Regulating retrotransposon activity through the use of alternative transcription start sites

International audienceRetrotransposons, the ancestors of retroviruses, have the potential for gene disruption and genomic takeover if not kept in check. Paradoxically, although host cells repress these elements by multiple mechanisms, they are transcribed and are even activated under stress conditions. Here, we describe a new mechanism of retrotransposon regulation through transcription start site (TSS) selection by altered nucleosome occupancy. We show that Fun30 chromatin remodelers cooperate to maintain a high level of nucleosome occupancy at retrotransposon-flanking long terminal repeat (LTR) elements. This enforces the use of a downstream TSS and the production of a truncated RNA incapable of reverse transcription and retrotransposition. However, in stressed cells, nucleosome occupancy at LTR elements is reduced, and the TSS shifts to allow for productive transcription. We propose that controlled retrotransposon transcription from a nonproductive TSS allows for rapid stress-induced activation, while preventing uncontrolled transposon activity in the genome

Checkpoint-Dependent and -Independent Roles of Swi3 in Replication Fork Recovery and Sister Chromatid Cohesion in Fission Yeast

Multiple genome maintenance processes are coordinated at the replication fork to preserve genomic integrity. How eukaryotic cells accomplish such a coordination is unknown. Swi1 and Swi3 form the replication fork protection complex and are involved in various processes including stabilization of replication forks, activation of the Cds1 checkpoint kinase and establishment of sister chromatid cohesion in fission yeast. However, the mechanisms by which the Swi1–Swi3 complex achieves and coordinates these tasks are not well understood. Here, we describe the identification of separation-of-function mutants of Swi3, aimed at dissecting the molecular pathways that require Swi1–Swi3. Unlike swi3 deletion mutants, the separation-of-function mutants were not sensitive to agents that stall replication forks. However, they were highly sensitive to camptothecin that induces replication fork breakage. In addition, these mutants were defective in replication fork regeneration and sister chromatid cohesion. Interestingly, unlike swi3-deleted cell, the separation-of-functions mutants were proficient in the activation of the replication checkpoint, but their fork regeneration defects were more severe than those of checkpoint mutants including cds1Δ, chk1Δ and rad3Δ. These results suggest that, while Swi3 mediates full activation of the replication checkpoint in response to stalled replication forks, Swi3 activates a checkpoint-independent pathway to facilitate recovery of collapsed replication forks and the establishment of sister chromatid cohesion. Thus, our separation-of-function alleles provide new insight into understanding the multiple roles of Swi1-Swi3 in fork protection during DNA replication, and into understanding how replication forks are maintained in response to different genotoxic agents

Etude de la regulation de la transcription chez la levure Saccharomyces cerevisiae : exemple du gene de l'iso-1-cytochrome c

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Etude de l'empreinte chromosomique au locus sexuel mat1 chez la levure Schizosaccharomyces pombe

L'empreinte chromosomique cartographiée au locus mat1contrôle le changement de type sexuel chez la levure Schizosaccharomyces pombe. La nature moléculaire de l'empreinte est sujet à polémique: cassure simple brin (CSB) de l'ADN ou modification de l'ADN sensible à la soude qui pourrait être un ARN. Nous avons ligaturé l'empreinte avec la ligase d'E. coli, reformant ainsi la séquence d'ADN parentale, suggérant fortement que l'empreinte est une CSB sans perte de nucléotide. La ligature complète nécessite un traitement par la polynucléotide kinase. Nous avons montré que les extrémités de la CSB sont 3'-OH et 5'-OH. La CSB est indépendante de la séquence, mais spécifique du site. Plusieurs éléments de séquence, nécessaires à la formation ou à la stabilité de la CSB ont été identifiés. Un des éléments est essentiel pour la pause de la fourche de réplication à proximité de mat1 et interagit in vivo avec Swi1p. Un autre élément est essentiel pour maintenir la CSB lors de la progression dans le cycle cellulaire. Nous avons montré que l'ADN polymérase du brin continu synthétise l'ADN jusqu'à l'extrémité de la CSB, formant un intermédiaire bout franc transitoire, capable d'initier le changement de type sexuel. Afin d'obtenir un système inductible pour le changement de type sexuel nous avons introduit un promoteur répressible par la thiamine à proximité de mat1. En utilisant ce système nous avons obtenu une population homogène de cellules sans CSB présentant un type sexuel stable. Nous avons montré que la CSB est formée pendant la première phase S, lorsque les intermédiaires de réplication apparaissent à mat1. La pause de la fourche de réplication à mat1 est simultanée à la formation de la CSB. Swi1p se fixe fortement sur le site de pause pendant la réplication. Les intermédiaires de conversion génique apparaissent une génération après la formation de la CSB. Ce système a permis de disséquer moléculairement les différentes étapes du changement de type sexuel chez S.pombe.A strand-specific imprint located at the mating-type (mat1) locus, controls mating-type switching in the yeast Schizosaccharomyces pombe. However, the molecular nature of the imprint had remained unclear and it was proposed to be a strand-specific DNA break (SSB) or a strand-specific, alkali labile DNA modification due to an RNA left behind after incomplete Okazaki fragment processing. In the present study, we showed that the imprint could be ligated by the E. coli DNA ligase to reform the parental DNA sequence, demonstrating that the imprint is a single stranded break (SSB), without a missing nucleotide. However, full ligation required the T4 polynucleotide kinase. We showed that the ends of the SSB are 3'-OH and 5'-OH. Mutation analysis of the SSB showed that the break is site but not sequence specific. Several new elements, required for SSB formation and stability, were identified. One of these elements was essential for a replication fort pause next to mat1 and interacted in vivo with the Swi1p. A second element was essential for maintaining the SSB during the cell cycle progression. We showed, that the leading strand DNA polymerase synthesized DNA to the edge of the SSB and formed a transitory blunt ended recombination intermediate, able to initiate mating type switching. We designed an inducible system for regulating mating-type switching by introducing a thiamine repressible promoter upstream of the mat1 locus. We were able to isolate homogenous population of cells that have not yet suffered a SSB or switched their mating type. We showed that the SSB was formed during the first S phase, when replication intermediates appeared at mat1. The replication fork pause and the SSB occurred simultaneously. During replication Swi1p bound strongly to the replication pause site. Intermediates of gene conversion were detected one generation after the SSB was formed. Thus, we have been able to dissect in molecular terms the genetic pedigree of the fission yeast.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Fission yeast cells switch mating-type in a directional manner, by gene conversions of the <i>mat1</i> locus.

<p>A recombinogenic DNA end is formed at <i>mat1</i> during DNA replication (black arrow) and the broken DNA invades a donor whose genetic information is copied into <i>mat1</i>. Wild-type M cells (<i>mat1-M</i> allele) use the <i>mat2-P</i> donor, while P cells (<i>mat1-P</i> allele) use the <i>mat3-M</i> donor, as depicted in the top drawing. The recombination enhancers SRE2 and SRE3 are central to these choices. Experiments in which donors and enhancers are swapped, alone or in combination, show that SRE2 and SRE3 are recognized in a cell-type specific manner to promote use of their adjacent donor. The heterochromatic structure of the <i>mat2-P–mat3-M</i> region is required for this differential recognition.</p

The Genetics of Quiescence

Maste

Rôle de SUMO dans l'(in)stabilité génétique chez S. pombe

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Etude la recombinaison au cours de la réplication du locus mat 1 de S. pombe

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF