As a topoisomerase II inhibitor, the major biological function of etoposide is to inhibit DNA synthesis, and thereby, resulting in accumulation of double-stranded DNA breaks (DSBs) [1
]. Consequently, as documented by previous studies in animal and human cells, etoposide constitutes a genotoxic stress which may also induce genomic instability indirectly by causing transpositional activation of otherwise quiescent transposable elements (TEs), and hence generating insertional mutagenesis [3
]. Most plant genomes harbor potentially active TEs which can be induced to become transpositionally active under specific conditions [4
]. Nonetheless, no information is available regarding whether the chemical etoposide may produce similar effects in plant cells as it does in animals [3
We have shown in this study that etoposide may indeed cause rampant transposition of a MITE transposon, mPing
, endogenous to the rice genome [25
]. However, the activation of mPing
occurred in a strictly genotype-dependent manner, as only one of the six tested genotypes showed this phenomenon. In addition, the transpositional events did not occur in somatic cells of the immediately treated plants (S0); rather, excision and reinsertion events were detected only in selfed progenies (S1 and S2) of the etoposide-treated S0 plants. This is consistent with developmental regulation of most plant TEs [26
]. Thus, for example, if the transpositions of mPing
occurred at the gametogenesis stage of the etoposide-treated S0 plants, then the events can not be detected in leaf tissue of treated S0 plants but should be detectable in any sporophyte tissues of the S1 plants.
Authenticity of transposition rather than genomic rearrangements was verified by multiple independent assays including gel-blotting, mPing
-specific TD, sequencing and locus-specific PCR amplification. The fact that only one of the eight analyzed TEs with transpositional potentiality was actually activated is in accord with the recent finding that the controlling mechanisms of TEs in plant genomes are highly individualized by diverse repressive epigenetic modifications [4
]. The mobilization of only mPing
without entailing concomitant transpositional activation of its TPase donors Ping
is also congruent with previous findings on this element [21
]. This is understandable as only transcriptional activation of its TPase donor should be sufficient for mPing
transposition. Sequencing of the newly inserted mPing
copies indicated that, on the one hand, the insertions were randomly distributed with regard to the 12 chromosomes of the rice genome, and on the other hand, the great majority of the insertion sites were landed within low-copy, genic regions. Both characteristics are in line with known propensity of newly induced mPing
insertions by various inductive conditions [19
A hallmark of epigenetic modifications lies in its metastability in response to internal or environmental perturbations. Importantly, alterations of at least some of the epigenetic modifications, e.g., cytosine DNA methylation, are known to produce transgenerational biological effects which could be relevant to coping with the particular stress condition [8
]. This is a particularly pertinent issue in plants, as altered DNA methylation patterns are more readily transmissible to organismal generations through meiosis in plants than in animals, and thereby, the biological effects they dictate [14
]. Therefore, it is apparently interesting to test whether the genotoxic effects of etoposide may also instigate epigenetic instabilities in addition to its genetic mutagenesis. Surprisingly, however, to our knowledge, no such information is available in any organism.
Thus, we have shown in this study for the first time in any organism that the topoisomerase II inhibitor etoposide is also epigenetically mutagenic in the sense that the chemical generated immediate changes in DNA methylation patterns in the leaf somatic cells of the treated S0 rice plants. However, this effect, at least in rice, is of variable penetrance with regard to genetic context, as only four out of the six studied genotypes showed evidence of methylation changes. Interestingly, nearly all the detected methylation changes by methylation-sensitive gel-blotting were found to occur only at the CHG sites, underscoring differential stability of CG vs. CHG methylation modifications in plants. It has been well-established that in plant genomes the three types of cytosine methylation patterns, CG, CHG and CHH, are maintained by distinct yet, to an extent, also overlapping DNA methyltransferases, with CG methylation being predominantly maintained by DNA Methyltransferase 1 (MET1), CHG methylation by Chromomethylase 3 (CMT3, a plant-specific DNA methyltransferase), and CHH methylation by Domains Rearranged Methyltransferase 2 (DRM2, a de novo
DNA methyltransferases which is also partly responsible for de novo
methylation of all cytosines) [29
]. Therefore, it can be envisioned that the etoposide treatments may differentially affect activity or titration of these DNA methyltransferases and result in the observed predominant CHG methylation changes. Nonetheless, based on the higher resolution analytical approach, i.e., bisulfite sequencing, which was capable of revealing methylation change of any single cytosine within the analyzed region, we have found that, for both analyzed loci, all three types of cytosine methylation patterns, CG, CHG and CHH, underwent changes due to the etoposide treatment. But taking both loci together, it still holds that CHG methylation appeared more prone to changing as a result of the etoposide treatment than CG methylation, which is consistent with the situation of other environmental stress-induced cytosine methylation changes in plants [18
The detected methylation changes included both decrease (hypo) and increase (hyper) in methylation, but with the former seemingly more predominant than the latter. This is consistent with the perturbation of otherwise fine-tuned activity of the various DNA methyltransferases that are responsible for faithfully maintaining the inheritable methylation patterns. However, it has been shown that loss of methylation of certain type (e.g., CG) may activate alternative back-up cellular systems, which in turn may catalyze hypermethylation of other types of methylation (e.g., CHH) and produce aberrant methylation patterns [30
]. This scenario may explain the detected hypermethylation in the bisulfite sequencing results.
It seems surprising that little difference was detected between the two concentrations (10 mg/L vs. 20 mg/L) of the etoposide treatment with regard to the induced DNA methylation changes. It has been established in human cells that each molecule of etoposide stabilizes only one single-stranded DNA break [1
]. Therefore, it can be deduced that if 10 mg/L has already reached the saturated concentration for the germinating rice seeds, then no difference should be expected with elevated concentrations.
Phenotypic examination under paddy-field condition of the etoposide-treated S0 rice plants and their selfed S1 and S2 generations showed that of the six genotypes, only S1 and S2 plants of RZ1 showed clear and heritable (at least between S1 and S2) variations in several phenotypic traits like fertility and kernel-shape. Because mPing transpositional activity was concomitantly detected in these plants, it suggests that the co-occurrence of the two phenomena may be more than coincidental. Additional investigations are needed to explore the correlative or causal relationships between the two phenomena.