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

Abstract

Dryad data: https://dx.doi.org/10.5061/dryad.d305qm1.abstract: The use of DNA demethylating agents has been popular in epigenetic studies. Recently, Cook and colleagues, in a 2015 American Naturalist article, claimed an effect of 5-aza-2 0 -deoxycytidine (5-aza-dC) on the sex ratio of a parasitoid wasp without verifying its effect on DNA methylation. We repeated the 5-aza-dC feeding treatment to test its effectiveness. We used bisulfite amplicon sequencing of 10 genes that either were heavily methylated, previously showed a response to 5-aza-dC, or were suggested to regulate fatty acid synthesis epigenetically, and we demonstrate that wasps fed 5-aza-dC did not show reduced DNA methylation at these loci. Therefore, the conclusion that demethylation shifts sex ratios upward needs reconsideration.

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

“…We agree with Ellers et al (2019) that quantifying the demethylation efficacy of orallyadministered 5-aza-dC in N. vitripennis will permit a more complete understanding of the relationship between DNA methylation and the sex allocation phenotype in Nasonia (see Cook et al 2015). More broadly, this will allow us to determine the extent to which genome-wide demethylation can be altered with 5-aza-dC in insect systems.…”

“…This result was consistent with predictions from genomic conflict over sex allocation theory (Wild and West 2009), suggesting that unmanipulated sex ratios are closer to the optimum for maternally-inherited alleles ( Figure 1). Ellers et al (2019), in their valuable comment on this study, raised the valid point that the 4 efficacy of 5-aza-dC in insect systems should be tested, and have suggested from their own experimentation that 5-aza-dC does not disrupt methylation in Nasonia vitripennis. To fully address their comments, we will begin by briefly reviewing the mechanism by which 5-aza-dC brings about the demethylation of DNA, before showing that assessing the role of 5-az-dC requires a whole genome approach.…”

“…The authors found no significantly differentially methylated CpGs at any of the 155 sites in association with 5-aza-dC exposure and conclude from this result that 5-aza-dC has no demethylating effect. To our knowledge, there is no evidence to suggest that the candidate genes chosen by Ellers et al (2019) are more (or less) likely to be influenced by 5-aza-dC -i.e. to have genomic contexts which favor or disfavor its action.…”

“…This was the first (albeit indirect) experimental evidence for genomic conflict over sex allocation. Ellers et al (2019) have since assayed the effects of 5-aza-dC on DNA methylation in ten Nasonia genes, finding no evidence of demethylation in these 10 genes, from which they conclude that 5-aza-dC has no demethylating capability in Nasonia vitripennis. Quantifying the efficacy of 5-aza-dC in terms of demethylation is indeed crucial to in-depth interpretation of studies using 5-aza-dC to link phenotypes to epigenetic regulation.…”

“…In their analysis of the involvement of DNA methylation in the photoperiodic diapause response in Nasonia, Pegoraro et al (2016) demonstrated that consumption of 5-aza-dC led to demethylation at specified CpG sites on a small scale. Ellers et al (2019) have examined the demethylating effect of 5-aza-dC at 155 CpG sites spread across 9 candidate genes in a slightly more comprehensive analysis. The authors found no significantly differentially methylated CpGs at any of the 155 sites in association with 5-aza-dC exposure and conclude from this result that 5-aza-dC has no demethylating effect.…”