| Resumo: | Polycomb Group (PcG) proteins are essential for orchestrating plant development by ensuring that each cell in an organism expresses the appropriate genes at the right time; however, the exact mechanisms by which they exert this regulation are still not fully understood. PcG proteins primarily assemble into two complexes: Polycomb Repressive Complex 1 (PRC1) and PRC2. In Arabidopsis thaliana, several studies have connected the recruitment of PRC2 to target genes with PRC1 activity and various transcription factors (TFs); however, how these TFs interact with PRC2 and whether they recruit PRC1 remains unclear. Moreover, while PRC2-mediated deposition of H3K27me3 is mainly associated with gene repression, PRC1 has a more versatile role in gene regulation, as it seems involved in gene repression and activation. Notably, PRC1 can monoubiquitinate both the canonical H2A histone and its variant H2A.Z. However, the conditions under which PRC1 differentiates between H2A and H2A.Z, and how the monoubiquitination of these histones influences gene expression remain unclear. This thesis aims to shed light in these fundamental questions. To investigate PcG recruitment mechanisms, we developed an in vivo system to tether selected TFs to a synthetic promoter, eliminating the complexity of endogenous PcG target promoters. We found that VIVIPAROUS1/ABSCISIC ACID INSENSITIVE 3-LIKE1 (VAL1) functions as a platform for the simultaneous recruitment of PRC1, PRC2, and histone deacetylase (HDAC) activities. While VAL1 directly interacts with HDAC and PRC1, the recruitment of PRC2 requires PRC1 activity and protein-protein interactions. In contrast, while other TFs could recruit PRC2 and HDAC activities, they failed to trigger PRC1 marking, highlighting a specific association between VAL1 and PRC1. Interestingly, all tested TFs, including VAL1, contained an Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) domain, which we found involved in recruiting both HDAC and PRC2 activities, thereby linking these two repressive mechanisms. Furthermore, we demonstrate that different TFs can enhance PRC2 recruitment in an additive manner, reinforcing PRC2 marking and repression, which may be necessary for long-term gene silencing. Our findings also clarify the role of PRC1 in gene regulation. We show that PRC1 monoubiquitinates H2A.Z, rather than canonical H2A, during the transition from embryonic to vegetative development. The incorporation of H2A.Zub facilitates PRC2 recruitment to repress seed development genes, but it also promotes the activation of vegetative development genes by preventing H3.1K27me1 incorporation. Moreover, we reveal that REF6-mediated demethylation of H3K27me3 is a prerequisite for H2A.Zub incorporation, positioning REF6 activity at the onset of gene activation. Together these data establish H2A.Zub as a central molecular “switcher” in gene expression reprogramming.
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