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Dissection of environmentally-mediated epigenetic silencing

Final Report Summary - ENVGENE (Dissection of environmentally-mediated epigenetic silencing)

The project exploited the plant epigenetic silencing system, vernalization, to uncover fundamental principles underpinning epigenetic regulation. We described the sequence of events whereby prolonged cold induced an epigenetic switch in expression states of a gene encoding a floral repressor (encoded by the Arabidopsis gene FLC). Through a combination of genetics, molecular biology and biochemical approaches with computational modelling we achieved our goal, a step change in our understanding of the mechanistic basis of epigenetic regulation. Our focused effort integrating the many facets of chromatin regulation on one target has meant that FLC has become widely recognized as a paradigm for understanding environmentally mediated epigenetic regulation.

We fully dissected the different phases of vernalization elaborating conserved features of epigenetic regulation generally. This involved characterization of chromatin regulation of FLC before cold, triggering of transcriptional silencing by prolonged cold, features of the nucleation of chromatin silencing during the cold, and spreading of the chromatin silencing in the post-cold growth but spatial restriction to FLC. A major success has been to uncover the importance of bistable chromatin states that determine the expression of FLC before and after cold and how the switching of these states underpins the quantitative epigenetic memory involved in vernalization. Detailed analysis and modeling of the dynamics of histone changes at FLC showed that the quantitative accumulation of the silencing was a consequence of an increasing proportion of cells switching epigenetic states. We detailed the accumulation of H3K27me3, first at one site in the gene during the cold, and then covering the whole gene after transfer back to warm. This was undertaken in wild-type and many mutants to understand the requirements for nucleation, spreading and spatial restriction of H3K27me3 at FLC. Using proteomics we also detailed the Polycomb complexes required for the H3K27me3 dynamics and determined that their localization and activity was defined by a set of PHD-containing proteins that influence the methyltransferase activity of the complex. H3K36me3 was also shown to be the most likely histone modification to act as the opposing histone modification to H3K27me3. Characterization of the dynamics of the major histone variants at FLC was also completed.

Significant progress in characterizing important cis sequences was achieved by exploiting natural variation in vernalization response. Arabidopsis accessions that require different lengths of cold to satisfy their vernalization requirement revealed FLC cis polymorphism that influenced FLC epigenetic silencing. A small number of nucleotide polymorphisms, located in the FLC intragenic site where H3K27me3 accumulates during the cold, were shown to underpin a requirement for longer periods of cold. Modelling of the changing histone modifications showed that these polymorphisms impact a mechanism important for the stable epigenetic memory of the silenced state.
We also made considerable progress in understanding the role of non-coding transcripts in initiation and maintenance of epigenetic silencing. Our analyses suggest that FLC antisense transcripts play multiple roles in the vernalization process, but not to recruit PRC2 complexes as proposed by many studies. We showed that an early cold sensor step involved up-regulation of expression of FLC antisense transcripts. Detailed analysis of this regulation led us into the finding that an R-loop limits FLC antisense production in vivo. Plants expressing an FLC defective in production of the antisense transcripts accumulate H3K27me3 normally, but show altered decreases in H3K36me3 and attenuated cold-induced FLC transcriptional silencing. FLC cis polymorphism in the A. thaliana natural variants was also shown to influence splicing and polyadenylation of the antisense transcripts and their functional significance was confirmed by transgenic analysis.

The interconnection of different layers of regulation of FLC was further investigated through analysis of changes in interaction of FLC intragenic sequences and changes in nuclear organization of the FLC locus, both of which are influenced by vernalization, and both of which ensure robust expression or silencing of FLC as the plant grows in a noisy environment.