L’identification de nouvelles activités chez les complexes Polycomb les lient aux structures d’ADN non-canoniques

Les protéines du groupe Polycomb (PcG) sont des protéines essentielles et conservées, qui forment deux complexes principaux, PRC1 et PRC2, qui sont recrutés au niveau de la chromatine et qui répriment stablement l’expression génique. Chez Drosophila melanogaster, les complexes Polycomb sont recrutés à des éléments d’ADN appelés éléments de réponse Polycomb (PREs) pour réprimer la transcription. PREs sont des éléments mémoires permutables qui peuvent maintenir la répression ou l’expression génique. Malgré des dizaines d’années d’étude, des questions fondamentales sur le fonctionnement du système PcG subsistent. 1) Comment les protéines PcG sont recrutées aux PREs uniquement lors du contexte développemental approprié, et comment les PREs peuvent conduire à la fois à l’activation et à la répression stable. 2) Comment les complexes PcG répriment la transcription, et si cela implique de nouvelles activités biochimiques et interactions. 3) Comment la répression dépendante des PcG peut-elle être propagé à travers le cycle cellulaire. La recherche de nouvelles activités biochimiques pour les complexes PcG pouvant répondre à ces questions fait l’objet de cette thèse. Les PREs sont transcrits en ARN qui pourraient donner la spécificité de contexte pour recruter les protéines PcG. Nous avons supposé que des R-loops puissent se former aux PREs, et être reconnues par les complexes Polycomb, ce que vous avons testé dans le chapitre 2. Nous avons identifié les séquences formant des R-loops dans des embryons et une lignée cellulaire de Drosophila melanogaster, et nous avons trouvé que ~30% des PREs forment des R-loops. Nous avons découvert que les PREs ayant formé des R-loops ont une plus forte probabilité d’être liés par les protéines PcG in vivo et in vitro. PRC2 lie des milliers d’ARN in vivo, mais aucune fonction claire n’y a été associée. En utilisant des expériences in vitro, nous avons identifié une activité d’invasion de brins pour PRC2 qui induit la formation d’hybride ARN-ADN, la partie principale d’une R-loop. Dans ce chapitre, nous avons trouvé que les PREs forment des R-loops, et sont impliquées dans le recrutement des protéines PcG qui induisent la répression génique stable. Nous avons découvert une activité d’invasion de brins pour PRC2 qui pourrait impliquer ce complexe Polycomb dans la formation de R-loops in vivo. Dans le chapitre 3, nous avons identifié une activité similaire à celle de la topoisomérase I associée avec PRC1 et la région C-terminale de sa sous-unité PSC (PSC-CTR). PRC1 et PSC-CTR peuvent relaxer un plasmide surenroulé négativement et ajouter des supertours négatifs à un plasmide relaxé en absence de topoisomérase. Cette activité suggère que la régulation de la topologie de l’ADN puisse être un nouveau mécanisme utilisé par les complexes PcG. PRC1 peut résoudre les R-loops formées sur un ADN négativement surenroulé in vitro. Une fonction possible pour cette activité de topoisomérase peut être la régulation des R-loops, dont la stabilité dépend à la fois de la séquence d’ADN et de la topologie de l’ADN environnant, in vivo. Dans cette thèse, nous avons identifié de nouvelles activités chez les complexes PcG : une activité d’invasion de brins pour PRC2 et une activité similaire à celle des topoisomérases pour PRC1. Ces deux activités impliquent les complexes PcG dans la formation et la résolution de R-loops. De plus, ces deux complexes peuvent reconnaitre les R-loops et sont recrutés aux PREs ayant formé ces structures. En conclusion, nous avons identifié de nouvelles activités pour les complexes Polycomb PRC1 et PRC2 qui les lient à la formation, la reconnaissance et la résolution de R-loops.Polycomb group (PcG) proteins are conserved, essential proteins, which assemble in two main complexes, PRC1 and PRC2, which are targeted to chromatin and stably repress gene expression. In Drosophila melanogaster, Polycomb complexes are targeted to DNA elements called Polycomb response elements (PREs) to repress transcription. PREs are switchable memory elements that can maintain either gene repression or gene activation. Despite decades of study, fundamental questions about how the PcG system functions remain. These include: 1) how PcG proteins are targeted to PREs only in the appropriate developmental context, and how PREs can mediate both stable activation and repression; 2) how PcG complexes repress transcription, and whether it involves novel biochemical mechanisms and interactions; 3) how PcG repression can be propagated through cell cycles. The search for new biochemical activities for PcG complexes that may answer these questions is the topic of this thesis. PREs are transcribed into RNAs which may give the context specificity to recruit PcG proteins. We hypothesized that R-loops may form at PREs, and be recognized by PcG complexes, which we tested in Chapter 2. We identified R-loop forming sequences in Drosophila melanogaster embryos and tissue culture cells, and found that ~30% of the PREs form R-loops. We found that PREs which have formed R-loops are more likely to be bound by PcG proteins both in vivo and in vitro. PRC2 binds to thousand RNA in vivo but no clear activity has been associated with it. Using in vitro assays, we identified a strand exchange activity for PRC2 which induces the formation of RNA-DNA hybrid, the main part of an R-loop. In this chapter, we have found that PREs form R-loops and are involved in the targeting of PcG proteins which induce stable gene repression. We have discovered an RNA strand exchange activity for PRC2 which may involve this Polycomb complex in the formation of R-loops in vivo. In Chapter 3, we identified a type I topoisomerase-like activity associated with PRC1 and the C-terminal region of its subunit PSC (PSC-CTR). PRC1 and PSC-CTR can relax a negatively supercoiled plasmid and add negative coils to a relaxed plasmid in absence of topoisomerase. This activity suggests regulation of DNA topology may be a novel mechanism used by PcG complexes. PRC1 can resolve R-loops formed on negatively supercoiled DNA in vitro. One role for the topoisomerase-like activity may be to regulate R-loops, whose stability of depends on both the DNA sequence and the topology of the surrounding DNA, in vivo. In this thesis, we identified new activities for Polycomb group complexes: an RNA strand exchange activity for PRC2 and a topoisomerase-like activity for PRC1. Both activities link PcG complexes to the formation and resolution of R-loops. In addition, both complexes can recognize R-loops and are recruited to PREs which have formed these structures. In conclusion, we have identified new nucleic acid-based activities for the Polycomb complexes PRC1 and PRC2, which link them to the formation, recognition and resolution of R-loops

RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2.

Polycomb Group (PcG) proteins form memory of transient transcriptional repression that is necessary for development. In Drosophila, DNA elements termed Polycomb Response Elements (PREs) recruit PcG proteins. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. PcG complexes modify chromatin, but also interact with both RNA and DNA, and RNA is implicated in PcG targeting and function. Here we show that R-loops form at many PREs in Drosophila embryos, and correlate with repressive states. In vitro, both PRC1 and PRC2 can recognize R-loops and open DNA bubbles. Unexpectedly, we find that PRC2 drives formation of RNA-DNA hybrids, the key component of R-loops, from RNA and dsDNA. Our results identify R-loop formation as a feature of Drosophila PREs that can be recognized by PcG complexes, and RNA-DNA strand exchange as a PRC2 activity that could contribute to R-loop formation

Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18-interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor (GEF) complex. 12 of these 28 interactions are supported by prior reports and we have directly validated novel interactions with SEC22A, TMCO4 and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites (MCSs), interactors included groups of microtubule/membrane-remodelling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We find that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Further, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated, or in which ORP2 expression is disrupted. Our data demonstrate that GEF-dependent Rab-interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder

RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2.

Polycomb Group (PcG) proteins form memory of transient transcriptional repression that is necessary for development. In Drosophila, DNA elements termed Polycomb Response Elements (PREs) recruit PcG proteins. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. PcG complexes modify chromatin, but also interact with both RNA and DNA, and RNA is implicated in PcG targeting and function. Here we show that R-loops form at many PREs in Drosophila embryos, and correlate with repressive states. In vitro, both PRC1 and PRC2 can recognize R-loops and open DNA bubbles. Unexpectedly, we find that PRC2 drives formation of RNA-DNA hybrids, the key component of R-loops, from RNA and dsDNA. Our results identify R-loop formation as a feature of Drosophila PREs that can be recognized by PcG complexes, and RNA-DNA strand exchange as a PRC2 activity that could contribute to R-loop formation