Nuclear organization of the Polycomb target FLC during the cold-induced epigenetic silencing of vernalization

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Epigenetic regulation is the maintenance of specific gene expression states in the absence of the initial regulator over many cell generations, and sometimes transgenerationally. It is profoundly important for correct growth, development and environmental response in most organisms. In many cases the molecular basis of epigenetic regulation is associated with modification of chromatin, the methylation of the DNA or post-translational modification of the associated histones. For example, an epigenetic silencing mechanism central to control of cell differentiation, morphogenesis and developmental timing involves the so-called 'Polycomb complexes', a set of protein complexes that add methylation groups to a particular residue on the amino-terminal tail of histone 3. This 'mark' then attracts other silencing proteins that keep the gene switched off. Non-coding RNAs often recruit the Polycomb proteins to their specific targets but the exact mechanisms determining the specificity of their action is still being actively dissected. The 'marks' laid down on the chromatin need to be copied to the daughter chromatin strand every time a cell divides. A feature that may be very important for maintenance of the epigenetically silenced state is 'storage' of the targets in certain places in the nucleus, where all similarly regulated genes are collected. This nuclear re-positioning upon epigenetic silencing has been extensively characterized in the case of inactivation of one of the X-chromosomes in female mammals, but emerging data in many other systems suggests this is generally very important for epigenetic regulation. How a change in nuclear position ties in with all the other changes that occur is still not clear because it is often hard to study all the questions on one epigenetically silenced target. Over the last 15 years the Dean laboratory has studied the epigenetic silencing of the gene (called FLC) encoding an important repressor of flowering in plants in a process called vernalization. The very long period of cold experience during winter slowly but surely shuts off expression of this gene and this shut-down is remembered during the subsequent spring and summer, allowing the plants to flower in favourable conditions. Unlike many mammalian systems the plant system has the advantage that extensive genetic analysis can be combined with molecular, computational and cell biology techniques. This has resulted in the regulation of FLC being considered as the paradigm for environmentally-mediated epigenetic regulation. We want to build on this advantage and exploit a new experimental technique to observe changes in the nuclear organization of FLC during the cold-induced epigenetic silencing process involved in vernalization. The new information on nuclear organization and chromatin interactions, at different phases of the vernalization process, will be integrated with the other mechanistic information through analysis of mutants and specific transgenic lines. The results emerging from this work will allow us to integrate the importance of changes in nuclear organization with dynamic changes in histone 'mark' accumulation, non-coding RNA production and the cell autonomous epigenetic switching that underlies vernalization. It will also provide important conclusions for understanding more generally how nuclear organization is interconnected with epigenetic regulation.