Thesis presented October 12, 2023

Abstract:
The development of multicellular organisms such as the model plant Arabidopsis thaliana relies on epigenetic factors that govern chromatin dynamics and regulate the transcription of specific genes by either repressing or activating them. Two groups of proteins function antagonistically in these regulations: the Polycomb group (PcG) and the Trithorax group (TrxG).
The Polycomb Repressive Complex 2 (PRC2) plays a crucial role in maintaining thousands of genes in a repressed state by depositing methylation marks on lysine 27 of histone 3 (H3K27me3). Conversely, proteins from the TrxG specifically counteract repression mediated by the PcG group, particularly on common gene targets. Although the PcG and TrxG proteins are progressively being identified and characterized, the molecular mechanisms that govern the transition of a gene from a PcG-repressed state to a TrxG-activated state remain poorly understood. In Arabidopsis, the ULTRAPETALA1 (ULT1) protein acts as an antagonist to the PRC2 complex, contributing to the conversion of an inactive state of PRC2 target genes into active state. Furthermore, in vivo interaction analyses (Y2H, BiFC, and IP-MS) have revealed that ULT1 interacts with the CLF and SWN methyltransferases of the PRC2 complexes. These observations indicate that ULT1 might directly disrupt the function of these complexes. However, the lack of biochemical and structural data on these proteins makes the study of their function challenging.
This PhD project aims to gain a better understanding of ULT1 and PRC2 antagonistic function using a structural biology and biochemistry approach. To achieve this, I characterized the ULT1 protein and the PRC2 complex from Arabidopsis in vitro, uncovering the molecular details of their interaction.
In the first section, dedicated to the biochemical characterization of the ULT1 protein, I successfully identified two structured domains after optimizing their expression and purification conditions. The resolution of the C-terminal domain of ULT1 by X-ray crystallography revealed an atypical CW domain that may recognize modifications on H3K36.
In the second part dedicated to the in vitro characterization of the interaction between ULT1 and PRC2 complexes, I demonstrated a physical interaction between ULT1 and the complex containing the methyltransferase SWN (PRC2-SWN complex). I also showed that this interaction was stable and that ULT1 binding did not dissociate the PRC2-SWN complex. Finally, a Cryo-EM study of the ULT1 and PRC2-SWN complex did not reveal density corresponding to ULT1, indicating that it might bind to a flexible region of the PRC2-SWN complex.
In conclusion, this PhD work enabled the development of essential tools and methods for in vitro study of ULT1 and PRC2 complexes in Arabidopsis, while also bringing new insights into the function of ULT1 and its antagonism with the PRC2-SWN complex. These findings direct our research toward a more precise characterization of the mechanisms that govern transition of chromatin from a repressed to an active state.

Keywords:
PRC2, Chromatin, Arabidopsis thaliana