Unravelling the networks that regulate seasonal rhythmicity in the epigenome

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

Two interacting rhythmical processes dominate the biology of most organisms on earth. The best studied is the circadian clock, which this has evolved to match the 24h rotation of earth. The second is the circannual clock driving ca 1-year rhythms, which has evolved in many life-forms to meet the profound environmental challenges of a seasonal planet. Although the two clock-work systems are interlocked, the precise mechanisms by which the circadian clock contributes to a seasonal response are yet to be established in any animal species, nor do we know how long-term circannual rhythm generation occurs.

The seasonal and circannual timing mechanisms have been studied in sheep. Our work and others has defined how the nocturnal hormone melatonin is used by the neuroendocrine system to provide an internal representation of external photoperiod, driving seasonal reproductive and metabolic responses. A key site of action is the pituitary gland, called pars tuberalis (PT), in a region immediately adjacent to the hypothalamus, where a local circadian clock-gene rhythm is entrained each night by the daily melatonin hormone signal in specialized thyroid-stimulating hormone (TSH) expressing cells. These PT thyrotrophs have been termed "calendar cells". Here, a key transcriptional co-activator (EYA3) is rhythmically regulated, and on long summer photoperiods (LP) is strongly augmented, leading to expression of PT TSH, which activates TSH-receptors and thyroid hormone (TH) metabolism in the adjacent hypothalamus. The TH-dependency for the seasonal response is a conserved feature of the biology of vertebrate species. We have now shown that hormone packaging protein (CHGA) is induced on short winter photoperiods (SP) and that specialised cells within the PT flip from a CHGA to an EYA3 state (binary switching) over the circannual cycle.

Our goal is to discover whether an "epigenetic" process underpins this binary switch and whether this switch drives long-term rhythm generation. Epigenetics is defined as the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states without changes to DNA sequence. It involves modification of histone proteins around which DNA is wrapped, and also the chemical nature (methylation) of one of the DNA nucleotides (cytosine). Our work builds in part on recently tested theoretical models developed by plant scientists studying vernalisation, which has revealed a critical role for histone-modifying enzyme complex (polycomb repressor-2, PRC2) in providing a memory of winter cold-exposure. PRC2 represses gene expression by acting on histone-3 proteins.

A key element of the PRC2 complex (EZH2) is activated on LP, and we will test whether this is involved in a global suppression of SP-expressed genes, including CHGA. We now have exciting new evidence that EZH2 also activates EYA3 on LP, via the circadian clock. This leads us to the hypothesis that chromatin modifying enzymes act as binary switches, activating LP-genes, and suppressing SP-genes. This could be the long-predicted biochemical switch mechanism driving circannual cycles in vertebrates. We investigate this switch mechanism in cell models, and also using cells and tissues from sheep over the circannual cycle. This will include studies of how protein modifications and partners of EZH2 are involved. This will allow us to test this hypothesis that changes in histone-3 protein methylation (driven by EZH2) drives the circannual cycle by mapping these to underlying changes the PT.

Finally, we will combine our findings with existing genomic data sets for domesticated and wild sheep breeds, and establish whether genetic circuits driving timing have been selected in course of domestication. Thus, we aim to unravel the central pathways driving the seasonal rhythm of life on our planet.

Technical Summary

We will investigate how seasonal clocks are regulated by epigenetic processes. This arises from our discovery that the PT thyrotroph (calendar cell) operates a binary switch mechanism over the circannual cycle. We aim to discover mechanisms involved. Our model is the seasonal sheep.

We will use ChIP-seq to define the relative contribution of the PcGs and TrxG complexes in driving the changing epigenetic landscape of the PT over the circannual cycle, and how the balance of these marks regulates a cellular binary switch. RNA-seq and Bis-seq data will also be collected in order to develop a full regulome of the PT over the circannual cycle.
The PcG element, EZH2 is the key component driving H3K27me3 and repression of target genes. Building on our recent studies, revealing an important co-activation role for EZH2 in driving EYA3 on long photoperiods, we will investigate a novel binary switch mechanism involving epigenetic repression and simultaneous co-activation by PcG, using a cellular model for EYA3 and CHGA switching. Our methods include used of engineered BACs , live single cell imaging, and bioinformatic models to test how dynamic methylation changes drive long-term transcriptional programmes. We will then test these outcomes in primary cells collected over the circannual cycle.

We will define mechanisms by which circadian clock elements regulate EYA3, CHGA and EZH2, and their interaction with PcG components, and BRET and mass spectrometry approaches (RIME), establish the role of post-translational modifications.

We will define morphological remodelling of the PT calendar cells, using 3-dimensional electron microscopic approaches and advanced optical (light-sheet) microscopy, to address how binary switching of individual calendar cell thyrotrophs is propagated across the PT tissue.

Finally, we will define the role of circannual timing circuits in the domestication and control of breeding seasons in different sheep breeds.

Planned Impact

This project addresses how seasonal timing mechanisms and circannual cycles are generated in mammals. Our studies aim to characterize the contributions of environmentally controlled transcriptional switches in the regulation of neuroendocrine control of seasonal timing, explore and test underlying epigenetic mechanisms involved. The studies will illustrate fundamental control mechanisms involved in growth, metabolism, immunity and reproduction in a livestock species and vertebrates more widely. This will provide insight into the genetic pathways that have been under selection during the evolution of livestock species, and how environmental signals are involved. It will also define the role for environmentally controlled epigenetic switches.

Involvement with industry: There is considerable interest within "pharma" in developing tools to regulate epigenetic processes, from cancer biology to psychiatry, and the existing close research links and formal cross-institutional memorandum of understanding with GSK and Manchester University, including a free exchange of research staff between the two institutions, will allow access to novel tool compounds to test in cellular and tissue assays. DB and staff at Roslin are part of the International Sheep Genome Consortium (ISGC). Together these projects and contacts provide a natural route to disseminate and exploit epigenetic information from within this project to industry and the wider community. Furthermore, links with the Sheep Genome Consortia and Breeding Societies is expected to lead to new opportunities and funding applications.

Implications for new understanding of domestication: This application will lead to an advance in knowledge of mechanisms regulating growth and reproduction in a livestock species. A stated long-term outcome of our project is to extend our studies to comparative genomics of different sheep breeds, and test the hypothesis that pathways, genes and circuits involved in seasonal timing have been selected for in the course of domestication.

Potential links to human health: Longer term, there are potential applications from our studies for the targeting of neuroendocrine pathways involved in growth and metabolism
In humans, shift work and of non-resonant/non-circadian feeding times have profound metabolic consequences. This represents a wide-spread problem in society as it likely underpins the health problems associated with long-term shift work patterns, and affecting 20% of the workforce in industrialized countries and greatly increasing the risk of metabolic diseases. The studies outlined in this application could define quite novel epigenetic mechanisms in which rhythmic signals may control metabolic processes, and help inform appropriate circadian management protocols with ultimate application to man, healthy living. Recent publications have demonstrated the effects of seasonal timing on human health, for example seasonal variation in human immunity has been reported (Dopico 2015 Nature communications, 6, 7000) and the effect of melatonin on the seasonality of multiple sclerosis relapses (Farez 2015, Cell, 162(6), 1338-1352). These publications suggest that the impact of seasonal timing may be similar to the impact of the circadian clock on human health and disease, emphasizing the potential importance and translational potential of investigating this timing mechanism in a well-established seasonal model organism (sheep).

Publications

10 25 50
 
Description The overarching aim of our project is to define how long-term seasonal rhythms of physiology and fertility are generated in a mammal, a problem of considerable agricultural importance, as it underpins virtually all production systems. We are the only group in the UK working on this problem in agricultural models, and have established an un-rivalled expertise in the background genomics and technology infra-structure to achieve these goals. Our goal is to discover whether an "epigenetic" process underpins this binary switch and whether this switch drives long-term rhythm generation. Epigenetics is defined as the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states without changes to DNA sequence. It involves modification of histone proteins around which DNA is wrapped, and also the chemical nature (methylation) of one of the DNA nucleotides (cytosine).
We use sheep as a model, as its robust seasonal phenotype, characterised genome and well-defined physiology, offers an outstanding model organism for such studies. Our focus of interest is a small region of the pituitary gland, the pars tuberalis (PT), as this is site where the hormone melatonin acts, to generate photoperiodic responses. Within the PT, long-term rhythms are generated, driving long-term "circannual" cycles of hormone secretion and fertility. We set 3 objectives in the current BBSRC-funded project. 1) Defining the circannual epigenome within the PT calendar cell 2) Establishing control mechanisms driving binary switching and 3) Defining implications of anatomical re-modelling by the circannual clock.
Our analytical lab-based platforms - some novel and developed by us in the project - have allowed us to map genomic sites of regulation combining ChIP-SEQ, and single-nuclear sequencing analysis. Our success in developing these techniques has allowed us to interrogate genome-wide remodelling of the PT clockwork and obtain a new understanding of how timing of breeding is controlled in an agriculturally important animal species.
Exploitation Route Understanding biological clocks and seasonal timing molecular mechanisms.
Sectors Agriculture, Food and Drink

 
Description Core Member BBSRC Panel A Response Mode
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
Description Identification of the photoreceptor for light detection in the avian brain
Amount £246,909 (GBP)
Funding ID RPG-2016-392 
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 09/2017 
End 09/2020
 
Title Comparing selection in breeds of sheep with long and short breeding season 
Description We sequenced 50 individuals from rare breeds of primitive sheep from phenotypic extremes regarding the timing and duration of breeding season. These were genotyped at 30x on the HiSeqX. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact We identified strong selection signatures on genes, many of which were associated with neurological development. 
 
Title Sheep Timeseries RNASeq 
Description We have used RNASeq to capture both daily and seasonal rhythms in the transcriptome of sheep pars tuberalis (PT). PT samples are from 60 sheep in controlled environments at long and short photoperiods. Samples were taken at ZT4 at days 1, 7, 28, 84, and 112 into the photoperiod. Two 24 hour times series were sampled in long and short photoperiods at 28 days every 4 hours. BioProject: PRJNA391103 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact It has enabled us to identify the circadian clock components that are seasonally regulated by photoperiod. 
 
Title Sheep methylation timeseries RRBS 
Description We have used RRBS to capture both daily and seasonal rhythms in the methylome of sheep pars tuberalis (PT). PT samples are from 60 sheep in controlled environments at long and short photoperiods. Samples were taken at ZT4 at days 1, 7, 28, 84, and 112 into the photoperiod. Two 24 hour times series were sampled in long and short photoperiods at 28 days every 4 hours. BioProject: PRJNA391103 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact We were able to identify targeted seasonal changes in methylation in response to photoperiod. 
 
Description Collaboration with the Rare Breed Survival Trust (RBST) to study the genetics of rare breeds of sheep 
Organisation Rare Breeds Survival Trust (RBST)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Sampling and resequencing DNA from rare breed sheep. This assists our research into identifying the genetics and selection of seasonal phenotypes and also has a wider impact for RBST, for UK agriculture, and globally in understanding and preserving the genetics of rare breeds. The collaboration also allows us to effectively disseminate the latest research to the rare breeds community via the RBST farmers network and publications.
Collaborator Contribution RBST provide specialist knowledge of the breeds that RSBT manage. An introduction to breeders for obtaining genetics samples. Feedback and consultation on design and ethical evaluation of the research. A network for the dissemination of research finding.
Impact We collected DNA for WGS from rare breed Portland sheep. RBST provided the introduction to relevant breeders and approval and feedback on the letter of introduction and informed consent. RBST recently extended an invitation to publish an article in their breeding magazine to update members on research outcomes.
Start Year 2016
 
Description Collaboration with the Shetland Sheep Society (SSS) to study the genetics of rare breeds of sheep 
Organisation Shetland Sheep Society
PI Contribution Sampling and resequencing DNA from rare breed sheep. This assists our research into identifying the genetics and selection of seasonal phenotypes and also has a wider impact for SSS, for UK agriculture, and globally in understanding and preserving the genetics of rare breeds. The collaboration also allows us to effectively disseminate the latest research to the rare breeds community via the SSS farmers network and publications.
Collaborator Contribution SSS provide specialist knowledge of the breeds that SSS manage. An introduction to breeders for obtaining genetics samples. Feedback and consultation on design and ethical evaluation of the research. A network for the dissemination of research finding.
Impact We collected DNA for WGS from rare breed Shetland breed sheep. SSS provided the introduction to relevant breeders.
Start Year 2016
 
Description 2017 Seminar: "Neuroendocrine and environmental interactions underlying reproductive behaviour in birds" The Institute of Biological & Environmental Sciences, The University of Aberdeen, Aberdeen, UK. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk given at the university and discussions with academics. Has inspired future collaborations with the University of Aberdeen.
Year(s) Of Engagement Activity 2017
 
Description BBSRC Japan Partnering Workshop on Neuroendocrine Control of Reproduction 
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 Japan Partnering Workshop. Invited speaker from Japan. Engaged with staff, post graduate students and undergraduate students. 20 People attended and presented. Much discussion and plans made for further collaboration and visits to Japan in 2019. In addition talks surrounding future careers for post docs and PhD students.
Year(s) Of Engagement Activity 2018
 
Description Industry Insights Day for Women in STEM 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact Opportunity for female undergraduate students considering a career in academia to be inspired by women in STEM. Talk and Q&A session.
Year(s) Of Engagement Activity 2018
 
Description Open doors 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 Public/other audiences
Results and Impact Over 200 members of the public attended which sparked questions and discussion regarding our research
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Projects Update Meeting regarding Circannual Clock in Lake District (With collaborators in University of Manchester, The Arctic University of Norway, and various interested emeritus professors) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Two days of talks, discussions and planning meetings with participants and scientific advisors regarding progress on sheep circannual related projects.
Year(s) Of Engagement Activity 2018
 
Description Seminar: "Neuroendocrine and environmental interactions underlying reproductive behaviour in birds" The Department of Zoology and Physiology, The University of Wyoming, Laramie, USA. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Seminar: "Neuroendocrine and environmental interactions underlying reproductive behaviour in birds" The Department of Zoology and Physiology, The University of Wyoming, Laramie, USA. Inspired women in STEm. This has sparked an exchange partnership between University of Wyoming and Roslin. I hosted a visiting group composed of undergraduate students in Jan 2018 from University of Wyoming.
Year(s) Of Engagement Activity 2017
 
Description Society for Research on Biological Rhythms (SRBR) meeting 2018 poster presentation (Amelia Island, FL, USA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation entitled: Genome-wide changes in DNA methylation mark the changing seasons in mammalian calendar cells. Reported on progress in seasonal changes in DNA CpH methylation determined by RRBS.
Year(s) Of Engagement Activity 2018
URL https://srbr.org/meetings/upcoming-meeting/
 
Description Talk ISGC workshop PAGXXV, San Diego, CA, USA, 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A talk on "Genome Wide Transcript and Methylation Changes in the Sheep Seasonal Clock" to the workshop organized by the International Sheep Genome Consortium. This included an update on the current progress on genotyping/resequencing exotic breeds with seasonal phenotypes. There were a number of questions and discussion as to how seasonal biology could be important in other agricultural traits (e.g. seasonal epigenetic control of salmon maturation).
Year(s) Of Engagement Activity 2017
URL http://www.intlpag.org/2017/program/workshop-list-xxv
 
Description Talk ISGC workshop PAGXXV, San Diego, CA, USA, 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A talk on "Genome Wide Transcript and Methylation Changes in the Sheep Seasonal Clock" to the workshop organised by the International Sheep Genome Consortium. There were a number of questions and discussion as to how seasonal biology could be important in other agricultural traits (e.g. seasonal epigenetic control of salmon maturation).
Year(s) Of Engagement Activity 2017
URL http://www.intlpag.org/2017/program/workshop-list-xxv
 
Description Talk at Cambridge Livestock Genomics Meeting, Cambridge, UK, 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk on "Genome Wide Transcript and Methylation Changes in the Sheep Seasonal Clock". The Livestock Genomics Meeting encompasses groups working across livestock species which provides invaluable incite into relevant work in other species and how our work is applicable to other traits of agricultural and economic importance. The talk prompted valuable discussion with groups working in the U.S. and Japan.
Year(s) Of Engagement Activity 2016
 
Description Talk at the Roslin Computational Genetics Discussion Group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Talk entitled "Genome Wide Transcript and Methylation Changes in the Sheep Seasonal Clock". There were useful questions on the status of EYA3 as a candidate seasonal regulator and discussion on the application of the work to breeding for seasonal phenotypes. An overview of the progress on resequencing exotic breeds was also discussed, which was of particular relevance to the genetics audience.
Year(s) Of Engagement Activity 2017
URL https://www.wiki.ed.ac.uk/display/RCG/Computational+Genetics+Discussion+Group
 
Description Talk on Clocks and Photoperiod: DNA methylation in the sheep pituitary (Babraham Institute, Cambridge) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk at Cross institute interdisciplinary workshop on Epigenetics. Very useful feedback and questions received regarding the talk and a number of scientists expressed interest in our findings.
Year(s) Of Engagement Activity 2018
 
Description Talk: Epigenetics and seasonal biology at Wild Clocks Conference (Munich, Germany) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited by session chair to talk about our work on circannual ovine DNA methylation in the PT. There were many questions on the talk and considerable interest in our findings.
Year(s) Of Engagement Activity 2018
URL https://www.orn.mpg.de/wild-clocks-conference
 
Description Transcriptional and Epigenetic Regulation of Seasonal Timing. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited lecture: Transcriptional and Epigenetic Regulation of Seasonal Timing. In "Evolution of seasonal timers". The 64th NIBB Conference, Okazaki Conference Centre, Okazaki, Japan. 22-24 April, 2016.
Year(s) Of Engagement Activity 2016