Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression

Background: DNA methylation plays a vital role in the cell, but loss-of-function mutations of the maintenance methyltransferase DNMT1 in normal human cells are lethal, precluding target identification, and existing hypomorphic lines are tumour cells. We generated instead a hypomorphic series in normal hTERT-immortalised fibroblasts using stably integrated short hairpin RNA. Results: Approximately two-thirds of sites showed demethylation as expected, with one-third showing hypermethylation, and targets were shared between the three independently derived lines. Enrichment analysis indicated significant losses at promoters and gene bodies with four gene classes most affected: (1) protocadherins, which are key to neural cell identity; (2) genes involved in fat homoeostasis/body mass determination; (3) olfactory receptors and (4) cancer/testis antigen (CTA) genes. Overall effects on transcription were relatively small in these fibroblasts, but CTA genes showed robust derepression. Comparison with siRNA-treated cells indicated that shRNA lines show substantial remethylation over time. Regions showing persistent hypomethylation in the shRNA lines were associated with polycomb repression and were derepressed on addition of an EZH2 inhibitor. Persistent hypermethylation in shRNA lines was, in contrast, associated with poised promoters. Conclusions: We have assessed for the first time the effects of chronic depletion of DNMT1 in an untransformed, differentiated human cell type. Our results suggest polycomb marking blocks remethylation and indicate the sensitivity of key neural, adipose and cancer-associated genes to loss of maintenance methylation activity.