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Do plants go further in deciding their cell fate: different target genes, different Polycomb Group mechanisms?

Final Report Summary - PLANTPCGSILENCING (Do plants go further in deciding their cell fate: different target genes, different Polycomb Group mechanisms?)

PcG-mediated repression constitutes a major epigenetic mechanism for controlling gene expression in higher eukaryotes. The PcG proteins are essential for many biological processes, including development, differentiation and proliferation. These proteins are present in multimeric complexes and mediate gene repression through the incorporation of histone modifying marks. In animals the two best characterized complexes are the PRC1 that mediates histone H2A lysine 119 monoubiquitination (H2Aub) of the target genes and PRC2 that has histone H3 lysine 27 (H3K27) trimethyltransferase activity. Numerous studies have demonstrated a tight link between PRC1 and PRC2; accordingly, the combined activity of the two complexes is required for stable repression of the target genes, but, how this is exactly accomplished is less clear.

In plants, detection of PRC1 components remained elusive for a long time. While the implication of the well-conserved plant PRC2 components in important developmental processes has for long been known, the lack of plant protein homologs to some of animal PRC1 components and the incorporation of plant-specific components have made difficult the identification and functional characterization of this complex. The main goal of this project was to determine the role of PRC1 in PcG repression throughout plant development. For this, we focused on the AtBMI1 proteins that have been proposed as putative PRC1 components.

Arabidopsis contains three AtBMI1s, AtBMI1A, AtBMI1B and AtBMI1C. Previous reports suggested that the AtBMI1s associate with AtRING1A or its paralog AtRING1B to constitute an E3 monoubiquitin ligase module responsible for the incorporation of H2AK121ub marks; however, little was known about the role of the AtBMI1s and this histone modification in plants. We found that the activity of the AtBMI1 proteins was crucial to stablish and maintain the repression of seed maturation genes after germination. We showed that the VP1/ABI3-LIKE (VAL) transcription factors through their interaction with AtBMI1 proteins direct H2AK121ub marking of seed maturation genes for their initial repression, whereas the H3K27me3 modification is a down-stream step. Since the recruitment of PcG complexes to specific targets in animals has been widely thought to be a sequential process in which first PRC2 incorporates H3K27me3 at a specific gene and then the PRC1 complex is recruited by its ability to bind to H3K27me3 to mediate H2A monoubiquitination, this was an unexpected result as it was indicating a reverse mechanism at least for the regulation of seed maturation genes.

By analyzing the transcriptome of single, double and triple atbmi1 mutants, we found that AtBMI1A and B display mainly redundant functions throughout development and that AtBMI1C only regulates a subset of AtBMI1A/B targets. Furthermore, we have identified a more comprehensive set of candidate genes regulated by AtBMI1 proteins. Our results indicated that in addition to switching off the seed maturation program after germination, AtBMI1s promote the transition from each developmental phase to the next throughout development and control cell proliferation during organ growth and development. By integrating transcriptomics datasets with previously published data, we show that AtBMI1-mediated gene repression requires different combinational modules of DNA binding factors always involving VAL proteins. We also analyzed the functional relationship of AtBMI1 proteins with other putative plant specific PRC1 components such as LHP1 and EMF1 as well as with PRC2. Our data pointed to different PRC1 functional networks in which genes may be regulated by AtBMI1 and/or EMF1 together with LHP1 and PRC2, and that additional proteins are required to regulate distinct subsets of genes.

A major issue to understand PcG repression is to determine the sequence of events in the mechanism. Our results have challenged the classical recruitment model of PcG complexes proposed in animals in which PRC2-mediated H3K27 trimethylation recruits PRC1 for H2A monoubiquitination by showing that PRC1 activity can also recruit PRC2 for the case of seed maturation genes. However, whether the two mechanisms took place in plants and in such case, what is their prevalence remained unanswered questions as the localization of H2AK121ub marks were examined at only a select number of PcG targets. To address these key questions, we first mapped the genome wide localization of H2AK121ub and H3K27me3 marks in wild-type Arabidopsis by chromatin immunoprecipitation followed by sequencing. We found that H2AK121ub marks were surprisingly widespread in Arabidopsis, often co-localizing with H3K27me3 but also occupying a set of non-canonical PcG targets that were devoid of H3K27me3 marks and were transcriptionally active. Furthermore, by profiling H2AK121ub and H3K27me3 marks in atbmi1a/b/c, lhp1 and in the PRC2 loss of function mutant clf28/swn7 we found that PRC2 activity is not required for H2AK121ub marking at most genes. In contrast, loss of AtBMI1 function impacts the incorporation of H3K27me3 marks at most PcG targets. Our findings argue against the classical hierarchical model for PcG mark deposition as the prevailing sequence of events in Arabidopsis.

In summary, the results obtained in this Marie Curie CIG funded project have strongly contributed to understand the role of plant PRC1 in PcG repression, unveiling similarities but also important differences to animals. Our findings represent a major step forward in understanding the PcG regulation, having important implications in the field of epigenetics, as they challenge the current view on the interdependency of the PcG complexes and provide new insights into the role of H2A monoubiquitination in the mechanism.