DNMT1 deficiency triggers mismatch repair defects in human cells through depletion of repair protein levels in a process involving the DNA damage response.

DNA methyltransferase 1 (DNMT1) maintains methylation at CpG dinucleotides, important for transcriptional silencing at many loci. It is also implicated in stabilizing repeat sequences: DNMT1 deficiency causes microsatellite instability in mouse embryonic stem cells, but it is unclear how this occurs, how repeats lacking CpG become unstable and whether the effect is confined to stem cells. To address these questions, we transfected hTERT-immortalized normal human fibroblasts (hTERT-1604) with a short hairpin RNA construct targeting DNMT1 and isolated stable integrants with different levels of protein. DNMT1 expression levels agreed well with methylation levels at imprinted genes. Knockdown cells showed two key characteristics of mismatch repair (MMR) deficiency, namely resistance to the drug 6-thioguanine and up to 10-fold elevated mutation rates at a CA(17) microsatellite reporter, but had limited viability. The likely cause of MMR defects is a matching drop in steady-state protein levels for key repair components in DNMT1 knockdown cells, affecting both the MutLα and MutSα complexes. This indirect effect on MMR proteins was also seen using a different targeting method in HT29 colon cancer cells and did not involve transcriptional silencing of the respective genes. Decreased levels of MMR components follow activation of the DNA damage response and blocking this response, and in particular poly(ADP-ribose) polymerase (PARP) overactivation, rescues cell viability in DNMT1-depleted cells. These results offer an explanation for how and why unmethylated microsatellite repeats can be destabilized in cells with decreased DNMT1 levels and uncover a novel and important role for PARP in this process.

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.