Epigenetic control of seasonal timing

Animals inhabiting seasonal environments need to adapt their physiology to survive to anticipate environmental change. To achieve this, sophisticated internal clockworks have evolved which drives annual cycles of behaviour and physiology. These can free-run in constant conditions ("circannual" rhythms) and are synchronised to the environment by a brain hormone Melatonin (MEL), produced at night from the pineal gland, the activity of which is regulated by the light-dark cycle. Accordingly, MEL targets are exposed to seasonal changes in duration of the MEL signal - long in winter and short in summer. The MEL signal acts on hormone secreting circuits (neuroendocrine), which in turn drive annual reproductive and metabolic cycles.

MEL acts on targets sites in a specific region at the base of the brain in the pituitary gland called the pars tuberalis (PT), which acts as a seasonal conductor - controlling hormone secretion such as prolactin (a hair growth regulator) in the main pituitary gland, and also within another immediately adjacent brain structure in the hypothalamus, where it regulates the thyroid hormone levels, controlling seasonal reproduction and metabolic cycles. We discovered a key gene regulating long-photoperiod (LP) responses in the PT called EYA3 which is rapidly activated by LP's and acts as a switch mechanism driving hypothalamic thyroid hormone changes.

We have also discovered many genes in the PT are regulated by a process called DNA methylation. Methylation suppresses activity of genes and is essential to the function of normal cells. Our studies show that both de-methylation - removal of the suppressive imprint - and methylation - imposition of the imprint - are remarkably dynamic. So much so, that each night MEL induces changes affecting the methylated state of over 1000 genes. Such DNA modifications are termed epigenetic - heritable changes caused by mechanisms other than changes in the underlying DNA sequence, and are crucial in many diseases, but also underpins normal physiology including ageing and differentiation of stem cells.

We aim to map all methylated sites in the sheep genome (the "methylome") in response to seasonal signals (the "seasonal-methylome"), simulated by changes in photoperiod using high-resolution bisulphite sequencing. This is important, as our current methylation screen was based on a low-resolution and less sensitive method that cannot identify specific methylated DNA nucleotides. We will now be able to define methylation changes that affect the function of specific DNA signals, such as binding sites for specific transcriptional regulators. We will compare material collected from animals maintained on short or long photoperiods, using tissue from the PT and also the ventral hypothalamus (where thyroid hormone metabolism is controlled), and examine how the methylome changes in different photoperiods. The pattern of methylation under different photoperiods will also be correlated with changes in the expression of genes, determined by whole genome RNA Sequencing (the "seasonal-transcriptome"). We will test the hypothesis that pre-exposure to specific photoperiods can affect an animal's response to changes in photoperiod, and how the "methylome" drives long-term seasonal rhythms.

These studies will provide the first insight into epigenetic control of genes in brain structures that control seasonal reproduction and growth in a mammal. Our studies may also reveal general features of the biology of livestock domestication, since components of the PT circuit (TSH-R) have been identified as being under strong selection in comparative studies of chickens. This may lead to new understanding of mechanisms controlling reproduction and growth, of commercial significance to the livestock industry in the UK, and reveal how animals could adapt to climate change through interaction of external signals in the environment, and DNA modifications.

We will investigate how seasonal clocks timing reproduction and metabolism are regulated by epigenetic processes. This arises from our discovery that melatonin - the seasonal timer hormone - controls dynamic changes in gene methylation state in the pars tuberalis (PT) of the pituitary gland, and may activate or suppress multiple pathways in rhythmic manner each day.

We will measure using high resolution bi-sulphite sequencing how genome-wide changes in methylation change in different photoperiods, defining modifications to specific CpG sites. In parallel we will use genome-wide RNA sequencing (RNA-Seq) to study expression of coding and non-coding genes, to develop predictive models of gene expression, including transcriptional (TF/TFBS's), post-transcriptional (microRNAs/target genes) and epigenetic (e.g. methylation/demethylation, histone modifications). These studies will be undertaken in both the PT and hypothalamus, where thyroid hormone metabolism is regulated by output from the PT. Long-term seasonal physiological responses may be regulated by gradual epigenetic changes, mapped as methylome changes in photorefractory animals. We will test the hypothesis that pre-exposure to different photoperiods sets the state of the methylome and define the extent of tissue and cellular remodelling.

We will also investigate how a key molecular switch (Eya3), driving long-day thyroid hormone pathways may be drive other neuroendocrine circuits, and explore coupling between this long-day switch and the underlying seasonal methylome.

Our approaches employ bioinformatics, modeling, whole genome sequencing approaches and extensive biochemical methodologies, combined with whole-animals studies. The project capitalizes from access to a recent sequenced sheep genome through our role in the International Sheep Genome Consortium (ISGC), and will be the first study of how epigenetic mechanisms may regulate growth and reproduction in a mammal and livestock species.

This is an ambitious project, which addresses the role of epigenetic modification in regulating growth and reproduction in a seasonal mammal. Our discovery of a major imprinting centre operating in the neuroendocrine axis of a seasonal species has potentially enormous scientific significance, as well as practical relevance for animal breeding, and may reveal how an animal can respond and adapt to climate change.

At Roslin we have strong links with all Animal Breeding sectors, including ruminants and poultry. A clear application of our studies lies in the identification of target genes for marker-assisted selection. Through a link project with Affymetrix and Aviagen we have developed the first 600K high density genotyping array for whole genome selection in poultry. This technology offers a natural route for exploitation of genetic information in other species, including the sheep. Roslin is part of the International Sheep Genome Consortium providing sequence and gene annotations. Together these provide a natural route to disseminate and exploit epigenetic information from within this project to industry and the wider community. For example, there is currently much excitement about the possibility of exploiting genetic variation in epigenetic imprints, which may underpin genotype x environmental interactions. Our work will define the first methylome for a seasonal species. Effective uptake of our research in livestock is facilitated by close links with the Biosciences KTN and presentations to industry. This project has the potential to impact on the sustainability of the livestock industries in the UK and thus to inform DEFRA policy. It falls within the remit of the BBSRC Sustainable Agriculture Strategy Board and priority for research on Global Food Security.

There is considerable interest within "Pharma" in developing tools to regulate epigenetic processes from cancer to psychiatry. In Manchester existing links with GSK, including a free exchange of research staff, will allow access to novel compounds to test. For example, there is considerable interest in the role that epigenetic changes may play in the ageing process, but there are few good models - we may have identified such a model, involved in both hypothalamic and pituitary function. The other major site in the brain where melatonin receptors are localized is in the hippocampal region - a key structure involved in memory consolidation; of particular relevance to ageing. In man, shift work and non-circadian feeding times have profound metabolic consequences. This represents a wide-spread problem in society as it likely underpins health issues associated with long-term shift work. Our research could define quite novel epigenetic mechanisms in which rhythmic signals may control metabolic processes and help inform appropriate circadian management protocols with applications in healthy living and healthy ageing.

Both Manchester and Edinburgh are very active in public engagement with science. Andrew Loudon is an active participant in Public Understanding Activities and regularly gives open public lectures within the University of Manchester and to outside bodies including Café Scientifique and numerous other UK groups, and many BBC interviews. David Burt actively promotes the genomics and genetics of livestock and its applications in industry and medicine. Roslin is active in public engagement with a number of events within the University each year. These consist of lectures, open days to the public, school visits and projects for schools, undergraduate and postgraduate students. The wider significance of research is disseminated through papers, web sites, press releases and interviews on radio. Julian Davis as a clinical endocrinologist has regular contact with patients and carers, works with the Pituitary Foundation and the Clinical Committee of the Society for Endocrinology to promote research through its media and public relations offices.