Modifiers of notch transcriptional activity identified by genome-wide RNAi

<p>Abstract</p> <p>Background</p> <p>The Notch signaling pathway regulates a diverse array of developmental processes, and aberrant Notch signaling can lead to diseases, including cancer. To obtain a more comprehensive understanding of the genetic network that integrates into Notch signaling, we performed a genome-wide RNAi screen in <it>Drosophila </it>cell culture to identify genes that modify Notch-dependent transcription.</p> <p>Results</p> <p>Employing complementary data analyses, we found 399 putative modifiers: 189 promoting and 210 antagonizing Notch activated transcription. These modifiers included several known Notch interactors, validating the robustness of the assay. Many novel modifiers were also identified, covering a range of cellular localizations from the extracellular matrix to the nucleus, as well as a large number of proteins with unknown function. Chromatin-modifying proteins represent a major class of genes identified, including histone deacetylase and demethylase complex components and other chromatin modifying, remodeling and replacement factors. A protein-protein interaction map of the Notch-dependent transcription modifiers revealed that a large number of the identified proteins interact physically with these core chromatin components.</p> <p>Conclusions</p> <p>The genome-wide RNAi screen identified many genes that can modulate Notch transcriptional output. A protein interaction map of the identified genes highlighted a network of chromatin-modifying enzymes and remodelers that regulate Notch transcription. Our results open new avenues to explore the mechanisms of Notch signal regulation and the integration of this pathway into diverse cellular processes.</p

Distinct contextual roles for Notch signalling in skeletal muscle stem cells.

International audienceNotch signalling acts in virtually every tissue during the lifetime of metazoans. Recent studies have pointed to multiple roles for Notch in stem cells during quiescence, proliferation, temporal specification, and maintenance of the niche architecture. Skeletal muscle has served as an excellent paradigm to examine these diverse roles as embryonic, foetal, and adult skeletal muscle stem cells have different molecular signatures and functional properties, reflecting their developmental specification during ontology. Notably, Notch signalling has emerged as a major regulator of all muscle stem cells. This review will provide an overview of Notch signalling during myogenic development and postnatally, and underscore the seemingly opposing contextual activities of Notch that have lead to a reassessment of its role in myogenesis

Le clonage et la caractérisation d'Enigma, une protéine mitochondriale impliquée dans la longévité chez la Drosophile

Un criblage génétique utilisant des mouches transgéniques à été réalisé dans notre laboratoire; ces mouches surexpriment deux formes de Notch constitutivement activé, l'une liée à la membrane et l'autre nucléaire. Seul un groupe de complémentation modifiant exclusivement le phénotype de l'oeil induit par le Notch nucleaire mais sans effet sur les mouches Notch membranaires a été identifié. Ce groupe a été appelé Enigma (Egm). Egm code une protéine présentant une homologie avec les enzymes de b-oxidation: la voie mitochondriale qui contrôle la dégradation des acides gras. J'ai observé que le dosage d'Egm disponible est critique pour un développement normal de l'organisme; les animaux " knock-out " pour Egm ne survivent pas au delà de l'état de larve; une basse quantité de protéine, tout juste suffisante pour la survie, ralentis la croissance de l'organisme, tandis que des niveaux intermédiaires d'Egm aboutissent à une augmentation significative de la durée de vie. Les mutants Egm ayant une longue durée de vie présentent une tolérance accentuée au stress oxidatif, une propriété caractéristique des mutations de longévité. Egm est localisée dans la mitochondrie, le site intracellulaire ou la beta-oxidation a lieu. De plus, les mouches mutantes pour Egm présentent des niveaux réduits de triglycérides et des corps gras mal formés, corroborant ainsi l'hypothèse que le locus est impliqué dans le métabolisme des acides gras. L'identification de Egm en tant que locus vieillissant est donc cohérente avec les études préalables, mais on peut aussi suggérer que la beta-oxydation est un nouveau mécanisme du contrôle de la longévité.A large-scale genetic screen was performed in our laboratory using transgenic flies overexpressing either a membrane tethered or a nuclear form of constitutively activated Notch receptor in the adult eye. Only one complementation group was identified that modified exclusively the N nuclear-induced eye phenotype, but had no effect on the N membrane flies. This group was named Enigma (Egm). Cloning of the locus revealed it encodes for a protein with homology to enzymes of b-oxidation, the mitochondrial pathway that controls fatty acid breakdown. I observed that the dosage of available Egm is critical for the normal development of the organism; knock-out animals lacking Egm do not survive beyond the larval stage; low levels of the protein, just sufficient for survival, decelerate the growth rate of the organism, whereas intermediate amounts of Egm result in a significant extension of adult lifespan. The long-lived Egm mutants also demonstrate increased tolerance to oxidative stress, a signature feature of longevity mutations. Egm is localized into the mitochondria, the intracellular site of b-oxidation activity. In addition, Egm mutant animals show reduced levels of triglycerides and malformed fat bodies, corroborating the notion that the locus is involved in fatty acid metabolism. The identification of Egm as an aging locus is consistent with previous studies, but at the same time it is uncovering b-oxidation as a putative novel mechanism of longevity control.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Stress relief: emerging methods to mitigate dissociation-induced artefacts

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The Notch signaling network in muscle stem cells during development, homeostasis, and disease

International audienceAbstract Skeletal muscle stem cells have a central role in muscle growth and regeneration. They reside as quiescent cells in resting muscle and in response to damage they transiently amplify and fuse to produce new myofibers or self-renew to replenish the stem cell pool. A signaling pathway that is critical in the regulation of all these processes is Notch. Despite the major differences in the anatomical and cellular niches between the embryonic myotome, the adult sarcolemma/basement-membrane interphase, and the regenerating muscle, Notch signaling has evolved to support the context-specific requirements of the muscle cells. In this review, we discuss the diverse ways by which Notch signaling factors and other modifying partners are operating during the lifetime of muscle stem cells to establish an adaptive dynamic network

Pax7 haploinsufficiency impairs muscle stem cell function in Cre-recombinase mice and underscores the importance of appropriate controls

International audienceAbstract Ever since its introduction as a genetic tool, the Cre-lox system has been widely used for molecular genetic studies in vivo in the context of health and disease, as it allows time- and cell-specific gene modifications. However, insertion of the Cre-recombinase cassette in the gene of interest can alter transcription, protein expression, or function, either directly, by modifying the landscape of the locus, or indirectly, due to the lack of genetic compensation or by indirect impairment of the non-targeted allele. This is sometimes the case when Cre-lox is used for muscle stem cell studies. Muscle stem cells are required for skeletal muscle growth, regeneration and to delay muscle disease progression, hence providing an attractive model for stem cell research. Since the transcription factor Pax7 is specifically expressed in all muscle stem cells, tamoxifen-inducible Cre cassettes (CreERT2) have been inserted into this locus by different groups to allow targeted gene recombination. Here we compare the two Pax7-CreERT2 mouse lines that are mainly used to evaluate muscle regeneration and development of pathological features upon deletion of specific factors or pathways. We applied diverse commonly used tamoxifen schemes of CreERT2 activation, and we analyzed muscle repair after cardiotoxin-induced injury. We show that consistently the Pax7-CreERT2 allele targeted into the Pax7 coding sequence (knock-in/knock-out allele) produces an inherent defect in regeneration, manifested as delayed post-injury repair and reduction in muscle stem cell numbers. In genetic ablation studies lacking proper controls, this inherent defect could be misinterpreted as being provoked by the deletion of the factor of interest. Instead, using an alternative Pax7-CreERT2 allele that maintains bi-allelic Pax7 expression or including appropriate controls can prevent misinterpretation of experimental data. The findings presented here can guide researchers establish appropriate experimental design for muscle stem cell genetic studies

A Critical Requirement for Notch Signaling in Maintenance of the Quiescent Skeletal Muscle Stem Cell State

Abstract Notch signaling plays a key role in virtually all tissues and organs in metazoans; however, limited examples are available for the regulatory role of this pathway in adult quiescent stem cells. We performed a temporal and ontological assessment of effectors of the Notch pathway that indicated highest activity in freshly isolated satellite cells and, unexpectedly, a sharp decline before the first mitosis, and subsequently in proliferating, satellite cell-derived myoblasts. Using genetic tools to conditionally abrogate canonical Notch signaling during homeostasis, we demonstrate that satellite cells differentiate spontaneously and contribute to myofibers, thereby resulting in a severe depletion of the stem cell pool. Furthermore, whereas loss of Rbpj function provokes some satellite cells to proliferate before fusing, strikingly, the majority of mutant cells terminally differentiate unusually from the quiescent state, without passing through S-phase. This study establishes Notch signaling pathway as the first regulator of cellular quiescence in adult muscle stem cells. Disclosure of potential conflicts of interest is found at the end of this article.</jats:p

Extraction and sequencing of single nuclei from murine skeletal muscles

International audienceSingle-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications. For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021)