Cell division drives DNA methylation loss in late-replicating domains in primary human cells

Abstract

DNA methylation undergoes dramatic age-related changes, first described more than four decades ago. Loss of DNA methylation at late-replicating regions of the genome attached to the nuclear lamina advances with age in normal tissues, and is further exacerbated in cancer. We present here the first experimental evidence that this DNA hypomethylation is directly driven by proliferation-associated DNA replication. Loss of DNA methylation at low-density CpGs in A:T-rich, partially methylated domains (PMD solo-WCGWs), tracks cumulative population doublings in primary cell culture. Cell cycle deceleration resulted in a proportional decrease in the rate of DNA hypomethylation. Blocking DNA replication via Mitomycin C treatment halted methylation loss. Loss of methylation continued unabated after TERT immortalization until finally reaching a severely hypomethylated equilibrium. Ambient oxygen culture conditions increased the rate of methylation loss compared to low-oxygen conditions, suggesting that some methylation loss may occur during unscheduled, oxidative damage repair-associated DNA synthesis. Finally, we present and validate a model to estimate the relative cumulative replicative histories of human cells, which we call "RepliTali" (Replication Times Accumulated in Lifetime).

A local sequence signature defines a subset of heterochromatin-associated CpGs with minimal loss of methylation in healthy tissues but extensive loss in cancer

“…By analyzing direct measurements of nascent DNA re-methylation during mitotic S-phase, we show that this process is mediated via re-methylation efficiency of this sequence signature. These results are consistent with recent work showing that PMD methylation loss accumulates linearly with cell divisions in various human cell types ( 14 ).…”

“…Since mitotic methylation loss is a type of molecular "clock", it is important to put our work in the context of other methylation clocks. Our PMD-based hypomethylation signature was recently confirmed as a mitotic clock in vivo in B-cell development and cancer (24) and in cell divisions in vitro in several human cell types (14). Each of these studies defines their own minimal versions of a hypomethylation clock, "epiCMIT" and "RepliTali", respectively.…”

“…An important limitation to this assay is that it was restricted primarily to measuring re-methylation within the most early-replicating DNA, which generally doesn’t include PMDs which are late-replicating. While we can not explicitly quantify the efficiency within PMD regions, this remains very strong evidence that re-methylation efficiency after replication is the direct cause of cumulative methylation loss within PMDs in aging and cancer, in addition to the genome wide profiling of methylation during serial passaging of cells in vitro ( 13, 14 ). The proposed process is illustrated in Figure 4B.…”

“…PMD methylation loss begins during normal aging in most tissues, and is associated with the chronological age of the individual (ZHOU). Long-term passaging in vitro has shown that methylation loss is linearly related to the number of cell divisions ( 13, 14 ), and the degree of hypomethylation in tumors is correlated with the number of somatic mutations (ZHOU). In non-cancer cells, all evidence to data suggests a mitotic clock-like process.…”

Revisiting the bad luck hypothesis: Cancer risk and aging are linked to replication-driven changes to the epigenome

“…Mutations are not the only -or perhaps even the most important -molecular events that result from cellular proliferation. We and others have shown that DNA methylation (DNAm) is also substantially altered as a direct function of cell division [6][7][8][9]. Further, the epigenome has been shown to undergo dramatic changes with aging and is implicated in establishing, driving and maintaining many cancers [10][11][12][13].…”