in ICF syndrome type 1 (ICF1). To study whether normal DNA methylation patterns may be restored in ICF1 cells, we corrected DNMT3B mutations in induced pluripotent stem cells from ICF1 patients. Focusing on repetitive regions, we show that in contrast to pericentromeric repeats, which reacquire normal methylation, the majority of subtelomeres acquire only partial DNA methylation and, accordingly, the telomeric ICF1 phenotype persists. Subtelomeres resistant to de novo methylation were characterized by abnormally high H3K4 trimethylation (H3K4me3), and short-term reduction of H3K4me3 by pharmacological intervention partially restored subtelomeric DNA methylation. These findings demonstrate that the abnormal epigenetic landscape established in ICF1 cells restricts the recruitment of DNMT3B, and suggest that rescue of epigenetic diseases with genome-wide disruptions will demand further manipulation beyond mutation correction. We examined the DNA methylation patterns of selected subtelomeric regions by performing targeted bisulfite sequencing. Samples used in this study correspond to iPSCs derived from fibroblasts of healthy individuals, individuals affected by ICF syndrome type1 (ICF1-iPSCs), ICF1-iPSCs corrected in the DNMT3B mutations following CRISPR/Cas9 editing, parental fibroblasts and fibroblast-like cells (FLs) differentiated from iPSCs. The study includes iPSCs treated with the small molecule OICR-9429 to pharmacologically reduce the H3K4me3 level.