Unravelling the networks that regulate seasonal rhythmicity in the epigenome

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.

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.

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).