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Final Report Summary - DGHOST (Ghosts in parthenogenetic daughters - epigenetic effects on clonal organisms can reveal the degree of phenotypic plasticity due to biotic cues)

The purpose of “dGhost”, European Union sponsored project at University of Birmingham, is to empirically access and characterise the epigenetic memory that transcends generations using a clonal model organism, and to quantify a reset coefficient in the ecoresponsive Daphnia. This organism arguably provides the most intimate knowledge about the interaction between ambient or environmental conditions with genetic processes that impact adaptive or plastic responses, genetic activity and metabolism. Its mode of reproduction includes asexuality; by simply allowing animals to reproduce clonally, there is a unique potential to discover transgenerational epigenetic memory, as a reset is hypothesized during germ cells formation and at early embryonic stages. As such, experiments using Daphnia can potentially shed light on new evolutionary processes where epigenetics is the major player. Dr Wojewodzic’s planned experiments, consisting of a large number of generations that were asexually propagated under different food qualities had the potential of understanding dietary associated epigenetic signatures across generations. However these lineages did not persist in Birmingham, due to various problems at the early stages of developing an animal facility. However, by conducting a transplant experiment using high quality (good) food and low quality (junk) food (a concept taken from the field of ecological stoichiometry) over 3 consecutive generations for two genetically distinct Daphnia isolates, Dr Wojewodzic had studied the epigenetic signatures across subsequent asexual generations to test for the role of epigenetic memory in the plastic responses of animals to food quality. Such an enormous experiment required many steps to be coordinated simultaneously. This is pioneering work that was successfully performed in Birmingham, leading to an on going characterization of multiple types of epigenomic signatures from the sequencing of chromatin immunoprecipitated DNA and associated RNA samples.
The second major goal of dGhost is to discover the epigenetic mechanism of male formation in Daphnia, by using a novel approach, where epigenetic mechanisms are disrupted by cancer drugs. Unlike most animals where sex is determined genetically, often by the inheritance of sex chromosomes, sex in Daphnia is determined by environmental factors, which cue females to switch from producing genetically identical daughters to producing genetically identical sons. dGhost proposes to develop a mechanistic understanding of how biotic stress can be propagated via epigenetics to produce functional males. Understanding this phenomenon can potentially have a future commercial implication for XXI century ecotoxicological tests and environmental health management, where methylation signatures can signal endocrine disruptors, or other toxins in the environment that can act upon the epigenome, thereby serving as an early warning system for human health. In this project, both methylation and histone modification were hypothesized to have an effect on Daphnia sex determination. Butyrate is a well-characterized metabolite that modulates the compaction of DNA. Dr Wojewodzic’s experiments concluded that this compound had no effect on the sex ratio of Daphnia embryos. However two cancer drugs that act on methylotransferases activity were also tested for their effects on shifting the sex ratio of Daphnia embryos. These have shown effects on the sex of Daphnia, both directly and indirectly. These observations were partly confirmed by confocal microscopic studies on early embroys.
This second project had attracted three EU sponsored technicians (Leonardo da Vinci and Erasmus Plus Program), two EU sponsored Spanish Student and three Masters students that were registered within the School of Biosciences. Dr Wojewodzic, together with Marie Skłodowska Curie fellowship also received “TOPP financiering grant” from Norwegian Research Council to compensate for the differences in his salary level were he had choosen to stay in Norway. He acknowledges the contributions of all agencies. The project should greatly influence Dr Wojewodzic’s scientific career upon completion of the work and the publications reporting on its discoveries, giving him the opportunity to apply for further EU grants (i.e., ERC establishment) for launching his own research group in Norway that will focus on Environmental Epigenetics.

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