Cortical Development and Brain Malformations: Insights From the Differential Regulation of Early Events of DNA Replication

During the development of the cortex distinct populations of Neural Stem Cells (NSCs) are defined by differences in their cell cycle duration, self-renewal capacity and transcriptional profile. A key difference across the distinct populations of NSCs is the length of G1 phase, where the licensing of the DNA replication origins takes place by the assembly of a pre-replicative complex. Licensing of DNA replication is a process that is adapted accordingly to the cell cycle length of NSCs to secure the timed duplication of the genome. Moreover, DNA replication should be efficiently coordinated with ongoing transcription for the prevention of conflicts that would impede the progression of both processes, compromising the normal course of development. In the present review we discuss how the differential regulation of the licensing and initiation of DNA replication in different cortical NSCs populations is integrated with the properties of these stem cells populations. Moreover, we examine the implication of the initial steps of DNA replication in the pathogenetic mechanisms of neurodevelopmental defects and Zika virus-related microcephaly, highlighting the significance of the differential regulation of DNA replication during brain development

Running title: Maximal loading of MCM2/4 in late G1

Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2-7 complex onto chromatin during G1 phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G1 phase. The immobile fraction of MCM2 and MCM4 increases during G1 phase, suggestive of reiterative licensing. In late G1 phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G1 phase progresses and do not colocalize with sites of DNA synthesis in S phase.Fil: Symeonidou, Ioanna Eleni. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Kotsantis, Panagiotis. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Roukos, Vassilis. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Rapsomaniki, Maria Anna. University of Patras. School of Medicine. Laboratory of General Biology; Grecia;Fil: Grecco, Hernan Edgardo. Max Planck Institute of Molecular Physiology. Department of Systemic Cell Biology; Alemania; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires; Argentina;Fil: Bastiaens, Philippe. Max Planck Institute of Molecular Physiology. Department of Systemic Cell Biology; Alemania;Fil: Taraviras, Stavros. University of Patras. School of Medicine. Laboratory of Physiology; Grecia;Fil: Lygerou, Zoi. University of Patras. School of Medicine. Laboratory of General Biology; Grecia

Evaluation of molecular diversity of databases of molecules having pharmaceutical interest, using chemical graph theory

La compétition entre les grands groupes pharmaceutiques dans la recherche de nouvelles substances actives a favorisé le développement de nouvelles techniques de synthèses (chimie combinatoire et synthèse parallèle) et de méthodes de tests rapides des molécules (High-throughput screening ou HTS). Contrairement aux méthodes classiques, ces techniques ont pour caractéristique commune d'opérer sur de très grands nombres de molécules. Malgré leur puissance, il est vite apparu nécessaire de sélectionner des sous-ensembles représentatifs de l'énorme éventail de molécules potentiellement actives. Cette exigence est à la base du concept de diversité moléculaire. Les structures chimiques sont usuellement caractérisées par des descripteurs moléculaires qui appartiennent à plusieurs classes distinctes. Les descripteurs topologiques, qui sont au nombre de plusieurs centaines, apparaissent bien adaptés pour aborder ce problème. Le but de ce travail est de proposer des méthodes pour choisir des ensembles optimaux de descripteurs, afin de bien échantillonner la diversité de l'espace chimique.NICE-BU Sciences (060882101) / SudocSudocFranceF

DNA replication control: Liquid-liquid phase separation comes into play

Liquid-liquid phase separation (LLPS) has been recently suggested as a new potential mechanism underpinning various organizational aspects of the cell, from the formation of sub-cellular, biomolecule enrichments to the assembly of organelles. In eukaryotes, DNA replication follows a strict temporal and spatial program that is majorly affected by the chromatin structure, the nuclear organization and the availability of limiting initiation factors, however the regulatory mechanisms driving the process have not been fully elucidated. Original data published lately revealed for the first time that the components of the pre-replicative complex, ORC, Cdc6 and Cdt1, are able to phase separate indicating a possible connection between LLPS and DNA replication. Here, we critically present these preliminary data and propose mechanistic models that could support this regulatory link and lead to new future research directions

Visualizing the dynamics of histone variants in the S-phase nucleus

Abstract Histone variants constitute a fundamental feature of the epigenome. However, their dynamics during normal and challenged DNA replication and their distribution in the three-dimensional space of the nucleus remain poorly characterized. A recent study employed stochastic optical reconstruction microscopy (STORM) to obtain a high-resolution view of the spatial distribution of H3 histone variants in the nucleus and related this to the timing of DNA replication

Advanced Gene-Targeting Therapies for Motor Neuron Diseases and Muscular Dystrophies

Gene therapy is a revolutionary, cutting-edge approach to permanently ameliorate or amend many neuromuscular diseases by targeting their genetic origins. Motor neuron diseases and muscular dystrophies, whose genetic causes are well known, are the frontiers of this research revolution. Several genetic treatments, with diverse mechanisms of action and delivery methods, have been approved during the past decade and have demonstrated remarkable results. However, despite the high number of genetic treatments studied preclinically, those that have been advanced to clinical trials are significantly fewer. The most clinically advanced treatments include adeno-associated virus gene replacement therapy, antisense oligonucleotides, and RNA interference. This review provides a comprehensive overview of the advanced gene therapies for motor neuron diseases (i.e., amyotrophic lateral sclerosis and spinal muscular atrophy) and muscular dystrophies (i.e., Duchenne muscular dystrophy, limb-girdle muscular dystrophy, and myotonic dystrophy) tested in clinical trials. Emphasis has been placed on those methods that are a few steps away from their authoritative approval

Additional file 5: of Geminin prevents DNA damage in vagal neural crest cells to ensure normal enteric neurogenesis

Figure S4. Efficient ablation of the Gem locus when combined with the inducible Sox10iCreER T2 line. Whole-mount gut preparations of control (A) and Sox10CreER(i8.5)|Gem (B) E12.5 embryos, immunostained for GFP to visualise the distribution of ENCCs within the gut. Red arrows indicate the position of the most caudally located ENCCs in the gut preparations. (C–D), Relative quantitation of Gem transcript levels in the FACS-purified ENCCs of Sox10CreER(i8.5)|Gem and Sox10CreER(i10)|Gem embryos normalised to the levels of b-actin. Unpaired t-test with Welch’s correction, **P value < 0.01, ***P value < 0.001. Scale bar: (A, B) 400 μm. (TIF 1130 kb

Whole transcriptome data analysis of mouse embryonic hematopoietic stem and progenitor cells that lack Geminin expression

We performed cDNA microarrays (Affymetrix Mouse Gene 1.0 ST Chip) to analyze the transcriptome of hematopoietic stem and progenitor cells (HSPCs) from E15.5dpc wild type and Geminin (Gmnn) knockout embryos. Lineage negative cells from embryonic livers were isolated using fluorescence activated cell sorting. RNA samples were used to examine the transcriptional programs regulated by Geminin during embryonic hematopoiesis. The data sets were analyzed using the GeneSpring v12.5 platform (Agilent). The list of differentially expressed genes was filtered in meta-analyses to investigate the molecular basis of the phenotype observed in the knockout embryos, which exhibited defective hematopoiesis and death. The data from this study are related to the research article “Geminin deletion increases the number of fetal hematopoietic stem cells by affecting the expression of key transcription factors” (Karamitros et al., 2015) [1].The microarray dataset has been deposited at the Gene Expression Omnibus (GEO) under accession GEO: GSE53056