Heterochromatin and cohesion protection at human centromeres: the final say of a long controversy?

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Assignment of linkage groups to the electrophoretically-separated chromosomes of the fungus Podospora anserina

International audienceAn electrophoretic karyotype of the filamentous fungus Podospora anserina has been obtained using contour-clamped homogeneous electric field gel electrophoresis. Six chromosomal bands were separated with one migrating as a doublet. The size of the chromosomes was estimated to be between 3.8 and 6.0 megabase pairs (mb) using the chromosomes of Schizosaccharomyces pombe as size standards, giving a total genome size of about 34 mb for the P. anserina genome. Homologous probes were used to assign five of the seven linkage groups (LGs) to chromosomal bands on the gel. Analysis of reciprocal translocation strains allowed us to complete the karyotype. In decreasing size order, the P. anserina chromosomes are LG I (6.0 mb); LG II (5.5 mb); LG V (5.1 mb); LG III (4.9 mb); LGs VI and VII (4.3 mb) and LG IV (3.8 mb)

Etude des elements chromosomiques chez le champignon filamenteux Podospora anserina

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 83201 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Régulation dynamique de l'association des cohésines aux chromosomes, établissement et maintien de la cohésion des chromatides sœurs

Le complexe cohésine maintient associées les chromatides sœurs depuis la réplication jusqu à leur ségrégation en mitose. Une question majeure est de comprendre comment la cohésion est établie lors de la phase S. Chez les mammifères et S. pombe, les cohésines sont associées de manière labile aux chromosomes pré-réplicatifs et l établissement de la cohésion en phase S s accompagne de la stabilisation de l association des cohésines aux chromosomes. L objectif de ce travail est de comprendre comment la dynamique des cohésines est régulée et comment son inhibition créée la cohésion.En G1 les cohésines associées aux chromosomes s échangent avec le pool soluble et leur dissociation dépend de Pds5 et Wapl. La première partie de ce travail présente les résultats d un crible génétique visant à identifier de nouveaux régulateurs de la dynamique des cohésines.L établissement de la cohésion nécessite l acétyltransférase Eso1 mais pas en contexte wpl1, indiquant que la seule mais essentielle fonction d Eso1 est de s opposer à celle de Wapl. L acétylation de Smc3 par Eso1 contribue mais n est pas suffisante pour contrecarrer Wapl, suggérant l existence d un autre événement dépendant d Eso1. En G1, Pds5 agit avec Wapl pour dissocier les cohésines des chromosomes mais après la phase S, Pds5 est requise pour leur maintien sur les chromosomes et pour la cohésion à long terme. Pds5 co-localise avec la fraction stable de cohésines mais pas Wapl. Nous suggérons un modèle dans lequel la cohésion est créée par deux événements d acétylation couplés à la progression de la fourche de réplication conduisant à l éviction de Wapl des cohésines destinées à produire la cohésion.Following DNA replication, sister chromatids are connected by cohesin to ensure their correct segregation during mitosis. How cohesion is created is still enigmatic. The cohesin subunit Smc3 becomes acetylated by ECO1, a conserved acetyl-transferase, and this change is required for cohesion. As in mammals, fission yeast cohesin is not stably bound to G1 chromosomes but a fraction becomes stable when cohesion is made. The aim of this work was to understand how cohesin dynamics is regulated and how the change in cohesin dynamics creates cohesion.In G1 chromatin bound cohesin exchange with the soluble pool and the unloading reaction relies in part on Wapl. The first part of this study reports on the identification of G1/S factors as new candidate regulators of cohesin dynamics.Following S phase a stable cohesin fraction is made. The acetyl-transferase Eso1 is not required for this reaction when the wpl1 gene is deleted. Yet, it is in wild-type cells, showing that the sole but essential Eso1 function is counteracting Wapl. Eso1 acetylates the cohesin sub-unit Smc3. This renders cohesin less sensitive to Wapl but does not confer the stable binding mode, suggesting the existence of a second Eso1-dependent event. The cohesin sub-unit Pds5 act together with Wapl to promote cohesin removal from G1 chromosomes but after S phase Pds5 is essential for cohesin retention on chromosomes and long term cohesion. Pds5 co-localizes with the stable cohesin fraction whereas Wapl does not. We suggest a model in which cohesion establishment is made by two acetylation events coupled to fork progression leading to Wapl eviction while keeping Pds5 on cohesin complexes intended to make cohesion.BORDEAUX2-Bib. électronique (335229905) / SudocSudocFranceF

Rôle des protéines Ssl3 et Ssl38 dans la transmission des chromosomes chez Schizosaccharomyces Pompe

La transmission fidèle des chromosomes lors des divisions mitotiques est essentielle à la stabilité du génome. En début de mitose, les chromosomes préalablement dupliqués en phase S condensent sous la forme de deux chromatides soeurs associées et les centromètres capturent les microtubulures du fuseau. Lors de la transition métaphase-anaphase, la cohésion des chromatides soeurs est éliminée, déclenchant leur migration vers les pôles opposés du fuseau. La cohésion est assurée par le complexe cohésine, chargé sur les chromosomes en phase G1 du cycle cellulaire et maintenu jusqu'en anaphase. Les mécanismes par lesquels les cohésines s'associent aux chromosomes et assurent la cohésion sont mal compris. Les centromères sont généralement constitués d'hétérochromatine, une forme compacte de la chromatine, dont l'assemblage fait intervenir la machinerie de RNAi. Des mutations affectant certains composants structuraux de l'hétérochromatine, comme Swi6/HP1 chez Saccharomyces pombe, altèrent la ségrégation des chromosomes en mitose. Afin d'appréhender les rôles de Swi6, ses partenaires fonctionnels ont été recherchés par un crible génétique. Ici, je présente l'étude des gènes ss13 et ss138 (swi6 synthétique létal). Le gène ss13 code un facteur de chargement des cohésines en phase G1 du cycle cellulaire, dont la fonction est conservée au cours de l'évolution. Etonnament, ss138 code un composant du spliceosome. Les résultats présentés montrent que Ss138 est nécessaire à l'intégrité de l'hétérochromatine centromérique. La signification biologique du lien entre Ss138, la maturation des ARNs, et l'assemblage de l'hétérochromatine centrométrique est discutée.In all eukaryotes, the genome stability relies on accurate chromosome segregation throughout mitotic divisions. During early mitosis, each duplicated chromosomes condense into a pair of tightly linked sister chromatids and centromeres capture spindle microtubules. At the metaphase to anaphase transition, cohesion between sister chromatids is removed, triggering their migration towards the opposite spindle poles. Sister chromatid cohesion is ensured by cohesin, a proteinaceous complex loaded onto chromosomes in G1 and maitained chromosomally bound until anaphase onset. The mechanisms through which cohesin is loaded onto chromosomes and ensures cohesion are ill defined.In most eukaryotes, centromeres are made of heterochromatin : a specialized form of chromatin whose assembly and maintenance rely on the RNAi pathway. Mutations affecting structural components of heterochromatin, such as Swi6/HP1 in fission yeast, impair chromosome segregation. In order to investigate the biological functions of Swi6, its functional partners were sought through a genetic screen. Here I report on the study of ss13 and ss138 (swi6 synthetic lethal). The ss13 gene encodes a cohesin loading factor, whose function in G1 in evolutionarily conserved. Unexpectedly, ss138 encodes a spliceosome component. Experimental data indicate that ss138 is essential for centrometric heterochromatin integrity and accurate chromosome segregation. The biological significance of a link between Ss138, RNA modifications and the assembly of centrometric heterochromatin is discussed.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

A second Wpl1 anti-cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP4

Cohesin mediates sister chromatid cohesion which is essential for chromosome segregation and repair. Sister chromatid cohesion requires an acetyl-transferase (Eso1 in fission yeast) counteracting Wpl1, promoting cohesin release from DNA. We report here that Wpl1 anti-cohesion function includes an additional mechanism. A genetic screen uncovered that Protein Phosphatase 4 (PP4) mutants allowed cell survival in the complete absence of Eso1. PP4 co-immunoprecipitated Wpl1 and cohesin and Wpl1 triggered Rad21 de-phosphorylation in a PP4-dependent manner. Relevant residues were identified and mapped within the central domain of Rad21. Phospho-mimicking alleles dampened Wpl1 anti-cohesion activity, while alanine mutants were neutral indicating that Rad21 phosphorylation would shelter cohesin from Wpl1 unless erased by PP4. Experiments in post-replicative cells lacking Eso1 revealed two cohesin populations. Type 1 was released from DNA by Wpl1 in a PP4-independent manner. Type 2 cohesin, however, remained DNA-bound and lost its cohesiveness in a manner depending on Wpl1-and PP4-mediated Rad21 de-phosphorylation. These results reveal that Wpl1 antagonizes sister chromatid cohesion by a novel pathway regulated by the phosphorylation status of the cohesin kleisin subunit

Position effect variegation at fission yeast centromeres

International audienceChromatin structure at Schizosaccharomyces pombe centromeres is unusual. The insertion of the ura4 gene within these centromeres resulted in genetically identical cells mosaic for its expression. Placement of the ade6 gene within cen1 or cen3 resulted in red-white sectored colonies, demonstrating the instability of gene expression. The occurrence of pink colonies implied that intermediate levels of repression were established. Repression of both genes within centromeres was temperature sensitive. The chromatin structure of the ura4 gene at centromeres was altered, suggesting that the unusual chromatin encroaches into the gene and inhibits normal expression. These repressive effects at S. pombe centromeres resemble the classical phenomenon of position effect variegation imposed by Drosophila heterochromatin on nearby genes. However, since the epigenetic states can be set at intermediate levels of expression, a purely euchromatin-heterochromatin dichotomy does not apply. A model for the epigenetic regulation of genes placed within S. pombe centromeres is presented

Kinetochore Targeting of Fission Yeast Mad and Bub Proteins Is Essential for Spindle Checkpoint Function but Not for All Chromosome Segregation Roles of Bub1p

Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a “wait anaphase” signal in cases of incomplete or improper kinetochore-microtubule attachment. Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores. We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochore-microtubule interactions. Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization. We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response. Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation

Splicing Factor Spf30 Assists Exosome-Mediated Gene Silencing in Fission Yeast▿

Heterochromatin assembly in fission yeast relies on the processing of cognate noncoding RNAs by both the RNA interference and the exosome degradation pathways. Recent evidence indicates that splicing factors facilitate the cotranscriptional processing of centromeric transcripts into small interfering RNAs (siRNAs). In contrast, how the exosome contributes to heterochromatin assembly and whether it also relies upon splicing factors were unknown. We provide here evidence that fission yeast Spf30 is a splicing factor involved in the exosome pathway of heterochromatin silencing. Spf30 and Dis3, the main exosome RNase, colocalize at centromeric heterochromatin and euchromatic genes. At the centromeres, Dis3 helps recruiting Spf30, whose deficiency phenocopies the dis3-54 mutant: heterochromatin is impaired, as evidenced by reduced silencing and the accumulation of polyadenylated centromeric transcripts, but the production of siRNAs appears to be unaffected. Consistent with a direct role, Spf30 binds centromeric transcripts and locates at the centromeres in an RNA-dependent manner. We propose that Spf30, bound to nascent centromeric transcripts, perhaps with other splicing factors, assists their processing by the exosome. Splicing factor intercession may thus be a common feature of gene silencing pathways