Periodic Reporting for period 4 - EPISWITCH (Mechanistic basis of nucleation and spreading underlying a Polycomb-mediated epigenetic switch)
Reporting period: 2024-03-01 to 2025-08-31
The EPISWITCH project investigated the epigenetic regulation of gene expression, central to development and environmental plasticity in many organisms. Epigenetic regulation generally involves regulation of chromatin – the organization of DNA and associated histone proteins in the nucleus. Chromatin states associated with different levels of gene expression have previously been extensively characterized, but how epigenetic states are switched from one to another was unclear. EPISWITCH exploited the process of vernalization, the cold-induced epigenetic silencing of Arabidopsis FLC to define the core events underpinning an epigenetic switch.
This is important for society since understanding gene regulation is a central problem in biology with many implications. Mis-expression of genes during development, or in response to environmental exposures, is the basis for many diseases in both plants and animals. It is also central to adaptation raising the possibility of generating next-generation, climate-proofed crop plants.
The objectives of the project were:
1) To test the hypothesis that the core of the epigenetic switch is an oligomerization event involvingaccessory proteins of the conserved chromatin silencing complex, Polycomb Repressive Complex 2 (Objective A).
2) To investigate whether this switch was promoted by local features in the FLC gene (Objective B).
3) To investigate the role of DNA replication/growth in long-term stable silencing (Objective C).
Our major conclusion is that the epigenetic switch underlying the cold-induced chromatin silencing in vernalization is a stochastic conformationally-induced oligomerization event. We demonstrated that oligomerization enhanced the functional affinity of multiple proteins at a specific region in the gene. This provided efficient retention of the proteins to deliver and inherit post-translational modifications to the chromatin resulting in the switch to stable Polycomb silencing. We also showed that local features of the gene were important and characterized a temperature-dependent arrangement in the nucleosomes (DNA wrapped around the histone octamers) that promotes silencing. We could show the requirement of reduced growth/DNA replication for stable silencing through analysis of natural variation in FLC silencing in Arabidopsis accessions adapted to latitudinal extremes.
This is important for society since understanding gene regulation is a central problem in biology with many implications. Mis-expression of genes during development, or in response to environmental exposures, is the basis for many diseases in both plants and animals. It is also central to adaptation raising the possibility of generating next-generation, climate-proofed crop plants.
The objectives of the project were:
1) To test the hypothesis that the core of the epigenetic switch is an oligomerization event involvingaccessory proteins of the conserved chromatin silencing complex, Polycomb Repressive Complex 2 (Objective A).
2) To investigate whether this switch was promoted by local features in the FLC gene (Objective B).
3) To investigate the role of DNA replication/growth in long-term stable silencing (Objective C).
Our major conclusion is that the epigenetic switch underlying the cold-induced chromatin silencing in vernalization is a stochastic conformationally-induced oligomerization event. We demonstrated that oligomerization enhanced the functional affinity of multiple proteins at a specific region in the gene. This provided efficient retention of the proteins to deliver and inherit post-translational modifications to the chromatin resulting in the switch to stable Polycomb silencing. We also showed that local features of the gene were important and characterized a temperature-dependent arrangement in the nucleosomes (DNA wrapped around the histone octamers) that promotes silencing. We could show the requirement of reduced growth/DNA replication for stable silencing through analysis of natural variation in FLC silencing in Arabidopsis accessions adapted to latitudinal extremes.
The work was published in the listed papers and described in many lectures given at international conferences. Overall, ee showed that the epigenetic switch underlying the cold-induced chromatin silencing in vernalization is a stochastic conformationally-induced oligomerization event. This concept is relevant to epigenetic switches in all organisms. In our work crystal structures of the oligomerization domains revealed a head to tail polymerization module conserved through evolution (Fiedler et al 2022). Transgenic Arabidopsis plants carrying mutated versions designed to disrupt the head to tail interface demonstrated that the oligomerization (VEL) domain was necessary for the epigenetic silencing. Oligomerization promoted multivalent chromatin association at the local nucleation region (Schulten et al 2025). Overall, this work revealed the functional specialization of VEL-proteins, and how they maintain the chromatin association of the Polycomb complex (Franco-Echevarria et al 2023), necessary to switch to an epigenetically silenced state.
Dissection of the local chromatin features that promote the epigenetic switch took us into the study of altered dynamics of the nucleosomes (DNA wrapped around the histone octamers) (Mikulski et al 2022, Montez et al 2025) and biomolecular condensates that alter transcription states (Zhu et al 2021). Overall, work in Objective B highlighted how local nucleosome dynamics and chromatin contacts link to chromatin structure transitions to integrate temperature inputs into epigenetic switching mechanisms in plants (Menon et al 2021, Nielsen et al 2024).
The role of DNA replication in the spreading of the silencing marks was investigated using the DNA fibre protocol, adopted extensively in yeast and mammals to analyse DNA replication (Baxter et al 2021). However, the low throughout of this technique meant we could not analyse replication specifically at FLC. A heterologous system was therefore used to analyse FLC sequences that affected DNA replication. A sequence was identified that strongly pauses the DNA replication machinery in a directional manner and this was associated with formation of RNA/ DNA hybrids (Fang et al 2020, Xu et al 2021a, Xu et al 2021b). A requirement for reduced growth/ DNA replication for stable FLC silencing was also revealed through analysis of natural variation in FLC silencing in Arabidopsis accessions adapted to latitudinal extremes (Zhu et al 2023, Yang et al 2022).
Dissection of the local chromatin features that promote the epigenetic switch took us into the study of altered dynamics of the nucleosomes (DNA wrapped around the histone octamers) (Mikulski et al 2022, Montez et al 2025) and biomolecular condensates that alter transcription states (Zhu et al 2021). Overall, work in Objective B highlighted how local nucleosome dynamics and chromatin contacts link to chromatin structure transitions to integrate temperature inputs into epigenetic switching mechanisms in plants (Menon et al 2021, Nielsen et al 2024).
The role of DNA replication in the spreading of the silencing marks was investigated using the DNA fibre protocol, adopted extensively in yeast and mammals to analyse DNA replication (Baxter et al 2021). However, the low throughout of this technique meant we could not analyse replication specifically at FLC. A heterologous system was therefore used to analyse FLC sequences that affected DNA replication. A sequence was identified that strongly pauses the DNA replication machinery in a directional manner and this was associated with formation of RNA/ DNA hybrids (Fang et al 2020, Xu et al 2021a, Xu et al 2021b). A requirement for reduced growth/ DNA replication for stable FLC silencing was also revealed through analysis of natural variation in FLC silencing in Arabidopsis accessions adapted to latitudinal extremes (Zhu et al 2023, Yang et al 2022).
The definition and structural characterization of a novel oligomerization domain is a significant discovery. The oligomerization fold is conserved throughout the tree of life so the implications for functional significance are enormous. The functional importance of this was demonstrated by mutation of the interface. We showed that single amino acid changes that block polymerization without affecting protein stability prevent epigenetic switching and loss of silencing. This domain is in Polycomb Repressive Complex 2 accessory proteins, so Arabidopsis VEL proteins can be considered evolutionary parallels of the polymerising Polycomb factors in animals, that function to assemble PRC1. This is a fascinating example of convergent evolution where different proteins with similar mechanistic principles have evolved in epigenetic silencing mechanisms. The next phase of the work will be to understand the self-templating mechanism that maintains VRN5-PRC2 oligomerization in the absence of the cold and the cold-induced VIN3. We defined a local chromatin structure at the nucleation region associated with transcriptional repression, a prerequisite for stable Polycomb nucleation. The exact multivalent interactions between the Polycomb and accessory proteins and the nucleosomes in that region remain to be fully defined- but this would require structural analysis via reconstitution experiments. Our work has shown a complex role of DNA replication – it acts as a major de-stabilizer of epigenetic silencing, but it is required to spread the silenced state. These antagonistic roles require further analyses.