DNA Methyltransferase 1 Knockdown Activates a Replication Stress Checkpoint

Abstract

DNA methyltransferase 1 (DNMT1) is an important component of the epigenetic machinery and is responsible for copying DNA methylation patterns during cell division. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming. Knockdown of DNMT1 leads to inhibition of DNA replication, but the mechanism has been unclear. Here we show that depletion of DNMT1 with either antisense or small interfering RNA (siRNA) specific to DNMT1 activates a cascade of genotoxic stress checkpoint proteins, resulting in phosphorylation of checkpoint kinases 1 and 2 (Chk1 and -2), ␥H2AX focus formation, and cell division control protein 25a (CDC25a) degradation, in an ataxia telangiectasia mutatedRad3-related (ATR)-dependent manner. siRNA knockdown of ATR blocks the response to DNMT1 depletion; DNA synthesis continues in the absence of DNMT1, resulting in global hypomethylation. Similarly, the response to DNMT1 knockdown is significantly attenuated in human mutant ATR fibroblast cells from a Seckel syndrome patient. This response is sensitive to DNMT1 depletion, independent of the catalytic domain of DNMT1, as indicated by abolition of the response with ectopic expression of either DNMT1 or DNMT1 with the catalytic domain deleted. There is no response to short-term treatment with 5-aza-deoxycytidine (5-azaCdR), which causes demethylation by trapping DNMT1 in 5-aza-CdR-containing DNA but does not cause disappearance of DNMT1 from the nucleus. Our data are consistent with the hypothesis that removal of DNMT1 from replication forks is the trigger for this response.Maintenance of the epigenome is crucial for normal gene expression and preservation of cell identity. The "epigenome" is the set of heritable properties encoded by elements other than DNA base sequence. The epigenome includes chromatin, which is fashioned by remodeling complexes and modification enzymes as well as a pattern of covalent modification of DNA by methylation. During cell division replication of both the genetic and epigenetic, information is faithfully conserved. Due to a preference for hemimethylated DNA, DNA methyltransferase 1 (DNMT1) is believed to be responsible for copying the DNA methylation pattern within the mother cell and maintaining the pattern of genomic methylation within the daughter cell (38). Several lines of evidence suggest a role for DNMT1 in cellular transformation (2,22,23,37,44): indeed, DNMT1 is upregulated in multiple human cancers (17, 33), and some colorectal cancers have been observed to bear mutations in DNMT1 (19). DNMT1 has therefore been proposed as a target for anticancer therapy (39). Indeed, preclinical studies using antisense to DNMT1 have already shown reversion of tumor growth both in vitro (9) and in vivo (29). Taken together, these data suggest an important role for DNMT1 in both maintaining the epigenome and controlling cell cycle.A loss of DNMT1 during replication would result in a loss of epigenetic information. It has therefore been proposed that cells have developed multiple ways...

DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos

“…UHRF1 depletion from cancer cells results in a variety of phenotypes, including cell cycle arrest (Li et al, 2011;Tien et al, 2011), apoptosis (Tien et al, 2011), loss of contact inhibition (Hopfner et al, 2002) and increased sensitivity to DNA-damaging agents (Arima et al, 2004;Mistry et al, 2010;Muto et al, 2002). Similar phenotypes have also been reported for DNMT1-deficient cells (Chen et al, 2007;Karpf and Matsui, 2005;Milutinovic et al, 2003;Unterberger et al, 2006;Vijayaraghavalu et al, 2013), suggesting DNA hypomethylation as the underlying cause of these phenotypes.…”

“…Indeed, topoisomerase 2a mutants have DNA damage and reduced cell proliferation (Dovey et al, 2009), and we found these mutants had very little BrdU incorporation (not shown), which is unlike the increase in DNA replication in uhrf1 and dnmt1 mutants. Other studies have reported that DNMT1 depletion from cancer cells causes an acute cell cycle arrest in S-phase (Milutinovic et al, 2003;Unterberger et al, 2006) and a recent study using Xenopus egg extracts shows a requirement for UHRF1 in origin licensing and processivity during DNA replication (Taylor et al, 2013). However, another study using Xenopus showed that UHRF1 depletion had no effect on DNA replication, but instead showed that blocking DNA replication abolished DNA methylation (Nishiyama et al, 2013).…”

“…One possibility is that re-replication or DNA hypomethylation itself could induce DNA damage or genomic instability (Truong and Wu, 2011), activating a checkpoint. A previous study demonstrated that loss of UHRF1 in colon cancer cells causes a cell cycle arrest by activating a DNA damage checkpoint (Tien et al, 2011), whereas in other cell types, Uhrf1 loss causes a G1/S arrest (Arima et al, 2004), reminiscent of the checkpoint response occurring as a consequence of DNMT1 loss (Chen et al, 2007;Milutinovic et al, 2004Milutinovic et al, , 2003Unterberger et al, 2006). It is possible that the hemi-methylated DNA that accumulates in dividing cells that lack Uhrf1 mimics damaged DNA and thus activates the DNA damage checkpoint.…”

“…Apoptosis is likely one mechanism underlying the small organ size in uhrf1 mutants, as both the liver (Sadler et al, 2007) and eye (Tittle et al, 2011) have more cell death than wild-type larvae. However, reduced proliferation, as found in Dnmt1-depleted cells in culture (Milutinovic et al, 2003;Unterberger et al, 2006) and Uhrf-deficient T-regulatory cells (Obata et al, 2014) may also contribute to the uhrf1 mutant phenotype.…”

“…An alternative hypothesis is that a surveillance mechanism for epigenetic damage elicits a cellular response to prevent propagation of cells with epigenetic damage, analogous to the DNA damage response (Milutinovic et al, 2003). This epigenomic stress response then induces genes to prevent cell cycle progression, by a mechanism that is not a direct result of loss of epigeneticmediated repression (Milutinovic et al, 2004), but instead to prevent passive loss of DNA methylation (Milutinovic et al, 2003;Unterberger et al, 2006). The mechanisms underlying the cellular response to UHRF1 loss or overexpression are not fully understood.…”