Identification et caractérisation de nouveaux partenaires protéiques du canal sodique cardiaque Nav1.5

La fonction du canal sodique cardiaque est cruciale pour l'initiation, la propagation et le maintien d'un rythme cardiaque normal. Le rôle crucial de Nav1.5 dans la fonction cardiaque a été mis en évidence par l'identification de mutations naturelles au niveau de son gène (SCN5A). Les mutations sur le gène SCN5A sont associées à de nombreuses maladies génétiques telles que: le syndrome de Brugada, le syndrome du QT long et les troubles de conduction. Le point commun à ces pathologies est les arythmies cardiaques qui peuvent mener à la mort subite. Ces mutations sur ce gène n'explique cependant que 20% des cas connus (cas du syndrome de Brugada) (Antzelevitch, 2006). D'où l'implication d'autres facteurs, comme les partenaires de Nav1.5, dans ces pathologies est une éventualité émergente. Objectif: Pour une meilleure compréhension de la physiopathologie de ces arythmies cardiaques, nous nous proposons dans ce projet d'identifier et de caractériser de nouveaux partenaires de Nav1.5. Méthodes: Les interacteurs potentiels de Nav1.5 sont identifiés par la méthode du double hybride chez la levure. Les interactions sont ensuite confirmées in vivo et in vitro par les méthodes de co-immunoprécipitation et de pull-down. Les effets de ces partenaires sur l'activité de Nav1.5 sont évalués par la méthode du patch clamp en configuration "cellule entière". La co-localisation tissulaire de Nav1.5 avec ses partenaires est obtenue par la microscopie confocale. Résultats: Deux nouveaux partenaires de Nav1.5 ont été identifiés: 1) l'a-actinine-2 qui interagit avec la porte d'inactivation et 2) la troponine I cardiaque (Tnlc) qui se lie à la boucle cytoplasmique I-II. L'a-actinine-2 se fixe via son domaine spectrine sur la porte d'inactivation de Nav1.5 et augmente ainsi son expression à la surface membranaire. L'a-actinine-2 se co-localise avec Nav1.5 dans les tubules T des cardiomyocytes humains. Quant à la troponine I, elle s'associe avec deux régions de la boucle I-II de Nav1.5 par le biais de sa région C-terminale. La Tnlc déplace la courbe d'inactivation de ce canal vers des valeurs plus hyperpolarisées sans affecter son expression à la surface membranaire. Toute fois, cet effet de la Tnlc disparaît en présence de la sous-unité pi de Nav1.5. Conclusion: L'identification de l'a-actinine-2 et la Tnlc représente une étape importante dans la compréhension des mécanismes régulant l'activité de Nav1.5 d'une part et d'autre part servira à décrypter les mécanismes moléculaires sous-tendant la complexité des arythmies cardiaques

The asymmetric distribution of RNA polymerase II and nucleosomes on replicated daughter genomes is caused by differences in replication timing between the lagging and the leading strand

International audienceChromatin features are thought to have a role in the epigenetic transmission of transcription states from one cell generation to the next. It is unclear how chromatin structure survives disruptions caused by genomic replication or whether chromatin features are instructive of the transcription state of the underlying gene. We developed a method to monitor budding yeast replication, transcription, and chromatin maturation dynamics on each daughter genome in parallel, with which we identified clusters of secondary origins surrounding known origins. We found a difference in the timing of lagging and leading strand replication on the order of minutes at most yeast genes. We propose a model in which the majority of old histones and RNA polymerase II (RNAPII) bind to the gene copy that replicated first, while newly synthesized nucleosomes are assembled on the copy that replicated second. RNAPII enrichment then shifts to the sister copy that replicated second. The order of replication is largely determined by genic orientation: If transcription and replication are codirectional, the leading strand replicates first; if they are counterdirectional, the lagging strand replicates first. A mutation in the Mcm2 subunit of the replicative helicase Mcm2-7 that impairs Mcm2 interactions with histone H3 slows down replication forks but does not qualitatively change the asymmetry in nucleosome distribution observed in the WT. We propose that active transcription states are inherited simultaneously and independently of their underlying chromatin states through the recycling of the transcription machinery and old histones, respectively. Transcription thus actively contributes to the reestablishment of the active chromatin state

Mechanics of DNA Replication and Transcription Guide the Asymmetric Distribution of RNAPol2 and New Nucleosomes on Replicated Daughter Genomes

Replication of the eukaryotic genome occurs in the context of chromatin. Chromatin is commonly thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication. In order to better understand the transmission of gene expression states from one cell generation to the next we have developed a method for following chromatin structure dynamics during replication-ChIP-NChAP-Chromatin Immuno-Precipitation-Nascent Chromatin Avidin Pulldown-which we used to monitor RNAPol2 and new nucleosome binding to newly-replicated daughter genomes in S. Cerevisiae. The strand specificity of our libraries allowed us to uncover the inherently asymmetric distribution of RNAPol2 and H3K56ac-a mark of new histones-on daughter chromatids after replication. Our results show a range of distributions on thousands of genes from symmetric to asymmetric with enrichment shifts from one replicated strand to the other throughout S-phase. We propose a two-step model of chromatin assembly on nascent DNAwhich provides a mechanistic framework for the regulation of asymmetric segregation of maternal histones, and discuss our model for chromatin assembly in the context of a mechanism for gene expression buffering without a direct role for H3K56ac

Dynamics of Nucleosome Positioning Maturation following Genomic Replication

Chromatin is thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication. Here, we measure a key aspect of chromatin structure dynamics during replication—how rapidly nucleosome positions are established on the newly replicated daughter genomes. By isolating newly synthesized DNA marked with 5-ethynyl-2′-deoxyuridine (EdU), we characterize nucleosome positions on both daughter genomes of S. cerevisiae during chromatin maturation. We find that nucleosomes rapidly adopt their mid-log positions at highly transcribed genes, which is consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in hir1Δ mutants reveal a role for HIR in nucleosome spacing. We also characterized nucleosome positions on the leading and lagging strands, uncovering differences in chromatin maturation dynamics at hundreds of genes. Our data define the maturation dynamics of newly replicated chromatin and support a role for transcription in sculpting the chromatin template

Dynamics of Nucleosome Positioning Maturation following Genomic Replication

International audienceChromatin is thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication. Here, we measure a key aspect of chromatin structure dynamics during replication-how rapidly nucleosome positions are established on the newly replicated daughter genomes. By isolating newly synthesized DNA marked with 5-ethynyl-2'-deoxyuridine (EdU), we characterize nucleosome positions on both daughter genomes of S. cerevisiae during chromatin maturation. We find that nucleosomes rapidly adopt their mid-log positions at highly transcribed genes, which is consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in hir1Δ mutants reveal a role for HIR in nucleosome spacing. We also characterized nucleosome positions on the leading and lagging strands, uncovering differences in chromatin maturation dynamics at hundreds of genes. Our data define the maturation dynamics of newly replicated chromatin and support a role for transcription in sculpting the chromatin template

In inflammatory myopathies, dropped head/bent spine syndrome is associated with scleromyositis: an international case–control study

Background Some myopathies can lead to dropped head or bent spine syndrome (DH/BS). The significance of this symptom has not been studied in inflammatory myopathies (IM).Objectives To assess the significance of DH/BS in patients with IM.Methods Practitioners from five IM networks were invited to report patients with IM suffering from DH/BS (without other known cause than IM). IM patients without DH/BS, randomly selected in each participating centre, were included as controls at a ratio of 2 to 1.Results 49 DH/BS-IM patients (DH: 57.1%, BS: 42.9%) were compared with 98 control-IM patients. DH/BS-IM patients were older (65 years vs 53 years, p<0.0001) and the diagnosis of IM was delayed (6 months vs 3 months, p=0.009). Weakness prevailing in the upper limbs (42.9% vs 15.3%), dysphagia (57.1% vs 25.5%), muscle atrophy (65.3% vs 34.7%), weight loss (61.2% vs 23.5%) and loss of the ability to walk (24.5% vs 5.1%) were hallmarks of DH/BS-IM (p≤0.0005), for which the patients more frequently received intravenous immunoglobulins (65.3% vs 34.7%, p=0.0004). Moreover, DH/BS-IM patients frequently featured signs and/or complications of systemic sclerosis (SSc), fulfilling the American College of Rheumatology/European Alliance of Associations for Rheumatology criteria for this disease in 40.8% of the cases (vs 5.1%, p<0.0001). Distribution of the myopathy, its severity and its association with SSc were independently associated with DH/BS (p<0.05). Mortality was higher in the DH/BS-IM patients and loss of walking ability was independently associated with survival (p<0.05).Conclusion In IM patients, DH/BS is a marker of severity and is associated with SSc (scleromyositis)