Nuclear organization of the Polycomb target FLC during the cold-induced epigenetic silencing of vernalization
Lead Research Organisation:
John Innes Centre
Department Name: UNLISTED
Abstract
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Technical Summary
We will exploit the genetic and molecular tools we have developed for the analysis of vernalization to explore the functional interconnection between nuclear organization and epigenetic silencing. This is an emerging area of investigation in the regulation of gene expression in many organisms. The target of vernalization, the FLC gene, has been tagged with a lacO array and detected using live cell imaging in root nuclei. We can combine analysis of the nuclear organization of FLC using this live imaging technique with the detailed genetic and molecular understanding of the cold-induced Polycomb silencing that we have previously characterized for FLC. This system should allow us to unpick the relative importance of these different aspects of gene silencing. We will further characterize the live imaging system for FLC-lacO and then undertake specific experiments to address the questions:
1) What genetic components are required for the FLC-lacO clustering?
2) Is clustering dependent on the different non-coding transcripts associated with FLC epigenetic silencing?
3) Is clustering a manifestation of increased nucleation during the cold?
4) Is the clustering epigenetically stable?
5) Do broader chromosomal interactions occur during vernalization?
The question of whether the observed clustering is a reflection of a broader set of chromosomal interactions at FLC will be addressed using 4C, a modification of the 3C chromatin conformation capture technique that allows high throughput analysis analyzing interactions of one locus with many others in the genome. This will inform about interactions of FLC with other genomic loci before, during and after the cold period, and add to our understanding of the relationship of chromosomal domains and changes in gene expression. This work will inform generally about how nuclear organization intersects with the dynamic sequence of events (eg. histone modifications and non-coding RNA) involved in epigenetic silencing mechanisms.
1) What genetic components are required for the FLC-lacO clustering?
2) Is clustering dependent on the different non-coding transcripts associated with FLC epigenetic silencing?
3) Is clustering a manifestation of increased nucleation during the cold?
4) Is the clustering epigenetically stable?
5) Do broader chromosomal interactions occur during vernalization?
The question of whether the observed clustering is a reflection of a broader set of chromosomal interactions at FLC will be addressed using 4C, a modification of the 3C chromatin conformation capture technique that allows high throughput analysis analyzing interactions of one locus with many others in the genome. This will inform about interactions of FLC with other genomic loci before, during and after the cold period, and add to our understanding of the relationship of chromosomal domains and changes in gene expression. This work will inform generally about how nuclear organization intersects with the dynamic sequence of events (eg. histone modifications and non-coding RNA) involved in epigenetic silencing mechanisms.
Planned Impact
unavailable
