A Systems Approach to Understanding Methylation Programming in Oocytes and its Consequences in Development

Lead Research Organisation: Babraham Institute
Department Name: Epigenetics

Abstract

The genetic information we receive from our parents is not only the DNA sequence of our genes. Genes are also marked in different ways in the egg and sperm from which we arise. These marks are referred to as 'epigenetic' marks, and one of the major types of epigenetic marking is a chemical tag on certain sites in the DNA called DNA methylation. DNA methylation marks are important because they can change the activity of the genes that they tag; dense DNA methylation on genes usually causes them to be shut down. In addition, DNA methylation marks can be faithfully copied on the DNA as cells divide, so that they can provide a form of memory on the gene to reflect earlier decisions about how that gene should be active. DNA methylation is a normal mechanism by which the body controls genes throughout our development and in response to changes in our environment or diet. However, because DNA methylation marks are easier to change than the underlying DNA sequence, DNA methylation patterns may also be more vulnerable to adverse environmental conditions and abnormal DNA methylation is commonly seen in diseases such as cancer.

In this research, we aim to understand the processes that control which of our genes are marked by DNA methylation in eggs and how these marks are transmitted after the egg is fertilised by the sperm. Eggs and sperm carry different patterns of DNA methylation, so parents can transmit different epigenetic information to their offspring and these could reflect environmental factors in their own history. DNA methylation undergoes particularly dramatic changes in the early embryo, and these transitions may be important for controlling how the embryo divides and the body plan is established and different tissues arise. We are particularly interested to discover whether the normal patterns of DNA methylation in the egg and early embryo are changed as a consequence of altered diet or processes involved in assisted reproduction techniques, such as superovulation. Poor diet - which might include protein deficiency in developing countries and fat-rich diets in the Western world - in mothers before or around the time of conception is known to increase the risk of developing diseases such as diabetes and cardiovascular disorders in later life. Knowing whether DNA methylation changes occur and why would help provide informed advice about nutrition and dietary supplements to women contemplating pregnancy. Assisted reproduction techniques, such as in vitro fertilisation, often involve harvesting eggs and culturing embryos during the time at which DNA methylation patterns are undergoing most rapid change and may be most susceptible. Therefore, it is important to know whether these techniques modify DNA methylation marks to be able to develop further improved fertility treatments and minimise the risk of creating epigenetic errors.

Technical Summary

We aim to provide an overarching account of DNA methylation in oocytes, its consequences and vulnerabilities. The work will be conducted mostly in the mouse as the model of choice for understanding epigenetic processes in gametes and preimplantation embryos. The connections we shall explore between transcription and methylation are based on known properties of Dnmt3a and Dnmt3L - which constitute the de novo methylation complex active in oocytes and which is believed to sense histone modifications - and histone modifying factors with evidence for transcription-coupled activity. Of particular interest to us is methylation of H3K4 and H3K36, which can be modified in response to transcription. We shall use conditional knock-outs for factors of the Kdm1 and Kdm5 families, which regulate H3K4 methylation, and we shall mutate Dnmt3a so that it is no longer binds the transcription-coupled mark H3K36me3. We shall map transcription units and promoter activity and how they change during oocyte growth, and correlate this with timing of DNA methylation in specific genes. We shall ablate specific transcription factors expressed in oocytes, and investigate whether this leads to predicted changes in gene body and intragenic CpG island methylation. This will be done with conditional alleles for transcription factors with known roles in oogenesis, but we shall also identify additional transcription factors with effects during the de novo methylation phase of oocyte growth by informatics analysis of expression and transcription start site datasets. We shall use our highly sensitive DNA methylation profiling (RRBS) as an endpoint in many of these mechanistic studies. We shall use RRBS (and other techniques we might develop) to profile changes in methylation in oocytes and embryos in response to manipulations in maternal diet and superovulation. We shall also profile DNA methylation in human preimplantation embryos to identify conserved sites and properties of DNA methylation.

Planned Impact

The work proposed in this application is mostly of a fundamental nature in a model organism aimed at understanding epigenetic processes in germ cells and preimplantation embryos. It seeks to provide an overarching account of how and where DNA methylation is established in female gametes, what the consequences of this mark is for how genes are expressed in the oocyte and how gene activity is regulated in the early embryo. Correct epigenetic programming in gametes and embryos has a lifelong impact on development and health, because some epigenetic marks once established or lost cannot be corrected at later times in development. Although the work will be carried out mostly in a genetically tractable and informative model organism, we shall also conduct comparative analysis in human preimplantation embryos to demonstrate whether insights gained in the model organism are relevant to human embryogenesis. As well as providing fundamental knowledge, we expect that the research could help identify risks in assisted reproduction methods, enhance knowledge in stem cell research, and identify whether epigenetic marks can be destabilised by poor maternal nutrition and could directly contribute to adverse health outcomes in later life. Therefore, the knowledge acquired from this research will likely to have major impacts in fundamental epigenetics and germ cell biology research, but also potential translational impacts in human nutrition, assisted reproduction and stem cell research.

We also expect an impact of our research in the technical advances we shall continue to make in developing methods for epigenetic profiling in small numbers of cells. Such advances could have widespread application in biomedical research. We have already been approached by several groups (from both academic and pharmaceutical sectors) from research areas unrelated to our field with an interest in applying our methods to their own biomedical questions. Therefore, we anticipate significant impact in training and knowledge exchange also at a technical level.

Publications

10 25 50
 
Description ESPE Research Fellowship
Amount € 124,000 (EUR)
Organisation European Society of Pediatric Endocrinology (ESPE) 
Sector Learned Society
Country European Union (EU)
Start 01/2014 
End 12/2015
 
Description Epigenome patterning in oocytes and its legacies in the embryo
Amount £1,964,775 (GBP)
Funding ID MR/S000437/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 08/2018 
End 07/2023
 
Description Post-doctoral travelling fellowship
Amount € 49,000 (EUR)
Organisation Academy of Finland 
Sector Public
Country Finland
Start 09/2013 
End 08/2015
 
Title High through-put single-cell DNA methylation profiling 
Description We have improved our previously published method of profiling DNA methylation genome-wide in single cells (Smallwood et al., Nat. Methods 2014), by implementing the method on a liquid handing robot (Clark et al. Nat. Protocols 2017), which is allowing much higher through-put applications. An extensive protocol is provided. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Allowing much higher through-put applications. 
 
Title Parallel analysis of DNA methylation, chromatin accessibility, RNA expression in single mammalian cells 
Description By combining previous single-cells we and others have developed, this new Method, term scNMT-seq, enables high-coverage, genome-wide profiling of DNA methylation, chromatin accessibility and RNA transcripts in parallel in single cells. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Very recently published; too soon to identify notable impacts 
 
Title Single-cell BS-seq 
Description We developed a method to profile DNA methylation genome-wide in single cells, which has now been published (Smallwood et al., Nat. Methods 2014). This capability could have widespread application in assessing DNA methylation in rare cells (e.g., human gametes, preimplantation embryos), to assess DNA methylation heterogeneity amongst cells, which could be an important mechanism in developmental decisions, aging and pathological states, notably in cancer. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact The development of this method has led to the establishment of new collaborations for our group. It has also been a key component underpinning the award of a Wellcome Trust Strategic Award by a consortium headed by my colleague Prof. W. Reik. 
 
Title Ultra-low cell histone modification profiling 
Description Optimised method for ultra-low cell native ChIP-seq for histone modifications, exemplified by use in 250 mouse oocytes (see Hanna et al. 2019 Nat. Struct. Mol. Biol.) 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact This is providing us with major new capability in profiling histone modifications reproducibly and quantitatively in biological samples such as mouse oocytes and mouse pre-implantation and early post-implantation embryos, and is now being applied for a variety of research questions by the group. We are also using the method in a number of collaborators and have trained others in the method. 
 
Title Chromatin modifications during mouse oogenesis 
Description High quality native ChIP-seq datasets for histone modifications H3K4me3, H3K27ac, H3K27me3 during oocyte growth and fully grown oocytes in the mouse. ChIP-seq datasets for H3K4me3 in oocytes genetically deficient in DNA methylation (Dnmt3a/Dnmt3b nulls). ChIP-seq datasets for oocytes genetically deficient in the major H3K4 methyltransferase MLL2 (KMT2B). Published in Hanna et al. Nat. Struct. Mol. Biol. 2018 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Recently released datasets, too early to identify impact outside of research group. 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93941GSE93941
 
Title DNA methylation during mouse oogenesis 
Description Genome-wide DNA methylation datasets by RRBS and PBAT from size-selected mouse oocytes during the oocyte growth phase. Published in Gahurova et al. 2017 Epigenetics and Chromatin. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact Only recently released, impact outside of research group too early to say. 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE86297
 
Title DNA methylation in Hira oocytes 
Description Genome-wide, single-cell DNA methylation data from mouse oocytes lacking the histone chaperone HIRA, and associated controls, as reported in Nashun et al. 2015 Molecular Cell 60, 611-625. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Too soon to define notable impacts, as the accompanying paper, Nashun et al. 2015 Molecular Cell, was published in October 2015. 
URL http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE66629
 
Title DNA methylation in oocytes deficient in histone modifier 
Description Genome-wide DNA methylation datasets (PBAT) and single-cell RNA-seq datasets from oocytes genetically deficient in the H3K4me3 methyltransferase MLL2 (KMT2B). Published in Hanna et al. 2018 Nat. Struct. Mol. Biol. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Recently released, too early to identify impact outside of research group. 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93941GSE93941
 
Title Gene expression during mouse oogenesis 
Description Deeply sequenced RNA-seq datasets from size-selected mouse oocytes during the oocyte growth phase. Published in Gahurova et al. 2017 Epigenetics and Chromatin. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact Only recently released, too early to identify impacts beyond research group. 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE86297
 
Title Human placental imprints 
Description DNA methylation analysis of newly identified human placental-specific imprinted genes; published in Hanna et al., 2016 Genome Research doi:10.1101/gr.196139.115. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Too soon to define notable impacts, as associated paper was published in Genome Research in January 2016. 
URL http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE76263
 
Title Oocyte ChIP-seq datasets 
Description ChIP-seq datasets for selected histone modifications in mouse growing oocytes. DNA methylation profiles of oocytes lacking the histone demethylases KDM1A, KDM1B. As reported in Stewart et al., 2015 Genes and Development, 29, 2449-2462 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Too soon to define notable impacts, as the associated manuscript was published in Genes and Development in December 2015 
URL http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE74549
 
Title Oocyte transcriptome, mouse 
Description Deep strand-specific RNA-seq to establish in-depth transcriptome of mouse oocytes, as reported in Veselovska et al., 2015 Genome Biology 16, 209 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact The publication in Genome Biology associated with these datasets has already attracted >30 citations, suggesting a good level of uptake of the data by the research community. 
URL http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE70116
 
Title Single-cell DNA methylation profiles 
Description Single-cell methylation datasets for mouse oocytes and ES cells were submitted to the Gene Expression Omnibus (GEO) under accession GSE56879. 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Too soon to comment 
 
Description Analysis of Mll2-deficient oocytes 
Organisation Technical University of Dresden
Department Biotechnology Center
Country Germany 
Sector Academic/University 
PI Contribution We framed the biological question and were responsible for experimental design and data analysis. We performed DNA methylation and histone modification analysis of oocytes conditionally deleted for Mll2 provided by the partner.
Collaborator Contribution Provided mice with conditional knock-out allele of Mll2 and Mll2-deficient oocytes.
Impact Joint publication: Hanna et al., Nat. Struct. Mol. Biol. 2018. Deposited sequence datasets at NCBI GEO: GSE93941.
Start Year 2013
 
Description Analysis of Setd1b-deficient oocytes 
Organisation Technical University of Dresden
Department Biotechnology Center
Country Germany 
Sector Academic/University 
PI Contribution We performed analysis of RNA-seq datasets in oocytes conditionally deleted for Setd1b generated by the partner, with reference to our mouse oocyte transcriptome assemblies (Veselovska et al., 2015 Genome Biol; Gahurova et al., 2017 Epigenetics & Chromatin).
Collaborator Contribution Partner was responsible for framing the project and experimental design and providing data from mice with conditional knock-out allele of Setd1b in oocytes
Impact Joint publication: Brici et al., Development 2017
Start Year 2013
 
Description Analysis of histone modifications in mice with loss-of-imprinting of Zac1 
Organisation Clermont Université
PI Contribution Our group was responsible for framing the project and experimental design; we provided tissues from mice with loss-of-imprinting of Zac1.
Collaborator Contribution Their group performed ChIP-PCR analysis in tissues from Zac1 loss-of-imprinting mice we provided.
Impact Joint publication Veselovska et al. 2015 Genome Biol.
Start Year 2013
 
Description Bioinformatic analysis of oocyte ChIP-seq data 
Organisation Helmholtz Zentrum München
Department Institute of Computational Biology
Country Germany 
Sector Private 
PI Contribution We provided ChIP-seq datasets for various histone modifications in mouse wild-type and genetic knock-out oocytes to enable partner to test new informatics tools they had developed.
Collaborator Contribution The partner provided new informatics methods, based on multivariate Hidden-Markov Models, to enable analysis of multiple ChIP-seq datasets, with focus on multivariate comparisons.
Impact Joint publication: Hanna et al., Nat. Struct. Mol. Biol. 2018. Deposited sequence datasets at NCBI GEO: GSE93941. Hosted visiting bioinformatics PhD student from collaborating lab. Inter-disciplinarity: partner provides modelling and informatics expertise, our group provides biological datasets.
Start Year 2016
 
Description Bioinformatic analysis of single-cell epigenomic datasets 
Organisation EMBL European Bioinformatics Institute (EMBL - EBI)
Country United Kingdom 
Sector Academic/University 
PI Contribution My group, together with the group of my colleague Wofl Reik, have developed methods for single-cell profiling of DNA methylation, upon which we have combined single-cell RNA-seq and single-cell profiling of chromatin accessibility. We have provided datasets from mouse ES cells and oocytes which the partner has explored and developed bioinformatic methods for investigating epigenetic heterogeneity.
Collaborator Contribution The partner has provided high level bioinformatic expertise.
Impact This has led to four published papers so far: Smallwood et al. 2014 Nat. Methods; Angermueller et al. 2016 Nat. Methods; Kelsey et al. 2017 Science; Clark et al. 2018 Nat. Comms. The multidisciplinarity is the combination of developmental epigenetics and bioinformatics.
Start Year 2014
 
Description DNA methylation analysis in oocytes genetically deficient for Kdm1a (Lsd1) and Kdm1b (Aof1) 
Organisation University of Texas
Department M. D. Anderson Cancer Center
Country United States 
Sector Academic/University 
PI Contribution We were responsible for framing the project and experimental design and performed genome-wide DNA methylation analysis in oocytes genetically deficient in the H3K4me2 demethylases KDM1A or KDM1B, provided by the collaborating group.
Collaborator Contribution The partner provided oocytes from mice genetically deficient in Kdm1a or Kdm1b; provided RNA-seq datasets for these oocytes.
Impact Joint publications Stewart et al., 2015 Genes & Dev; Gahurova et al. 2017 Epigenetics & Chromatin. Deposited sequence datasets at NCBI GEO: GSE73803 and GSE74549.
Start Year 2011
 
Description DNA methylation analysis in oocytes genetically deficient in Hira 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre (CSC)
Country United Kingdom 
Sector Academic/University 
PI Contribution We performed single-cell, genome-wide bisulphite sequencing of oocytes deficient in H3.3 chaperone HIRA, provided by the collaborating group; using our newly established single-cell BS-seq method (Smallwood et al., 2014 Nat. Meths).
Collaborator Contribution The partner was responsible for framing the project and experimental design and provided mouse oocytes conditionally deleted in Hira.
Impact Joint publication Nashun et al. 2015 Mol. Cell. Deposited sequence datasets at NCBI GSE66629.
Start Year 2014
 
Description Identification of polymorphically imprinted loci in human placenta 
Organisation University of British Columbia
Country Canada 
Sector Academic/University 
PI Contribution Our team analysed 450k array datasets from human placenta samples provided by the collaborating group. We also used targeted bisulphite sequencing to validate candidate imprinted loci identified by the array analysis.
Collaborator Contribution Their team was responsible for framing the project and experimental design and provided human placental DNA samples.
Impact Joint publication Hanna et al. 2016 Genome Res. Deposited sequence datasets at NCBI GEO GSE74738 and GSE76273
Start Year 2015
 
Description Babraham Schools Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Annual event in which local GCSE and A-level students are hosted in my group and many other groups at the Institute for a day of practical demonstrations.
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,
 
Description Babraham Schools Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact As part of the annual Babraham Schools' Day, my group hosted 5 GCSE and 5 A level students for half-day practical sessions in the lab.
Year(s) Of Engagement Activity 2017
 
Description Babraham Schools' Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact As part of the annual Babraham Schools' Day, my group hosted 5 GCSE and 5 A level students for half-day practical sessions in the lab.
Year(s) Of Engagement Activity 2018
 
Description Babraham Schools' Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Hands-on demonstration to secondary school children, most of whom interested in pursuing biomedical sciences as undergraduates.
Year(s) Of Engagement Activity 2018
 
Description Cambridge Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Poster Presentation
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Presentation of of the Babraham Institute Epigenetics exhibit at the Cambridge Science Festival March 2013. A large number of interested public attended, viewed the exhibit and asked questions of me and the other presenters during the weekend event.

Some very interesting and insightful questions from some visitors to the exhibit.
Year(s) Of Engagement Activity 2013
 
Description GenomeWeb interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Interview with GenomeWeb on the publication of our single-cell combined DNA methylation and transcriptome sequencing method published in Nature Methods (Angermueller et al. 2016).
Year(s) Of Engagement Activity 2016
 
Description Local community visit - Pampisford Society 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact A society from the local community - the Pampisford Society - visited the Institute because of interest in finding out about the nature of the research conducted at the Institute. As well as a tour of some of the research facilities, including a virtual tool of the animal facilities, and presentations on the Institute, there were two research talks given, one by myself. The talk covered how we profile epigenetic marks in gametes and early embryos, and how we seek to understand possible effects of lifestyle (diet) and age on the transmission of epigenetic marks from parents to offspring. There was considerable interest in this area of research.
Year(s) Of Engagement Activity 2017
 
Description Museums Week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Many very stimulating discussions with members of the public at many levels who approached the exhibit.

No tangible impacts beyond the perception that many of the people who visited the exhibit appeared to be interested to find out about epigenetics and will have left the exhibit more informed; there is substantial interest in epigenetics amongst many people, particularly after television programmes that have covered some of the ground.
Year(s) Of Engagement Activity 2014
 
Description Press release - Keeping eggs fresh with epigenetics 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release from Babraham to accompany publication of paper 'MLL2 conveys transcription-independent H3K4me3 in the oocyte', by Hanna et al. Nat. Struct. Mol. Biol. 2018. Picked up by 9 news outlets and blogs.
Year(s) Of Engagement Activity 2018
URL https://www.babraham.ac.uk/news/2018/01/keeping-egg-cells-fresh-with-epigenetics
 
Description Press release - Molecular Cell paper 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release associated with publication Nashun et al. 2015 Molecular Cell on role of nucleosome replacement in oocyte development.
Year(s) Of Engagement Activity 2015
 
Description STEM teacher visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact As part of a week-long visit to the Institute, my group hosted 3 biology A-level teachers from local schools for involvement in practical work in the lab over a 2-day period.
Year(s) Of Engagement Activity 2016
 
Description Talk to journalism students 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact Presentation about the topic of this research award to ~15 journalism students from City University hosted at the Babraham Institute, March 2013.

Some genuinely interested and insightful and informed questions from students interested in pursuing a career in science journalism.
Year(s) Of Engagement Activity 2013
 
Description Teacher workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Talk to sixth-form teachers as part of 'Twilight Teacher Session', held at the Wellcome Trust Sanger Institute, Hinxton. The remit of the event was to inform teachers about state-of-the-art DNA sequencing technologies and their applications. It comprised a tour of sequencing facilities at the Sanger and a talk from a Sanger scientist and from myself, in which I presented current progress in single-cell sequencing methods and their applications in reproductive medicine. The workshop also discussed teaching aids that could be used to illustrate current sequencing capabilities.
Year(s) Of Engagement Activity 2017