Ontogeny-Driven rDNA Rearrangement, Methylation, and Transcription, and Paternal Influence

Gene rearrangement occurs during development in some cell types and this genome dynamics is modulated by intrinsic and extrinsic factors, including growth stimulants and nutrients. This raises a possibility that such structural change in the genome and its subsequent epigenetic modifications may also take place during mammalian ontogeny, a process undergoing finely orchestrated cell division and differentiation. We tested this hypothesis by comparing single nucleotide polymorphism-defined haplotype frequencies and DNA methylation of the rDNA multicopy gene between two mouse ontogenic stages and among three adult tissues of individual mice. Possible influences to the genetic and epigenetic dynamics by paternal exposures were also examined for Cr(III) and acid saline extrinsic factors. Variables derived from litters, individuals, and duplicate assays in large mouse populations were examined using linear mixed-effects model. We report here that active rDNA rearrangement, represented by changes of haplotype frequencies, arises during ontogenic progression from day 8 embryos to 6-week adult mice as well as in different tissue lineages and is modifiable by paternal exposures. The rDNA methylation levels were also altered in concordance with this ontogenic progression and were associated with rDNA haplotypes. Sperm showed highest level of methylation, followed by lungs and livers, and preferentially selected haplotypes that are positively associated with methylation. Livers, maintaining lower levels of rDNA methylation compared with lungs, expressed more rRNA transcript. In vitro transcription demonstrated haplotype-dependent rRNA expression. Thus, the genome is also dynamic during mammalian ontogeny and its rearrangement may trigger epigenetic changes and subsequent transcriptional controls, that are further influenced by paternal exposures

Cohesin Proteins Promote Ribosomal RNA Production and Protein Translation in Yeast and Human Cells

Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and 35S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the human cohesinopathies

Longevity by RNA polymerase III inhibition downstream of TORC1

Three distinct RNA polymerases (Pols) transcribe different classes of genes in the eukaryotic nucleus1. Pol III is the essential, evolutionarily conserved enzyme that generates short, non-coding RNAs, including transfer RNAs (tRNAs) and 5S ribosomal RNA (rRNA)2. Historical focus on transcription of protein-coding genes has left the roles of Pol III in organismal physiology relatively unexplored. The prominent regulator of Pol III activity, Target of Rapamycin kinase Complex 1 (TORC1), is an important longevity determinant3, raising the question of Pol III’s involvement in ageing. Here we show that Pol III limits lifespan downstream of TORC1. We find that a reduction in Pol III extends chronological lifespan in yeast and organismal lifespan in worms and flies. Inhibiting Pol III activity in the adult worm or fly gut is sufficient to extend lifespan, and in flies, longevity can be achieved by Pol III inhibition specifically in the intestinal stem cells (ISCs). The longevity phenotype is associated with amelioration of age-related gut pathology and functional decline, dampened protein synthesis and increased tolerance of proteostatic stress. Importantly, Pol III acts downstream of TORC1 for lifespan and limiting Pol III activity in the adult gut achieves the full longevity benefit of systemic TORC1 inhibition. Hence, Pol III is a pivotal output of this key nutrient signalling network for longevity; Pol III’s growth-promoting, anabolic activity mediates the acceleration of ageing by TORC1. The evolutionary conservation of Pol III affirms its potential as a therapeutic target

Ca V

Membrane depolarization activates the multisubunit CaV1.2 L-type calcium channel initiating various excitation coupling responses. Intracellular trafficking into and out of the plasma membrane regulates the channel's surface expression and stability, and thus, the strength of CaV1.2-mediated Ca2+ signals. The mechanisms regulating the residency time of the channel at the cell membrane are unclear. Here, we coexpressed the channel core complex CaV1.2α1 pore-forming and auxiliary CaVβ subunits and analyzed their trafficking dynamics from single-particle-tracking trajectories. Speed histograms obtained for each subunit were best fitted to a sum of diffusive and directed motion terms. The same mean speed for the highest-mobility state underlying directed motion was found for all subunits. The frequency of this component increased by covalent linkage of CaVβ to CaV1.2α1 suggesting that high-speed transport occurs in association with CaVβ. Selective tracking of CaV1.2α1 along the postendocytic pathway failed to show the highly mobile state, implying CaVβ-independent retrograde transport. Retrograde speeds of CaV1.2α1 are compatible with myosin VI-mediated backward transport. Moreover, residency time at the cell surface was significantly prolonged when CaV1.2α1 was covalently linked to CaVβ. Thus, CaVβ promotes fast transport speed along anterograde trafficking and acts as a molecular switch controlling the endocytic turnover of L-type calcium channels

Ca V β controls the endocytic turnover of Ca V 1 .2 L‐type calcium channel

Membrane depolarization activates the multisubunit CaV1.2 L-type calcium channel initiating various excitation coupling responses. Intracellular trafficking into and out of the plasma membrane regulates the channel's surface expression and stability, and thus, the strength of CaV1.2-mediated Ca2+ signals. The mechanisms regulating the residency time of the channel at the cell membrane are unclear. Here, we coexpressed the channel core complex CaV1.2α1 pore-forming and auxiliary CaVβ subunits and analyzed their trafficking dynamics from single-particle-tracking trajectories. Speed histograms obtained for each subunit were best fitted to a sum of diffusive and directed motion terms. The same mean speed for the highest-mobility state underlying directed motion was found for all subunits. The frequency of this component increased by covalent linkage of CaVβ to CaV1.2α1 suggesting that high-speed transport occurs in association with CaVβ. Selective tracking of CaV1.2α1 along the postendocytic pathway failed to show the highly mobile state, implying CaVβ-independent retrograde transport. Retrograde speeds of CaV1.2α1 are compatible with myosin VI-mediated backward transport. Moreover, residency time at the cell surface was significantly prolonged when CaV1.2α1 was covalently linked to CaVβ. Thus, CaVβ promotes fast transport speed along anterograde trafficking and acts as a molecular switch controlling the endocytic turnover of L-type calcium channels

A short polybasic segment between two conserved domains of the ß(2a)-subunit modulates the rate of inactivation of R-type calcium channel

Besides opening and closing, high voltage-activated calcium channels transit to a nonconducting inactivated state from which they do not re-open unless the plasma membrane is repolarized. Inactivation is critical for temporal regulation of intracellular calcium signaling and prevention of a deleterious rise in calcium concentration. R-type high voltage-activated channels inactivate fully in a few hundred milliseconds when expressed alone. However, when co-expressed with a particular β-subunit isoform, β(2a), inactivation is partial and develops in several seconds. Palmitoylation of a unique di-cysteine motif at the N terminus anchors β(2a) to the plasma membrane. The current view is that membrane-anchored β(2a) immobilizes the channel inactivation machinery and confers slow inactivation phenotype. β-Subunits contain one Src homology 3 and one guanylate kinase domain, flanked by variable regions with unknown structures. Here, we identified a short polybasic segment at the boundary of the guanylate kinase domain that slows down channel inactivation without relocating a palmitoylation-deficient β(2a) to the plasma membrane. Substitution of the positively charged residues within this segment by alanine abolishes its slow inactivation-conferring phenotype. The linker upstream from the polybasic segment, but not the N- and C-terminal variable regions, masks the effect of this determinant. These results reveal a novel mechanism for inhibiting voltage-dependent inactivation of R-type calcium channels by the β(2a)-subunit that might involve electrostatic interactions with an unknown target on the channel's inactivation machinery or its modulatory components. They also suggest that intralinker interactions occlude the action of the polybasic segment and that its functional availability is regulated by the palmitoylated state of the β(2a)-subunit

La construction de territoires micro-régionaux : les pays comme institutions politiques en charge du développement local

National audienceLe travail de recherche pluridisciplinaire est consacré à la mise en place de périmètres de développement local en France depuis 1999 : les Pays. Il s'agit de structures décidées par les acteurs politiques locaux qui visent l'élaboration d'un projet de développement. Ces structures ont deux caractéristiques marquées dans le contexte institutionnel français : elles sont conçues à l'échelle micro-régionale, sur un mode volontaire par libre association des communes. Il en résulte, pour les Pays effectivement constitués, une grande diversité de taille et de structure. Le travail présenté dresse un état des lieux de cette diversité, en proposant une typologie de l'ensemble des Pays recensés en France. Il développe ensuite une double analyse, sociologique et économique, des territoires correspondants. L'analyse sociologique est centrée sur le rôle des élus dans la construction des Pays et sur la place qui est dévolue aux autres catégories d'acteurs ; l'analyse économique pose la question de la pertinence des périmètres ainsi délimités pour le développement régional