Nucleation of Polycomb silencing at FLC

The Dean group studies the control of flowering time in the reference plant, thalecress (Arabidopsis thaliana). Several flowering pathways converge to regulate a gene called FLC and variation in expression of this gene contributes enormously to natural variation in flowering time of Arabidopsis types collected from around the world. Winter conditions switch off expression of the FLC gene, removing the brake to flowering, and thus accelerating the flowering process. This induction of a plant's flowering process by exposure to the prolonged cold of winter is termed "vernalization." Genetic, molecular and computational analysis of vernalization in thalecress has shown it involves a cellular memory mechanism, one that also occurs during development of our own bodies. In the plant, cold switches off expression of FLC and this repressed state is maintained by Polycomb proteins that add methylation groups to proteins associated with FLC DNA. These modifications are passed from the mother to the daughter DNA strand to maintain the repressed state of the gene through the development as epigenetic regulation and similar mechanisms keep many thousands of genes switched off (silenced) during vertebrate development. A key question in the epigenetic field is what determines whether a specific gene becomes silenced or not.

Recent work in the Dean lab has made progress in this question through characterization of a change in the sequence of the FLC gene that blocks the silencing by cold. This then enabled the factor that binds to that specific region to be identified. Use of a tagged version of the factor then enabled isolation of a range of proteins that were in many cases unexpected and which suggested what might be involved in the silencing switch. This proposal aims to follow up these findings to fully describe the multiple factors required to specify a gene for epigenetic silencing. Understanding from a study such as this can provide concepts important to epigenetic regulation across many genomes.

This proposal will investigate a central question in epigenetics -what factors specify a gene for Polycomb silencing? It will build on the recent identification of a single nucleotide polymorphism within the target gene that blocks Polycomb silencing in the well-characterized Polycomb switching system, cold-induced Arabidopsis FLC silencing. Knowledge of the DNA sequence required enabled identification of the binding protein and then proteomic identification of proteins that associate with that binding protein. This opens up experimental approaches to elucidate the mechanisms underlying what specifies Polycomb targets and what is required to epigenetically switch a locus from one chromatin state to another.

We will test the hypothesis that multiple factors determine Polycomb target selection and that their combined action synergize to nucleate Polycomb complexes, and thus switch the gene from an epigenetically active to a silent state. The specific DNA binding protein is VAL1 and its interacting partners include components of the Polycomb Repressive Complex 1 and the Apoptosis and Splicing Associated Protein (ASAP) complex. In turn the ASAP components interact with PHD proteins previously associated with the PRC2 complex, required at FLC for epigenetic switching. These protein interactions thus directly link DNA sequence specificity through to epigenetic switching via ASAP and PRC1.

The work will involve four interconnected experimental avenues aimed at mechanistically understanding how these functions link together. We will define the minimal sequence and VAL factor requirement for nucleation; establish the role of the ASAP complex; investigate how ASAP links through to VRN5 and VIN3 and test if Polycomb Repressive Complex 1 functions directly in FLC silencing. Our aim will be to fully integrate all the functions and understand how they lead to robust epigenetic switching.

Dissection of the mechanisms underlying epigenetic regulation is a very active area of biology. Indeed the global epigenetics market size was valued at USD 3.98 billion in 2014. Increasing prevalence of cancer is the key driver propelling the growth of the epigenetics market." (http://www.grandviewresearch.com/industry-analysis/epigenetics-market). Research on plant systems may also provide opportunities in agriculture and different mechanistic insights from biomedical research.

Important concepts relevant to gene expression generally are emerging from very different systems. The Dean lab has identified an excellent system in which to explore chromatin-based epigenetic mechanisms and the interaction of multiple components that synergize functionally to effect a switch. FLC regulation represents a system where different approaches can be combined to give a comprehensive view of the complexity and plasticity of epigenetic regulation. This will be of broad relevance to the biotechnology community, the pharmaceutical industry, particularly those involved in cancer research - and possibly also ageing research.

A clearer understanding of the molecular basis of switching mechanisms underpinning flowering/vernalization will inform strategies as to how to manipulate heading date, a key trait in breeding of many crops. The John Innes Centre is very unusual in combining basic biological research with crop-based studies. Groups interact on a daily basis, so results emerging from model species are quickly applied to crops such as Brassicas and cereals. Prof. Dean, collaboratively with Dr Judith Irwin (Dept. Crop Genetics, John Innes Centre) also has very good links with the plant breeding and biotechnology industries (Elsoms Seeds Ltd, Bejo Zaden BV and Bayer Crop Science), and there are frequent visits in both directions. The focus in the Dean/Irwin labs is manipulation of vernalization requirement and response - a key process in the production of many vegetable crops, broccoli, cauliflower, parsnips and carrots. Varieties of these vegetables are bred to ensure year round supply but the vagaries of winter temperatures and climate uncertainty tend to lead to gluts or shortages in production. Development of varieties less influenced by temperature but still producing in different seasons of the year would considerably reduce waste, potentially open up new production areas and increase efficiency of delivery. Ongoing funded collaborations with breeding companies (Elsoms Seeds Ltd) aim to translate this understanding into practical benefits.

The work will also contribute fundamental information that provides a framework of understanding for manipulation of developmental processes generally in less tractable but strategically important plants. The understanding emanating from epigenetic analysis in Arabidopsis is a good paradigm for how fundamental information informs generally and influences experimental strategies of a wide community of plant biologists.