Thesis presented July 08, 2022

Abstract:
During the development of multicellular organisms, switches in gene expression allow different cell types to emerge from a same undifferentiated cell. These switches involve protein complexes of the Polycomb (PcG) and Trithorax (TrxG) groups that modify the chromatin structure with repressive and activating effects, respectively.
In the model plant Arabidopsis thaliana, CURLY LEAF (CLF) and SWINGER (SWN), two histone methyltransferases (HMT) of the Polycomb Repressive Complex 2 (PRC2), repress gene expression via the deposition of a trimethyl mark on lysine 27 of histone H3 (H3K27me3). The histone demethylase (HDMT) RELATIVE OF FLOWERING6 (REF6) can counteract this repression by actively removing the H3K27me3 mark. The ATPase remodeler BRAHMA (BRM) can also counteract the PRC2-mediated repression, by displacing nucleosomes and opening the chromatin. All these chromatin effectors contribute to the regulation of many genes, each of them being specific of various contexts. The mechanisms that confer locus and spatial or temporal context specificity to the activity of CLF/SWN, REF6 and BRM are not fully elucidated, and the factors that allow the switch of genes from a repressed to an active state (and vice versa) are not known.
The SAND domain protein ULTRAPETALA1 (ULT1) induces the switch of more than 900 target genes from a repressed to an active state, by counteracting the action of CLF, thereby reducing the level of H3K27me3 marks at their locus. These targets include the floral homeotic genes AGAMOUS (AG), APETALA3 (AP3) and SEPALLATA3 (SEP3), which ULT1 enables to be activated in appropriate floral tissues. Because ULT1 lacks enzymatic domains that could be responsible for a direct alteration of the chromatin structure, its mode of action could thus involve chromatin effectors.
Therefore, the goal of my thesis was to investigate how ULT1 could contribute to the regulation of its target genes via an interaction with enzymes that add (HMTs) or remove (HDMTs) the H3K27me3 mark on one hand, and with the remodeler BRM on the other hand.
First, I studied the genetic interaction between ULT1 and REF6, and demonstrated that ULT1 and REF6 act independently from one another on the de-repression of homeotic floral genes. I then studied in vivo the interaction between ULT1 and the two methyltransferases CLF and SWN. I demonstrated by yeast two-hybrid assay that the SAND and CW-like domains of ULT1 and the N-terminal domains of the catalytic domain of CLF and SWN were sufficient to allow the physical interaction between these partners. These results indicate that ULT1 could directly inhibit CLF/SWN activity rather than contributing to an active demethylation with REF6.
Second, I investigated the molecular and genetic interactions between ULT1/2 and the remodelers BRM and its homolog SPLAYED (SYD). I could detect a physical interaction in vivo and I showed that the N-terminal parts of BRM and SYD catalytic domains were sufficient to mediate physical interaction with ULT1. A genetic analysis showed that the interaction between the ULT1/2 and BRM pathways is antagonistic regarding the regulation of flowering time and organ number within the three first floral whorls, while synergetic for the control of floral determinacy. Furthermore, ULT1 can still activate floral homeotic genes in a brm loss-of-function context, indicating that the ULT1 and ATPase remodeler pathways are independent in this process. Altogether, these results show that ULT1 and BRM interact via different modalities depending on the considered developmental process.

Keywords:
chromatin, histone marks, PRC2, Arabidopsis thaliana