Genome-wide disruption of DNA methylation by 5-aza-2’-deoxycytidine in the parasitoid waspNasonia vitripennis

Abstract

25DNA methylation of cytosine residues across the genome influences how many genes and phenotypes 26 are regulated. As such, understanding the role of DNA methylation and other epigenetic mechanisms 27 has become very much a part of mapping genotype to phenotype, a major question in evolutionary 28 biology. Ideally, we would like to manipulate DNA methylation patterns on a genome-wide scale, to 29 elucidate the role of epigenetic modifications in phenotypic expression. Recently, the demethylating 30 agent 5-aza-2'-deoxycytidine (5-aza-dC; commonly used in the epigenetic treatment of certain 31 cancers), has been deployed to explore the epigenetic regulation of a number of traits of interest to 32 evolutionary ecologists. Recently, we showed that treatment with 5-aza-dC shifted patterns of sex 33 allocation as predicted by genomic conflict theory in the parasitoid wasp Nasonia vitripennis. This was 34 the first (albeit indirect) experimental evidence for genomic conflict over sex allocation facilitated by 35 DNA methylation. However, this work lacked confirmation of the effects of 5-aza-dC on DNA 36 methylation, drawing commentary on the efficacy of 5-aza-dC in a novel system. Here, using whole-37 genome bisulphite sequencing, we demonstrate unequivocally that 5-aza-dC disrupts methylation 38 across the Nasonia vitripennis genome. We show that disruption leads to both hypo-and hyper-39 methylation, may vary across tissues and time of sampling, and that the effects of 5-aza-dC are 40 context-and sequence specific. We conclude that 5-aza-dC has the potential to be repurposed as a 41 tool in evolutionary ecology for studying the role of DNA methylation. 42 43 44 45 46 47 48 49 50 51

The Importance of Validating the Demethylating Effect of 5-aza-2′-deoxycytidine in Model Species

“…Boldfacing indicates statistical significance. study in Nasonia vitripennis, also showing a lack of uniform demethylating effect of 5-aza-dC treatment across sites (Cook et al 2019). We observed the same result for each of the three categories of genes we tested: there was no reduction in DNA methylation in highly methylated genes, nor in genes previously shown to have varying methylation levels, nor in genes involved in lipogenesis, a metabolic process that has been suggested to be epigenetically regulated.…”

“…In bumble bees, 5-aza-dC has an effect on overall DNA methylation, but its effect is erratic, as hyper-and hypomethylation were both observed after 5-aza-dC treatment (Amarasinghe et al 2014). A similar variable effect of 5-aza-dC treatment on methylation status of individual CpG sites was also found in a genome-wide study of methyl-ation in N. vitripennis (Cook et al 2019). Our study adds to this growing body of literature by showing a lack of consistent demethylating effect of 5-aza-dC in N. vitripennis.…”

Validating the Demethylating Effects of 5-aza-2′-deoxycytidine in Insects Requires a Whole-Genome Approach

“…Ellers et al (2019) raised the valid concern that the anti-metabolic activity of 5-aza-dC could result in a decline in the physical condition of Nasonia vitripennis females, indirectly producing observed phenotypic differences through a mechanism independent of DNA demethylation. Whilst we have shown that 5-aza-dC at the dosage used in our original experiment produces altered methylation patterns (above; Cook et al 2018), the possibility of confounding side-effects should not be ignored. We do note, however, that an effect of condition-dependent sex allocation has yet to be elucidated in Nasonia vitripennis, although pathological side-effects of any given chemical exposure cannot be discounted.…”

“…Recently we carried out a genome-wide analysis of the demethylating capacity of 5-aza-dC in Nasonia vitripennis (Cook et al 2018). Briefly, female wasps were exposed to one of three 5-aza-dC exposure regimes: 1) 20% sucrose for 24h, 2) 20% sucrose supplemented with 10 µM 5-aza-dC for 24h and 3) 20% sucrose supplemented with 10 µM 5-aza-dC for 48h.…”

“…It is possible that drug-induced demethylation in one region of the genome may have altered the regulation of the DNA methylation machinery itself, such that hypermethylation occurs. Nonetheless, it is clear that 5-aza-dC alters methylation patterns in the N. vitripennis genome and that this is associated with the allocation of offspring sex ratio (Cook et al 2018. Ellers et al (2019) raised the valid concern that the anti-metabolic activity of 5-aza-dC could result in a decline in the physical condition of Nasonia vitripennis females, indirectly producing observed phenotypic differences through a mechanism independent of DNA demethylation.…”