Identification of intergenerational Cd-induced differentially methylated regions in zebrafish life-long exposed to cadmium

Despite still being a matter of debate, notably in vertebrates, there are growing evidences that pollutant-induced epigenetic changes can be propagated across generations. Whereas such modifications could have long lasting effects on organisms and even on population viability, environmentally relevant data from long-term exposure combined with follow-up through multiple generations remain scarce for non-mammalian species. In this work, we performed a transgenerational experiment comprising 4 successive generations of zebrafish, which enabled the study of inheritance of epigenetic changes across generations. Only fish from the first generation (F0) were exposed throughout their life to an environmentally realistic concentration of cadmium (Cd), a well-known dangerous environmental pollutant. In order to identify without a priori the DNA regions that could be differentially methylated (called Differentially Methylated Regions, DMRs) in response to Cd exposure, we first used a MeDip-Seq (Methylated DNA immunoprecipitation coupled with high throughput pyrosequencing on NovaSeq 6000) approach on larvae and adult zebrafish of the F0 and F1 generation. Coupled with phenotypic data, we then analyzed the methylation level of several Cd-induced DMRs that were common to individuals of the F0 and F1 generation in zebrafish from all the 4 generations by means of a sequence targeted and base resolution method, i.e. bisulfite pyrosequencing.