Flatworm DNA methylation: deciphering the mark and characterising the machinery.

We are all familiar with the subject of genetics, which describes how the fundamental unit of inheritance (the gene) is passed on from parent to offspring. Changes in the underlying DNA sequence that make up our inherited genes help explain why differences (eye colour, eye shape, chin size, etc.) in our outward appearance (phenotype) occur.

However, genetics cannot fully explain the wide-ranging phenotypic diversity exhibited by all organisms on this planet. To do so in a systematic manner, we also have to consider another type of inheritance system called epigenetics. Epigenetics is the study of inherited changes in gene function (leading to different phenotypes) that cannot be explained by changes in the underlying DNA sequence of the gene. In other words, epigenetics attempts to explain everything that genetics cannot.

One particular type of epigenetic process responsible for inherited changes in phenotype is facilitated by DNA methylation. Loss of DNA methylation regulation has been extensively studied in humans and is associated with cancer, obesity, immunodeficiencies and intellectual disabilities. Recent studies have additionally demonstrated that heritable DNA methylation patterns are influenced by our interaction with environmental (chemicals, drugs, etc.) factors. However, very little is known about DNA methylation or the processes that regulate it in other animal systems, especially invertebrates.

Flatworms are a tremendously important invertebrate group (within the phylum Platyhelminthes) responsible for many economically- and biomedically-relevant parasitic diseases. These parasitic invertebrates undergo extensive developmental changes throughout their complicated lifecycles, which often involves interaction with more than one host or environment. Does environmentally influenced DNA methylation contribute to the success of parasites like these? If so, how does this occur and what genes are targeted by this epigenetic mechanism?

Working in Aberystwyth, we will apply state of the art molecular biology tools to try and understand how DNA methylation regulates flatworm development. We have already found that DNA methylation is present in all three classes (Trematoda, Cestoda and Monogenea) of parasitic platyhelminth and that the enzymes responsible for these genome modifications are highly conserved. Using a model platyhelminth (Schistosoma mansoni), our project aims to further characterise the enzyme responsible for flatworm DNA methylation and to identify the genes specifically targeted by the DNA methylation machinery. By doing so in a synergistic manner, we expect to discover new roles for this epigenetic process during animal evolution. This information may eventually lead to novel ways to combat parasitic diseases.

DNA methylation, catalysed by DNA methyltransferases (Dnmts), is an epigenetic process that regulates metazoan gene expression, repetitive element silencing, allelic exclusion and development. However, within the economically and biomedically important Platyhelminthes (flatworms), virtually nothing is known about the role of this regulatory mediator. Studies of flatworm DNA methylation systems will, therefore, be essential to our understanding of how epigenetic processes have evolved and how DNA methylation specifically contributes to platyhelminth lifecycle diversity, host interactions and disease pathogenesis.

Our laboratory is using complementary techniques to identify and characterise flatworm DNA methylation systems. Through RNAi and drug inhibition studies, we have demonstrated that Schistosoma mansoni (a parasitic flatworm) contains a Dnmt (SmDnmt2) responsible for DNA methylation and oviposition. Follow-up investigations now show that DNA methylation is not restricted to schistosomes, but is also present in the two other parasitic platyhelminth classes.

To further understand the significance of a representative platyhelminth DNA methylation system, this proposal aims to fully characterise schistosome SmDnmt2 and identify genome-wide DNA methylation patterns in schistosome larvae and adults. We will first identify sub-cellular localisations, interacting partners, additional enzymatic activities and DNA binding loci of SmDnmt2 using cell transfections, yeast 2 hybrid screens, recombinant protein expression/RNA methyltransferase activities and CHiP assays. Secondly, we will utilise 2-D proteomics to identify the schistosome gene products affected by SmDnmt2 inhibition or knockdown. These collective studies will fully define the schistosome DNA methylation mediator. Finally, using newly generated Illumina sequence data obtained from WGBS or MeDIP libraries, we will thoroughly characterise the DNA methylome of schistosome larvae and adults.

The goal of this research is to provide: i) functional information concerning an enzyme (SmDnmt2) responsible for schistosome DNA methylation and oviposition (i.e. potential drug target) and ii) sequence datasets describing a representative platyhelminth DNA methylome. The main impact of the research will be to advance basic scientific knowledge related to evolutionary epigenetics and DNA methylation. As a result, the primary beneficiaries will be the scientific community and the general public. However, the longer-term implications of the proposed research might lead to commercial/drug development opportunities for the pharmaceutical industry/commercial drug companies.

Public impact/engagement
The public is fascinated with parasites and the ways in which they can affect our everyday lives, as can be seen by interest shown in the recent high profile TV series 'Monsters Inside Me' (Discovery Channel) and 'Embarrassing Bodies' (Channel 4). With the additional positive press given to the health benefits of 'Helminthic Therapy' and the recent announcement of the UK government's investment in controlling neglected tropical diseases, we are now experiencing an unprecedented era in public awareness related to parasitology. Our experiences in various public engagement activities throughout the years has demonstrated that the general public are surprised to hear that molecular mechanisms (mediated by genetics and epigenetics-based processes) responsible for generating human phenotypic alterations (e.g. cancer, etc.) are also operating in these deadly parasites. Moreover, the public are increasingly interested in how modern day molecular approaches and cutting-edge technologies will contribute to the study, control and prevention of these widespread infectious diseases. Therefore, the potential outcomes of our proposed research will impact on the general public by providing insight into/enhancing knowledge of genomes, epigenomes, DNA methylation and platyhelminth biology. This will contribute to medical/health knowledge related to parasitic diseases and epigenetics. Furthermore, our research will raise the general public's awareness into the molecular processes that are responsible for age-related diseases such as cancer.

Commercial impact
We are also aware of the commercial impact of our research and have under taken steps to ensure this activity remains firmly embedded within our laboratory. The study of helminth developmental biology, which aspects of this proposal touch upon, have led to collaborative research with Pfizer Animal Health, Intervet Innovations and Phytoquest Ltd. Here, with support from our Commercialisation and Consultancy Services Department (CCS) and the BBSRC Follow on Fund (FoF), we are currently developing technologies useful for assessing worm phenotype, developmental state and viability. While these techniques are useful for experiments planned within our Case for Support, they are equally valuable to collaborative drug companies involved in anthelmintic screening. We have, therefore, patented aspects of these technologies that may be commercially valuable (subject of PCT patent application 0916240.5). Specific findings of this research will likely be of interest to commercial drug companies and, thus, we will continue to engage with CCS to discuss further IP possibilities throughout the project.