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The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of GTP nucleotides. Among post-translational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER structure remains unclear. Here, we show that Exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of the ER morphology and dynamics. We have integrated multi-omics data and super-resolution imaging to characterize the broad effect of EXT1 inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Finally, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome-structure axis, with implications in biotechnology and medicine.

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of guanosine triphosphate nucleotides. Among posttranslational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER structure remains unclear. Here, we show that exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multiomics and superresolution imaging to characterize the broad effect of EXT1 inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Last, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome structure axis, with implications in biotechnology and medicine

Viral infections are known to hijack the transcription and translation of the host cell. However, the extent to which viral proteins coordinate these perturbations remains unclear. Here we used a model system, the human T-cell leukemia virus type 1 (HTLV-1), and systematically analyzed the transcriptome and interactome of key effectors oncoviral proteins Tax and HBZ. We showed that Tax and HBZ target distinct but also common transcription factors. Unexpectedly, we also uncovered a large set of interactions with RNA-binding proteins, including the U2 auxiliary factor large subunit (U2AF2), a key cellular regulator of pre-mRNA splicing. We discovered that Tax and HBZ perturb the splicing landscape by altering cassette exons in opposing manners, with Tax inducing exon inclusion while HBZ induces exon exclusion. Among Tax- and HBZ-dependent splicing changes, we identify events that are also altered in Adult T cell leukemia/lymphoma (ATLL) samples from two independent patient cohorts, and in well-known cancer census genes. Our interactome mapping approach, applicable to other viral oncogenes, has identified spliceosome perturbation as a novel mechanism coordinated by Tax and HBZ to reprogram the transcriptome., SCOPUS: ar.j, info:eu-repo/semantics/published

Adaptation to variations in oxygen concentration is a conserved mechanism in all metazoans as this process is central for the maintenance of cell and tissue homeostasis. Two major and highly conserved processes contribute to hypoxia-induced gene reprogramming. The first one relies on the transcriptional activation of gene expression by the Hypoxia Inducible Factors (HIFs) leading to the upregulation of a large panel of genes. The second one corresponds to a strong modification of the translation program. While the mechanisms underlying HIF-dependent transcriptional activation have been well characterized, the ones governing translation reprogramming are only partially understood. To uncover how mRNA translation takes place at low oxygen tension, we used Drosophila as our research model since both Drosophila flies and S2 cells are highly resistant to low O2. We first demonstrate that several genes are efficiently translated under hypoxia in Drosophila S2 cells. By a gene reporter-based approach, we demonstrate that Ldh mRNA 3’UTR is sufficient to promote reporter mRNA association to polysomes in hypoxia. A deletion analysis of Ldh 3’UTR leads to the identification of a ACAAA-rich sequence important for polysomal association and translation in hypoxia.Cap-binding factors play a key role in controlling translational initiation. We have shown that the cap-binding translation initiation factor eIF4EHP (4EHP) plays a dual role on translation under hypoxic conditions. Despite having a general repressive function on global translation under normoxic and hypoxic conditions, we demonstrated that 4EHP also positively controls the translation of specific mRNAs under hypoxia. Inactivation of 4ehp reduces LDH protein synthesis and impairs reporter mRNA translation in hypoxia. Deletion of 4ehp inhibites the translation of several candidate genes harboring a ACAAA motif in 3’UTR under hypoxia, suggesting that 4EHP is required for hypoxic translation of mRNAs carrying ACAAA-rich mo, L'adaptation aux variations de la concentration en oxygène est un mécanisme conservé chez tous les métazoaires car ce processus est central pour le maintien de l'homéostasie cellulaire et tissulaire. Deux processus hautement conservés contribuent à la reprogrammation génétique induite par l'hypoxie. Le premier repose sur l'activation transcriptionnelle de l'expression génique par les facteurs inductibles de l'hypoxie (HIF) conduisant à l’induction d'un large panel de gènes. Le second correspond à une forte modification du programme traductionnel. Alors que les mécanismes sous-jacents à l'activation transcriptionnelle dépendante de HIF ont été bien caractérisés, ceux qui régissent la reprogrammation de la traduction ne sont que partiellement compris.Pour découvrir comment la traduction de l'ARNm se déroule à faible tension d'oxygène, nous avons utilisé la drosophile comme modèle d’étude, car les mouches Drosophila melanogaster et les cellules S2 issue de cet organisme sont très résistantes à de faibles teneurs en O2. Nous avons tout d’abord démontré que plusieurs gènes sont efficacement traduits en hypoxie dans les cellules S2 de drosophile. Par une approche basée sur l’utilisation de gènes rapporteurs, nous avons démontré que la région 3’ Non traduite (3’UTR) de l’ARNm Ldh est suffisante pour promouvoir l’association de l’ARNm du rapporteur aux polysomes en conditions hypoxiques. Une analyse par délétion de la région 3’UTR de l’ARNm Ldh a conduit à l’identification d’une séquence riche en ACAAA importante pour l’association polysomale et la traduction en hypoxie. La reconnaissance de la coiffe joue un rôle clé dans le contrôle de l'initiation de la traduction Nous avons montré que le facteur d'initiation de la traduction eIF4EHP (4EHP), qui se lie à la coiffe, joue un double rôle sur la traduction dans des conditions hypoxiques. Bien qu'il ait une fonction répressive sur la traduction générale dans des conditions normoxiques et hypoxiques, nous avons démontré que 4E, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

HOX proteins are homeodomain transcription factors mainly known for their implications in animal embryogenesis. While their functions have been well documented, the molecular mechanisms regulating HOX protein activity remain scarcely characterized. To identify proteins susceptible to modulate HOX activity, our group has proceeded with interactomic screenings focusing on two proteins among which HOXA2. This allowed to reveal that the transcriptional activity of HOXA2 can be regulated by its cytoplasmic relocation mediated by the ubiquitination-promoting protein KPC2. Here, we found out that HOXA2 can interact with three novel proteins involved in transcription factor activity modulation, namely the phosphatase PPP1CB, the kinase MSK1 and the chromodomain DNA-binding CHD4. At the functional level, our data support a novel regulatory model according to which PPP1CB increases the nuclear export of HOXA2 initiated by KPC2. This export is correlated with a decrease in HOXA2 transcriptional activity. Once in the cytoplasm, both PPP1CB and KPC2 act together to de-ubiquitinate HOXA2 and stabilize the protein. PPP1CB and KPC2 would therefore contribute to establish a cytoplasmic store of HOXA2 in a way to modulate HOXA2 activity. This activity regulation model including post-translational modifications and subcellular relocation had not been highlighted before for a HOX protein. About the HOXA2-CHD4 interaction, we observed that CHD4 can also decrease the HOXA2 transcriptional activity in vitro. However, while our data support that CHD4 could bind to target loci shared with HOXA2 in mouse embryos, the possible involvement of CHD4 in HOXA2 target gene selection and regulation requires additional investigation. Similarly, although the MSK1 kinase is known to target transcription factors, we failed to observe a direct effect of MSK1 toward HOXA2 behavior in vitro., (SC - Sciences) -- UCL, 2019

RésuméLa voie de l'immunodéficience (Imd) joue un rôle crucial dans la défense immunitaire de Drosophila melanogaster contre les infections par les bactéries Gram- négatives. La liaison du peptidoglycane de type DAP, produits de la membrane bactérienne à des protéines de reconnaissance, les récepteurs PGRPs, présents à la surface des cellules immunitaires déclenchent l'activation d'un processus de signalisation intracellulaire conduisant à l'activation du facteur de transcription RELISH qui favorise l'expression robuste des peptides antimicrobiens (AMP). En combinant des approches pharmacologiques et génétiques, nous avons montré que la déacétylase SIRT1, consommatrice de NAD, contrôle l’activation de la voie Imd dans les cellules S2. Nos données indiquent que l’inactivation de Sirt1 régule à la fois l’expression du gène Pgrp-lc ainsi que le clivage de la protéine IMD induit par l’homologue de la caspase-8, DREDD. De plus, nos données montrent qu’IMD est une protéine acétylée. L’ensemble de nos résultats met en évidence SIRT1 comme nouveau facteur régulateur de la réponse immune innée de Drosophila melanogaster.AbstractThe Immunodeficiency (Imd) pathway plays a crucial role in the immune defense of Drosophila melanogaster against infection by Gram-negative bacteria. Binding of bacterial membrane product DAP-type peptidoglycan to peptidoglycan recognition proteins (PGRP) present at the surface of immune cells triggers the activation of an intracellular signaling process leading to the activation of RELISH transcription factor which promotes robust expression of anti-microbial peptides (AMP). Combining pharmacological and genetic approaches, we show that the NAD- consuming deacetylase sirtuin 1 (SIRT1) controls the activation of Imd pathway in S2 cells. Our data indicate that Sirt1 inactivation strongly impairs both the expression of Pgrp-lc signaling receptor and downstream IMD cleavage by the caspase-8 homolog DREDD. In addition, our data show that IMD is an acetyla, Doctorat en Sciences, info:eu-repo/semantics/nonPublished