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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - AMBITION (Unravelling the molecular Basis of epigenetic silencing: what factors define a gene as a Polycomb target?)

Teaser

Summary

Establishment and maintenance of gene expression states is central to development and differentiation in any eukaryotic organism. An important component of maintenance of developmentally specified gene expression is gene silencing mediated by Polycomb group (PcG) proteins. Polycomb group proteins are conserved from vertebrates to plants and are associated with at least two chromatin-modifying complexes: Polycomb repressive complex 1 and 2 (PRC1, PRC2), which establish transcriptional silencing of target genes. Polycomb regulation maintains correct gene expression states throughout development and perturbations lead to developmental abnormalities and disease. Knowledge about the composition of individual PRC complexes and their biochemical modes of action has advanced in the recent years. However, one of the central questions in the field of chromatin biology remains unanswered: which factors specify a gene for Polycomb silencing?

An ideal system to study Polycomb targeting is a locus where epigenetic silencing can be induced externally, and the maintenance of silencing can be easily recorded during subsequent development. In the model plant Arabidopsis thaliana, the integration of complex developmental and environmental signals determining flowering time occurs via tight regulation of the floral repressor gene FLOWERING LOCUS C (FLC). At FLC, specific DNA binding proteins (VAL1, VAL2) and their partners interact in a not yet fully understood regulatory network with Polycomb proteins, which consequently converts environmental cues (prolonged cold) into stable epigenetic memory (silencing of the gene).

Building on previously identified proteomic interactions, the AMBITION project hypothesised that FLC regulation involves components of the Apoptosis and Splicing Associated Protein (ASAP) complex. ASAP functions in RNA processing and quality control, thus putatively linking VAL1 DNA sequence specificity with co-transcriptional regulation directly to Polycomb mediated epigenetic silencing. The project combined several interconnected molecular, biochemical and genetic avenues to provide novel and detailed mechanistic insights into the epigenetic regulation of Polycomb target genes.

Work performed

The AMBITION project investigated both the required DNA sequence and protein factors which enable the FLC locus to switch from a transcriptionally â€˜onâ€™ to an epigenetically â€˜offâ€™ state. The DNA sequence motifs recognised by the VAL binding proteins are located in the first intron of FLC in tandem orientation and 27bp apart (and this is conserved in all Brassicaceae FLC orthologues analysed to date). This genomic region has been targeted by CRISPR-Cas9 mutagenesis, generating random mutations and small insertions/deletions to address questions like: whether there is co-operative interaction between the two VAL proteins, whether one or two motifs are involved in the epigenetic switch or whether the distance and sequence between the motifs is important.

In addition, the AMBITION project addressed the role of the Apoptosis and Splicing Associated Protein (ASAP) complex on FLC regulation during vernalization. Transgenic mutant lines have been created and analysed before, during and after cold treatment to attribute the effect of individual ASAP components on the transcriptional output and epigenetic state of FLC. Interestingly, single mutants show only subtle effects during vernalisation, but seem to regulate FLC transcript levels in the warm, similar to known co-transcriptional regulators of the autonomous pathway. Genetic studies have been conducted to understand potential cooperativity and functional interaction of ASAP proteins and in repressing FLC transcription and epigenetic silencing of the locus.

The results from the AMBITION project have not been yet been fully disseminated to the public. A manuscript is in preparation and will result in a timely open access publication in a peer-reviewed scientific journal according to the H2020 guidelines.

Final results

Epigenetics is one of the fastest growing areas of biological sciences. But although epigenetic silencing has become a central issue in studies of development and disease, precise knowledge of the molecular mechanisms involved remains rudimentary. The AMBITION project aimed at investigating these regulatory mechanisms in the context of flowering via the vernalisation pathway. Flowering time regulation is of utmost importance for agricultural implications, especially in the rapidly changing climate we face today. The ability to engineer a locus to be a target of Polycomb silencing is therefore essential if we are to be able to manipulate epigenetic regulation for crop improvement in the future.