Methyltransferases mediate cell memory of a genotoxic insult

Abstract

Characterization of the direct effects of DNA-damaging agents shows how DNA lesions lead to specific mutations. Yet, serum from Hiroshima survivors, Chernobyl liquidators and radiotherapy patients can induce a clastogenic effect on naive cells, showing indirect induction of genomic instability that persists years after exposure. Such indirect effects are not restricted to ionizing radiation, as chemical genotoxins also induce heritable and transmissible genomic instability phenotypes. Although such indirect induction of genomic instability is well described, the underlying mechanism has remained enigmatic. Here, we show that mouse embryonic stem cells exposed to γ-radiation bear the effects of the insult for weeks. Specifically, conditioned media from the progeny of exposed cells can induce DNA damage and homologous recombination in naive cells. Notably, cells exposed to conditioned media also elicit a genome-destabilizing effect on their neighbouring cells, thus demonstrating transmission of genomic instability. Moreover, we show that the underlying basis for the memory of an insult is completely dependent on two of the major DNA cytosine methyltransferases, Dnmt1 and Dnmt3a. Targeted disruption of these genes in exposed cells completely eliminates transmission of genomic instability. Furthermore, transient inactivation of Dnmt1, using a tet-suppressible allele, clears the memory of the insult, thus protecting neighbouring cells from indirect induction of genomic instability. We have thus demonstrated that a single exposure can lead to long-term, genome-destabilizing effects that spread from cell to cell, and we provide a specific molecular mechanism for these persistent bystander effects. Collectively, our results impact the current understanding of risks from toxin exposures and suggest modes of intervention for suppressing genomic instability in people exposed to carcinogenic genotoxins.

Figure 1. Persistent and transmissible induction of genomic instability

a, Irradiated (or mock irradiated) target ES cells are cultured for three weeks. During the three weeks, cells were passaged three times a week at densities of 0.5-2×10⁶ cells per 55mm² dish. After three weeks, 6×10⁴ naive WT ES cells subsequently shared media with the progeny of target ES cells for 5 days (to create primary bystanders). Primary bystanders were then cultured for another three weeks. Naive WT ES cells then shared media with the progeny of primary bystanders (to create secondary bystanders). Media was shared via co-culture, employing 1 μm transwell inserts (Corning), or by exposure to conditioned media (filtered [0.25μm]; 1:1, fresh media:conditioned media). ES cells were exposed to ionizing radiation (3 Gy) using a Co-60 source (73 cGy/min). DNA damage was assessed by the alkaline comet assay (Olive, 2006) in primary (b), and secondary (c), bystanders. For all comet analysis, >100 nucleoids were analyzed per condition using Komet 5.5 (Andor Technology, Ireland) and P values were produced by a two-tailed Mann-Whitney. For comet studies, boxes represent the quartiles, whiskers mark the 10^th and 90^th percentiles, and the median is indicated. For all studies, data was combined from three or more independent experiments.

Figure 2. γIR does not lead to the persistent induction of genomic instability in primary bystanders to Dnmt1-/- Dnmt3a-/- Dnmt3b-/- cells

DNA damage by comet assay (a) and SCEs (b) in naive WT ES cells exposed to media from WT and Dnmt1-/- Dnmt3a-/- Dnmt3b-/- cells. See Fig. 1 for experimental design. SCEs were counted for ≥80 spreads/condition as previously described (Engelward et al., 1996). For SCE studies, median with interquartile range is shown and P values were produced by a two-tailed t-test.

Figure 3. Dnmt1 and Dnmt3a are required for persistent induction of homologous recombination in naïve, primary bystander ES cells

SCEs in naive WT ES cells exposed to media from γIR (and mock irradiated) WT, Dnmt1-/-, Dnmt3a-/-, and Dnmt3b-/- target ES cell populations. See captions from Figs. 1 & 2 for design and analysis.

Figure 4. Transient suppression of Dnmt1 protects against radiation-induced genomic instability

a, Doxycycline-dependent repression of Dnmt1 in mouse ES cells assessed by Western blot. b, SCEs in primary bystanders to normal and Dnmt1 transiently-deficient cells. c, SCEs in naive (secondary bystander) cells exposed to media from primary bystanders to normal and Dnmt1 transiently-deficient cells. Data analysis as per caption for Fig. 2.