Gènes du cluster alc (voie d'utilisation de l'éthanol) et protéines membranaires de la famille GPR1/FUN34/YaaH chez le champignon filamenteux Aspergillus nidulans

L aptitude d Aspergillus nidulans à utiliser l éthanol comme seule source de carbone nécessite deux gènes de structure, alcA et aldA (codant les deux enzymes nécessaires à l oxydation de l éthanol en acétate via l acétaldéhyde), ainsi qu'un gène régulateur, alcR, codant l'activateur transcriptionnel spécifique de cette voie (système alc). L induction par AlcR nécessite la présence dans le milieu de culture d un co-inducteur tel que l éthanol. D autres gènes sont regroupés en cluster avec alcR et alcA et sont induits par des inducteurs du système alc, mais ne sont pas nécessaire à l'utilisation de l'éthanol : alcM, alcS, alcO et alcP. Nous avons d abord procédé à l'analyse fonctionnelle du gène alcS qui est le plus fortement exprimé. Ce gène est co-régulé avec alcA et code une protéine de la membrane plasmique qui appartient à une nouvelle famille de protéines membranaires appelée GPR1/FUN34/YaaH. Une analyse comparative du génome d A. nidulans a révélé l existence de deux autres gènes induits par des inducteurs connus du système alc et dont les protéines, AN5226 et AN8390, appartiennent à cette même famille. La caractérisation de ces gènes, nous a permis de montrer qu'AN5226 est essentiel au transport de l'acétate, ce que nous avons confirmé par des expériences de transport d'acétate marqué au 14C. Parallèlement, l analyse fonctionnelle d alcP a permis la mise en évidence de l'existence d'un second système de régulation, indépendant de celui du système alc, qui répond aux alcools, aux cétones et aux esters.The ethanol utilization pathway (alc system) of Aspergillus nidulans requires two structural genes, alcA and aldA, which encode the two enzymes allowing conversion of ethanol into acetate via acetaldehyde, and a regulatory gene, alcR, encoding the pathway-specific autoregulated transcriptional activator. The AlcR-specific induction requires the presence of a co-inducer such as ethanol or other primary alcohols. The alcR and alcA genes are closely linked to other genes that are transcriptionally induced by specific inducers of the alc system although they are dispensable for growth on ethanol: alcM, alcS, alcO and alcP. In this study, we first characterized alcS, the most abundantly transcribed of these genes. alcS is strictly co-regulated with alcA and encodes a plasma membrane protein which belongs to the novel GPR1/FUN34/YaaH membrane protein family. Blast analysis against the A. nidulans genome led to the identification of two novel ethanol- and ethylacetate-induced genes encoding other members of this family, AN5226 and AN8390. Functional characterization of the AN5226 gene, together with 14C-Acetate uptake experiments demonstrated an essential role for AN5226 in mediated acetate transport. Through the functional analysis of an other gene of the alc cluster, alcP, we demonstrated the existence of a second regulatory system responsive to alcohols, ketones and ester (ake), independant of AlcR.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

The loading of condensin in the context of chromatin.

International audienceThe packaging of DNA into chromosomes is a ubiquitous process that enables living organisms to structure and transmit their genome accurately through cell divisions. In the three kingdoms of life, the architecture and dynamics of chromosomes rely upon ring-shaped SMC (Structural Maintenance of Chromosomes) condensin complexes. To understand how condensin rings organize chromosomes, it is essential to decipher how they associate with chromatin filaments. Here, we use recent evidence to discuss the role played by nucleosomes and transcription factors in the loading of condensin at transcribed genes. We propose a model whereby cis-acting features nestled in the promoters of active genes synergistically attract condensin rings and promote their association with DNA

A genetic screen for functional partners of condensin in fission yeast

Mitotic chromosome condensation is a prerequisite for the accurate segregation of chromosomes during cell division, and the conserved condensin complex a central player of this process. However, how condensin binds chromatin and shapes mitotic chromosomes remain poorly understood. Recent genome-wide binding studies showing that in most species condensin is enriched near highly expressed genes suggest a conserved link between condensin occupancy and high transcription rates. To gain insight into the mechanisms of condensin binding and mitotic chromosome condensation, we searched for factors that collaborate with condensin through a synthetic lethal genetic screen in the fission yeast Schizosaccharomyces pombe. We isolated novel mutations affecting condensin, as well as mutations in four genes not previously implicated in mitotic chromosome condensation in fission yeast. These mutations cause chromosome segregation defects similar to those provoked by defects in condensation. We also identified a suppressor of the cut3-477 condensin mutation, which largely rescued chromosome segregation during anaphase. Remarkably, of the five genes identified in this study, four encode transcription co-factors. Our results therefore provide strong additional evidence for a functional connection between chromosome condensation and transcription

The Yeast Polo Kinase Cdc5 Regulates the Shape of the Mitotic Nucleus

Abnormal nuclear size and shape are hallmarks of aging and cancer. However, the mechanisms regulating nuclear morphology and nuclear envelope (NE) expansion are poorly understood. In metazoans, the NE disassembles prior to chromosome segregation and reassembles at the end of mitosis. In budding yeast, the NE remains intact. The nucleus elongates as chromosomes segregate and then divides at the end of mitosis to form two daughter nuclei without NE disassembly. The budding yeast nucleus also undergoes remodeling during a mitotic arrest; the NE continues to expand despite the pause in chromosome segregation, forming a nuclear extension, or "flare," that encompasses the nucleolus. The distinct nucleolar localization of the mitotic flare indicates that the NE is compartmentalized and that there is a mechanism by which NE expansion is confined to the region adjacent to the nucleolus. Here we show that mitotic flare formation is dependent on the yeast polo kinase Cdc5. This function of Cdc5 is independent of its known mitotic roles, including rDNA condensation. High-resolution imaging revealed that following Cdc5 inactivation, nuclei expand isometrically rather than forming a flare, indicating that Cdc5 is needed for NE compartmentalization. Even in an uninterrupted cell cycle, a small NE expansion occurs adjacent to the nucleolus prior to anaphase in a Cdc5-dependent manner. Our data provide the first evidence that polo kinase, a key regulator of mitosis, plays a role in regulating nuclear morphology and NE expansion

Nucleosome eviction in mitosis assists condensin loading and chromosome condensation

International audienc