Periodic Reporting for period 3 - EPISWITCH (Mechanistic basis of nucleation and spreading underlying a Polycomb-mediated epigenetic switch)
Periodo di rendicontazione: 2022-09-01 al 2024-02-29
Epigenetic regulation of gene expression is central to development and environmental plasticity generally. Conserved chromatin mechanisms maintaining epigenetic states have been characterized, but how states are switched is still unclear. We exploit the epigenetic silencing of Arabidopsis FLC, where switching is controlled by prolonged cold, to define the core events underpinning an epigenetic switch. Our hypothesis is that an oligomerization event involving PRC2-accessory proteins, promoted by chromatin features at the local site, constitutes the epigenetic switch. The resulting oligomer self-templates incoming proteins providing the feedback mechanisms that confer metastable memory. Spreading of a subset of the chromatin proteins and H3K27me3 across the body of the gene, in a process intimately connected with DNA replication, then provides long-term epigenetic memory.
We are testing this hypothesis in a programme with three interconnected objectives:
A. Functional analysis of protein oligomerization in the epigenetic switching mechanism
B. Elucidating the local chromatin features that promote the switch
C. Dissecting the transition from metastable to long-term epigenetic silencing
This work combines molecular genetics, DNA replication expertise with structural biology and biophysical analysis.
We are testing this hypothesis in a programme with three interconnected objectives:
A. Functional analysis of protein oligomerization in the epigenetic switching mechanism
B. Elucidating the local chromatin features that promote the switch
C. Dissecting the transition from metastable to long-term epigenetic silencing
This work combines molecular genetics, DNA replication expertise with structural biology and biophysical analysis.
Overall, we have made significant progress in our three objectives. We have defined and structurally characterized a novel oligomerization domain, shown its functional importance in epigenetic switching, defined the characteristics of the chromatin at the nucleation site, and improved our understanding of the transition to long-term silencing. During the first 18 months the level of staffing was lower than initially anticipated. In order to catch up on our deliverables post-covid we hired an experienced technician to expand the plant transformation capability and recruited an additional post-doctoral scientist to supplement activities in Objective B, so all is now back on track.
In Objective A, our work has characterized a novel polymerization domain on the Arabidopsis PRC2-accessory proteins VIN3 and VRN5. Mutations blocking polymerisation of this VEL domain prevent PRC2 silencing at FLC, demonstrating its functional importance in epigenetic memory. Plant VEL proteins can therefore be considered evolutionary parallels of the polymerising Polycomb factors in animals, facilitating assembly of dynamic multivalent complexes that underpin protein-templated memory of the silenced state.
For Objective B, we have progressed our understanding of what local chromatin features are important for nucleation by development of a novel technique to assess DNA accessibility in vivo. This has revealed a relatively inaccessible three nucleosome region covering the nucleation site where VIN3 and VRN5 associate, potentially reflecting a secondary structure in the chromatin. We have also completed a structural analysis of the chromatin-associating domains in VIN3 and VRN5, defining a composite domain whose PHD finger does not bind histone modifications but nucleic acids.
For Objective C, we have used the DNA fibre protocol and a heterologous system to analyse how the R-loop structure at the 3’ end of FLC can stall the replisome. However, the low throughput of the fibre protocol has meant we are unable to do fibre-FISH at FLC. We are therefore continuing investigation of the question using a genetic analysis to study the interaction of DNA replication with the Polycomb and other silencing factors needed for long-term silencing.
In Objective A, our work has characterized a novel polymerization domain on the Arabidopsis PRC2-accessory proteins VIN3 and VRN5. Mutations blocking polymerisation of this VEL domain prevent PRC2 silencing at FLC, demonstrating its functional importance in epigenetic memory. Plant VEL proteins can therefore be considered evolutionary parallels of the polymerising Polycomb factors in animals, facilitating assembly of dynamic multivalent complexes that underpin protein-templated memory of the silenced state.
For Objective B, we have progressed our understanding of what local chromatin features are important for nucleation by development of a novel technique to assess DNA accessibility in vivo. This has revealed a relatively inaccessible three nucleosome region covering the nucleation site where VIN3 and VRN5 associate, potentially reflecting a secondary structure in the chromatin. We have also completed a structural analysis of the chromatin-associating domains in VIN3 and VRN5, defining a composite domain whose PHD finger does not bind histone modifications but nucleic acids.
For Objective C, we have used the DNA fibre protocol and a heterologous system to analyse how the R-loop structure at the 3’ end of FLC can stall the replisome. However, the low throughput of the fibre protocol has meant we are unable to do fibre-FISH at FLC. We are therefore continuing investigation of the question using a genetic analysis to study the interaction of DNA replication with the Polycomb and other silencing factors needed for long-term silencing.
The definition and structural characterization of a novel oligomerization domain is the major achievement beyond the state of the art. 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 PRC2 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 will also further define the local chromatin structure at the nucleation region, its cross talk with the VEL proteins and how it changes upon transition to long-term silencing.