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

Lead Research Organisation: Aberystwyth University
Department Name: IBERS

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description 1) We have developed entirely new software for identifying where DNA methylation occurs in any genome (found at DOI: 10.1186/s12859-016-1158-7 and OMICtools). We have used this software to identify where low levels of DNA methylation occur in the pathogenic blood fluke worm Schistosoma mansoni (manuscript being written).
2) We have significantly expanded our understanding of invertebrate epigenetic processes by providing evidence linking DNA methylation to chromatin modifications (manuscript to be submitted this year to PLoS Pathogens). The regulatory role of schistosome epigenetic components in stem cell proliferation and maintenance was also obtained in this grant (data included in the same manuscript).
3) We have characterised the DNA methylation machinery of Biomphalaria glabrata (the intermediate host responsible for transmitting Schistosoma mansoni) as well as identified new symbiotic micro-organisms living within this mollusc. We also demonstrated that the B. glabrata DNA methylation machinery is influenced by interactions with parasites.
Exploitation Route The software can be used by any scientist involved in DNA methylation analyses (independent on target species) and is especially useful for those species that have low levels of non-CpG methylation.

The linkage of DNA methylation to histone modifications within an invertebrate species may push others to look for this association in other BBSRC-targetted invertebrate species.

Parasite influenced changes to host DNA methylation machinery components may be applicable to other host/parasite systems.

The symbiotic micro-organisms identified in the intermediate host B. glabrata may be useful for further investigations of biological control.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology,Other

URL https://omictools.com/dismiss-tool
 
Description Flatworm Functional Genomics Initiative (FUGI)
Amount £1,355,599 (GBP)
Funding ID 107475/Z/15/Z 
Organisation Wellcome Trust 
Department Wellcome Trust Strategic Award
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2020
 
Title DISMISS: Detection of Stranded Methylation in MeDIP-Seq Data 
Description We developed a new software package that detects strand-associated DNA methylation from existing MeDIP-Seq datasets. DISMISS is currently under review at BMC Bioinformatics. 
Type Of Material Data analysis technique 
Provided To Others? No  
Impact This will allow other investigators to identify where strand specific DNA methylation occurs (+ or - DNA strands) from datasets derived from MeDIP-Seq DNA methylation analyses. 
 
Description BVGH facilitated Alnylam collaboration 
Organisation Alnylam Pharmaceuticals
Country United States 
Sector Private 
PI Contribution With knowledge provided by Alnylam, we were able to demonstrate that chemically modified siRNAs were more efficient in mediating RNAi in schistosomes.
Collaborator Contribution Alnylam provided us with knowledge related to the specific types of chemical modifications to use in maximising RNAi.
Impact Out collaboration was featured in BVGH's second collaboration storybook (October 2015).
Start Year 2013
 
Description Biomphalaria glabrata genome consortium 
Organisation University of New Mexico
Country United States 
Sector Academic/University 
PI Contribution We have contributed to the analysis of the Biomphalaria glabrata genome and have specifically identified a functional DNA methylation machinery as well as an endosymbiont present in this snail's genome.
Collaborator Contribution The University of New Mexico led the Biomphalaria glabrata genome project (many other contributors).
Impact A manuscript submitted to Nature Communications describing the B. glabrata genome is under review (Feb 2016). A manuscript to be submitted to PLoS NTDs (a companion paper to the genome paper) will be submitted soon.
Start Year 2013
 
Description Epigenetic probe compound collection 
Organisation University of Oxford
Department Structural Genomics Consortium (SGC)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Drug screening SGC epigenetic compounds against schistosome worms
Collaborator Contribution The SGC has provided us with ~40 compounds to screen against schistosome worms
Impact We have identified several compounds that target epigenetic machinery components as potential new anti-schistosomals. This has resulted in a new PhD studentship award (Welsh Government and IBERS, AU) worth ~£60K.
Start Year 2014
 
Description Epigenetics of schistosome biology and CHiP-Seq using the anti-SmMBD2/3 Ab 
Organisation University of Peradeniya
Country Sri Lanka 
Sector Academic/University 
PI Contribution We have raised a polyclonal antibody in rabbits directed against recombinant SmMBD2/3. We also have characterised the schistosome histone methylation machinery as a follow-up to our studies on DNA methylation.
Collaborator Contribution Our partners will try and develop CHiP-Seq technologies to use this polyclonal Ab to identify methylated regions in the schistosome genome. We have jointly published a review on the significance of histone modifying enzymes in regulating schistosome development and lifecycle transmission.
Impact Trends Parasitol. 2016 Dec 28. pii: S1471-4922(16)30224-0. doi: 10.1016/j.pt.2016.12.002. [Epub ahead of print] (Epi)genetic Inheritance in Schistosoma mansoni: A Systems Approach. Cosseau C1, Wolkenhauer O2, Padalino G3, Geyer KK3, Hoffmann KF3, Grunau C4.
Start Year 2016
 
Description Measuring DNA Modifications 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Preparation and supply of B.glabrata S.mansoni DNA samples to Markus Ralser at the Crick Institute.
Collaborator Contribution Photometric measurement of DNA modifications in material supplied and further method refinement (towards development of a DNA modification atlas).
Impact Several clear modifications in the supplied material was evident. Refinement of the method of measurement is ongoing and further DNA material may be supplied.
Start Year 2018
 
Description Small non-coding RNA identification 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Identification of sncRNAs excreted/secreted by schistosomes. Some of these sncRNAs may be targeted by DNA methylation and are being analysed now.
Collaborator Contribution Thorough training in the isolation of sncRNAs from excreted/secreted products and NGS library production.
Impact presentation at international meetings
Start Year 2013
 
Description Streamlining the production of recombinant proteins 
Organisation Research Complex at Harwell
Department Oxford Protein Production Facility-UK (OPPF-UK)
Country United Kingdom 
Sector Academic/University 
PI Contribution Work with Oxford Protein Production Facility (OPPF, UK) to express, purify and crystallize (solve structures) putative helminth drug targets. During this reporting period, we have hosted OPPF's Director (Ray Owens) at Aberystwyth University and have organised a short-term laboratory placement for a research team member to the OPPF-UK (June-August 2017).
Collaborator Contribution The OPPF-UK hosted a placement of a Hoffmann Laboratory member for training in high throughput expression, purification and crystallization of proteins. During the placement, the best conditions for protein expression were optimised and collection of crystallographic data allowed a subsequent study of the 3D structure of the proteins under different conditions (in the presence or absence of cofactor, substrate and small molecules).
Impact The data collected during the period contributed to collective knowledge regarding schistosome proteins and will prove extremely valuable for further drug discovery work.
Start Year 2017
 
Title Anthelmintic compounds 
Description The invention relates to compounds and their use in therapy, including in pharmaceutical compositions and combinations, especially in the treatment and/or prophylaxis of the tropical diseases schistosomiasis and/or fascioliasis, e.g. in humans or in non-human land mammals such as sheep, cattle, other ruminants, or goats. 
IP Reference PCT/GB2015/054078 
Protection Patent application published
Year Protection Granted 2014
Licensed No
Impact None
 
Title DISMISS 
Description An R script, which as an additional step in MeDIP-Seq data analysis workflow, enables the allocation of strands to methylated DNA regions. DISMISS does this by analyzing the proportions of first mate reads aligning to the methylated locus from the plus and minus strands. 
Type Of Technology Webtool/Application 
Year Produced 2017 
Open Source License? Yes  
Impact DNA methylation is an important regulator of gene expression and chromatin structure. Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is commonly used to identify regions of DNA methylation in eukaryotic genomes. Within MeDIP-Seq libraries, methylated cytosines can be found in both double-stranded (symmetric) and single-stranded (asymmetric) genomic contexts. While symmetric CG methylation has been relatively well-studied, asymmetric methylation in any dinucleotide context has received less attention. Importantly, no previously available software for processing MeDIP-Seq reads was able to resolve these strand-specific DNA methylation signals. DISMISS is now a new capability for detecting strand-associated DNA methylation from existing MeDIP-Seq analyses. 
URL https://omictools.com/dismiss-tool
 
Description 3rd Annual Drug Discovery Congress 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The Life Sciences Research Network Wales (LSRNW), which supports world class science within Wales, hosted its 3rd Annual Drug Discovery Congress on 30th November and 1st December 2016 at St David's Hotel, Cardiff.

Ser Cymru funded researchers were invited to share their research progress, discuss the challenges faced in developing the next generation of therapeutics, strengthen collaborations and find new partnerships across the academic community, both internationally and in Wales. The event, which was opened by Welsh Government's Minister for Skills and Science, Julie James, with an address on the growth of the Life Sciences sector in Wales, welcomed attendees including students and leading academics whose work has successfully led to commercialisation, as well as industry representatives with substantial experience in the drug development pathway.

Selected PhD students and postdoctoral researchers were invited to present an update on their research; including the development of the next generation of drugs to tackle major societal health issues such as, cancer, antimicrobial resistance and neurodegenerative diseases.
Year(s) Of Engagement Activity 2016
URL http://www.cardiff.ac.uk/news/view/531465-lsrnws-3rd-annual-drug-discovery-congress-awards-ccmrc-stu...
 
Description Aberystwyth Science Week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Between 100-500 regional primary school students visited Aberystwyth University for the UK's Science Week. Parasitology was presented to students and teachers; this activity led to discussions and questions.
Year(s) Of Engagement Activity 2013,2014,2015
 
Description British Society for Parasitology (BSP) Annual Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact BSP presentations delivered on direct and indirect outputs of BBSRC funded grant. In 2013, a plenary talk was given by Hoffmann to summarise the results of the BBSRC grant to date. In 2014, BSP presentations were specifically focused on small non-coding RNA discovery (presented by F. Nowacki) as well as identifying a compound that induced similar phenotypes as 5-AzaC (J. Edwards) and complemented the activities of the BBSRC funded project. In 2015, two talks were given (K Geyer and K Whatley) to update the progress of DNA methylation analyses and epigenetic drug discovery in schistosomes.
Year(s) Of Engagement Activity 2013,2014,2015
 
Description British Society for Parasitology (BSP) Annual Spring Meeting 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Aberystwyth University hosted the British Society for Parasitology Spring Meeting. 432 delegates attended from around the world. Over 50 presentations were delivered across the different specialist subject streams.The meeting generated significant media and local interest and many constructive discussions and possible new collaborations were initiated.
Year(s) Of Engagement Activity 2018
URL http://bsp.uk.net/2018/05/03/bsp-spring-meeting-2018/
 
Description Drug Discovery Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Drug discovery 'mini symposium' hosted by Hoffmann Laboratory, 13 July 2018. Attended by AU staff and postgraduate students and associates from the School of Pharmacy and Pharmaceutical Sciences at Cardiff University (Professor Andrew Westwell, Professor Andrea Brancale and Dr Salvatore Ferla). 1-day programme of presentations, questions and discussions to explore new development opportunities in drug discovery.
Year(s) Of Engagement Activity 2018
 
Description International Flatworm Biology Symposium 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Event attended by K Geyer and K Whatley both presenting data connected with BBSRC-related activities on DNA methylation and epigenetic drug discovery in Schistosoma mansoni.
Year(s) Of Engagement Activity 2015
 
Description Keystone Symposia: Drug Discovery for Parasitic Diseases (Tahoe, Nevada) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Knowledge shared on the current status of anti-parasitic drug discovery and advances in our understanding of the mechanisms of resistance with a final discussion on the future direction of research (within and outside epigenetic targets).
Year(s) Of Engagement Activity 2016
 
Description Laboratory Visit (Cardiff) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Two meetings (January and April 2015) conducted between Aberystwyth University and Cardiff University representatives to share experiences in compound design, computer-aided drug discovery (CADD) and to explore examples of CADD implementation for epigenetic targets.
Year(s) Of Engagement Activity 2015
 
Description Laboratory Visit (Edinburgh) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Knowledge sharing between laboratories on small non-coding RNA libraries (Aberystwyth University represented by Fanny Nowacki).
Year(s) Of Engagement Activity 2013
 
Description Penglais School Visit and lecture on Genomics as part of the Genetics Roadshow 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact 80 students and 10 teachers attended a talk about the power of genomics in shaping our lives. This sparked questions and discussion afterwards.

A sixth form student visited our lab as part of their work experience week.
Year(s) Of Engagement Activity 2013
URL http://www.walesgenepark.cardiff.ac.uk/2013/04/10/genetics-roadshow-for-schools-2013/
 
Description Twitter feed 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Twitter is being used to disseminate our research-related activities to between 100-500 individuals.
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description Visit and Talk - LUMC 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Scientific seminar delivered to staff and students at Leiden University Medical Center, the Netherlands, with questions and associated follow-up discussions. Possibilities explored for future collaboration opportunities.
Year(s) Of Engagement Activity 2017
 
Description Visit to Liverpool School of Tropical Medicine 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Scientific seminar delivered to staff and students at LSTM with questions and associated one-to-one follow-up meetings. Possibilities explored for future collaboration to further research.
Year(s) Of Engagement Activity 2016
 
Description Visit to Manchester University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Seminar on host-parasite interactions and vaccine development delivered to staff and students at Manchester Collaborative Centre for Inflammation Research (MCCIR), Faculty of Life Sciences, University of Manchester. Visit included series of smaller group discussions to explore future collaborative opportunities to further DNA characterisation research.
Year(s) Of Engagement Activity 2016
 
Description WWAYS Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact As part of a programme of lectures organised by the West Wales Association of Young Scientists (WWAYS), Professor Karl Hoffmann presented a lecture to local school children (Year 10-13 students from the Aberystwyth area) on his career path and how parasites and Schistosomes in particular, became the focus of his work. Prof. Hoffmann's lecture focused on the fundamentals of parasitology, the vast numbers of parasites inhabiting the Earth and the enormous impact they have on human health. A number of well-considered questions followed, indicating that the session has sparked a genuine curiosity and has perhaps inspired the next generation of parasitologists.
Year(s) Of Engagement Activity 2018