Study of topoisomerase VI and its partners in barrier insulator function and photooxidative stress response in Arabidopsis thaliana

La Topoisomérase VI (TopoVI) est composée des sous unités TopoVI A et TopoVI B, présentes chez les archées, les protistes et les viridiplantae. Chez les plantes, TopoVI possède deux sous unités additionelles nommées BIN4 et RHL1, et est impliquée dans diverses fonctions telles que l’endoréplication, la skotomorphogenèse et la signalisation hormonale. Lors d’un crible génétique, un mutant ponctuel de TopoVI A (caa39) a été caractérisé comme activant de manière constutive une réponse au stress photo-oxydant. Les études approfondies de ce mutant suggèrent que TopoVI contrôle l’expression des gènes de réponse à l’oxygène singulet.Les travaux de cette thèse ont participé à démontrer que TopoVI assure également une fonction isolatrice barrière en protègeant les ilots d’euchromatine (IEs) localisés dans les régions péricentromériques contre l’invasion de la marque hétérochromatinienne H3K9me2. Dans le mutant caa39, l’invasion de marques répressives dans les IEs est corrélée avec la baisse d’expression des gènes résidant dans ces mêmes ilots.Ces travaux montrent également que TopoVI intéragit avec la sous unité de cohésine SMC3. L’obtention de lignées supprimant l’expression de SMC3 a permis d’étudier l’impact d’un disfonctionnement du complèxe cohésine dans la plante. Comme chez le mutant caa39, la baisse du niveau de SMC3 conduit à l’activation de l’expression des gènes de réponse au stress oxydant ainsi qu’a une résistance au traitement par forte lumière. Cependant, la perte de SMC3 ne provoque pas de changement uniforme dans l’expression des gènes d’IEs, suggérant que le complèxe cohésine n’est pas impliqué dans la fonction barrière assurée par TopoVI.The Topoisomerase VI (TopoVI) is composed of the TopoVI A and TopoVI B core subunits, and is present in archaea, protists and viridiplantae. In plants, TopoVI also includes two additional subunits named BIN4 and RHL1, and is involved in various biological process such as endoreduplication, skotomorphogenesis or hormone signalling. Moreover, a previous genetic screen revealed that plants carrying a mutant allele of TopoVI A (caa39) constitutively activate a response to photooxidative stress. Extensive studies of caa39 suggest that TopoVI controls the expression of singlet oxygen responsive genes. The work reported in this manuscript helped demonstrating that TopoVI complex also provides a barrier insulator function by protecting euchromatin islands located in pericentromeric regions against spreading of the heterochromatin mark H3K9me2. In caa39, invasion of repressive marks into euchromatin islands correlates with decreased expression of genes localized inside euchromatin islands.In this study, we also demonstrated that TopoVI interacts with the cohesin subunit SMC3. We obtained two independent lines silencing the expression of SMC3. These lines allowed to investigate the consequences of a defective cohesin complex in plants. As observed in caa39, decreased SMC3 level leads to overexpression of oxidative stress response genes as well as resistance to high light stress. However, loss of SMC3 does not cause a uniform change in the expression of euchromatin island genes, suggesting that the cohesin complex is not involved in TopoVI-dependent barrier function

You shall not pass! A Chromatin barrier story in plants

International audienceAs in other eukaryotes, the plant genome is functionally organized in two mutually exclusive chromatin fractions, a gene-rich and transcriptionally active euchromatin, and a gene-poor, repeat-rich, and transcriptionally silent heterochromatin. In Drosophila and humans, the molecular mechanisms by which euchromatin is preserved from heterochromatin spreading have been extensively studied, leading to the identification of insulator DNA elements and associated chromatin factors (insulator proteins), which form boundaries between chromatin domains with antagonistic features. In contrast, the identity of factors assuring such a barrier function remains largely elusive in plants. Nevertheless, several genomic elements and associated protein factors have recently been shown to regulate the spreading of chromatin marks across their natural boundaries in plants. In this minireview, we focus on recent findings that describe the spreading of chromatin and propose avenues to improve the understanding of how plant chromatin architecture and transitions between different chromatin domains are defined

MoBiFC: development of a modular bimolecular fluorescence complementation toolkit for the analysis of chloroplast protein-protein interactions

International audienceThe bimolecular fluorescence complementation (BiFC) assay has emerged as one of the most popular methods for analysing protein-protein interactions (PPIs) in plant biology. This includes its increasing use as a tool for dissecting the molecular mechanisms of chloroplast function. However, the construction of chloroplast fusion proteins for BiFC can be difficult, and the availability and selection of appropriate controls is not trivial. Furthermore, the challenges of performing BiFC in restricted cellular compartments has not been specifically addressed. Here we describe the development of a flexible modular cloning-based toolkit (MoBiFC) for chloroplast BiFC and proximity labelling using synthetic biology principles. The approach facilitates the cloning process for chloroplast-targeted proteins, allows robust ratiometric quantification, and the toolkit comes with model positive and negative controls. Our study also highlights many potential pitfalls including the choice of fluorescent protein (FP) split, negative controls, cell type, and reference FP. Finally, we provide an example of how users can enrich the toolset by providing functional proximity labelling modules, and we discuss how MoBiFC could be further improved and extended to other compartments of the plant cell

Topoisomerase VI participates in an insulator-like function that prevents H3K9me2 spreading into euchromatic islands

The organization of the genome into transcriptionally active and inactive chromatin domains requires well-delineated chromatin boundaries and insulator functions in order to maintain the identity of adjacent genomic loci with antagonistic chromatin marks and functionality. In plants that lack known chromatin insulators, the mechanisms that prevent heterochromatin spreading into euchromatin remain to be identified. Here, we show that DNA Topoisomerase VI participates in a chromatin boundary function that safeguards the expression of genes in euchromatin islands within silenced heterochromatin regions. While some transposable elements are reactivated in mutants of the Topoisomerase VI complex, genes insulated in euchromatin islands within heterochromatic regions of the Arabidopsis thaliana genome are specifically downregulated. H3K9me2 levels consistently increase at euchromatin island loci and decrease at some TE loci. We further show that Topoisomerase VI physically interacts with S-adenosylmethionine (SAM) synthase MAT3, which is required for H3K9me2 deposition. Topoisomerase VI promotes MAT3 occupancy on heterochromatic elements and its exclusion from euchromatic islands, thereby providing a mechanistic insight into the essential role of Topoisomerase VI in the delimitation of chromatin domains

Topoisomerase VI participates in an insulator-like function that prevents H3K9me2 spreading

International audienceThe organization of the genome into transcriptionally active and inactive chromatin domains requires well-delineated chromatin boundaries and insulator functions in order to maintain the identity of adjacent genomic loci with antagonistic chromatin marks and functionality. In plants that lack known chromatin insulators, the mechanisms that prevent heterochromatin spreading into euchromatin remain to be identified. Here, we show that DNA Topoisomerase VI participates in a chromatin boundary function that safeguards the expression of genes in euchromatin islands within silenced heterochromatin regions. While some transposable elements are reactivated in mutants of the Topoisomerase VI complex, genes insulated in euchromatin islands within heterochromatic regions of the Arabidopsis thaliana genome are specifically down-regulated. H3K9me2 levels consistently increase at euchromatin island loci and decrease at some transposable element loci. We further show that Topoisomerase VI physically interacts with S-adenosylmethionine synthase methionine adenosyl transferase 3 (MAT3), which is required for H3K9me2. A Topoisomerase VI defect affects MAT3 occupancy on heterochromatic elements and its exclusion from euchromatic islands, thereby providing a possible mechanistic explanation to the essential role of Topoisomerase VI in the delimitation of chromatin domains

Post-translational regulation of photosynthetic activity via the TOR kinase in plants

Chloroplasts are the powerhouse of the plant cell, yet they are resource-intensive and will cause photooxidative damage if their activity overshoots the demands of growth. The adjustment of chloroplast activity to match growth is therefore vital for stress acclimation. Here we identify a novel post-translational mechanism linking the conserved eukaryotic TOR kinase that promotes growth and the guanosine tetraphosphate (ppGpp) signaling pathway of prokaryotic origin that regulates chloroplast activity, and photosynthesis in particular. We show that RelA SpoT Homologue 3 (RSH3), a nuclear-encoded chloroplastic enzyme responsible for ppGpp biosynthesis, interacts directly with the TOR complex via a plant-specific N-terminal region (NTR) which is hyper-phosphorylated in a TOR-dependent manner. Downregulation of TOR activity reduces NTR phosphorylation, enhances ppGpp synthesis by RSH3, and causes a ppGpp-dependent decrease in photosynthetic capacity. Altogether we demonstrate that the TOR-RSH3 signaling axis is a novel and direct post-translational mechanism that allows chloroplast activity to be matched with plant growth, setting a new precedent for the regulation of organellar function by TOR. One sentence summary The TOR kinase post-translationally controls guanosine tetraphosphate signaling to regulate plant photosynthetic activity