A major program of evolutionary and comparative genomics of yeasts has been in progress in my laboratory for many years (see publications). In the next few months (before summer 2015) I need to finish a few comparisons about a new clade to publish as soon as possible.

In wild life, yeast cells are able to survive in severe conditions, without nutriment for a long period of time because the cell is able to enter in stationary phase. During this phase, the cell can transform varied sources of energy and pause its growth to preserve the cells from death. It is known that most of genes are downregulated in stationary phase and the cell activity is globally reduced to its strict minimum, while a subset of “specialized” genes is induced to promote survival in extreme conditions. We are analysing the transcriptome of exponentially grown or stationary phase yeasts, investigating different level of regulation.

Meiosis is the specialized, highly regulated process at the basis of the sexual reproduction of eukaryotes. During this process, a diploid cell undergoes a single round of DNA replication and two successive rounds of chromosome segregation, halving the chromosome set to generate four haploid products (gametes). Homologous (i.e. paternal and maternal) chromosomes during the prophase of the first division of meiosis undergo a highly regulated succession of events that include recognition, pairing, and synapsis along their length. An important aspect of chromosome organization, besides pairing and synapsis, consist in the condensation step: axial elements form between sister chromatids, bridging distant axis sites comprising cohesins, and resulting in the extrusion of chromatin loops away from the axis. The precise organization of these chromatin loops remain unclear, and is notably impaired by the sequence similarities of the two homologs. Our aim is to characterize the fine organization of chromatin on both homologs during meiotic prophase, and how this organization is functionally related to a recombination event.

Genomic DNA is hierarchically packed within the living cells and genome duplication requires the concerted effort of many thousands of individual replication units. As such, to ensure the integrity of transmission of the genetic information, both eukaryotes and prokaryotes have evolved sophisticated mechanisms to monitor DNA replication. Some of these mechanisms aim to maintain both a temporal and a spatial organization of the replication program, leading to multiple replication time regions and the compartmentalization into replication foci, subnuclear sites which accumulate numerous DNA replication factors. It should be noted that Saccharomyces cerevisiae represents an exception to the standard eukaryotic strategy for genome duplication. Similar to bacteria, S. cerevisiae possess well-defined replication origin sequences that can fire at a very efficient rate during S phase, leading to a very homogenous pattern of DNA replication. A common mo del suggests that, once replication starts dynamic events take place since co-regulated replication forks, having similar replication timing, cluster within a discrete number of foci that show distinct patterns of nuclear localization over the S-phase. Once initiated, the DNA synthesis might be compromised if the replication fork encounters an RFB (Replication Fork Barrier) such as DNA lesions, tightly bound protein-DNA complexes etc. The RFBs are considered a potential source of genetic instability and may lead to many chromosomal rearrangements. As a consequence, eukaryotes employ a complex DNA damage response against RFBs, which aims to maintain the stability of the stalled forks and provides the time required to repair and resume replication. Recent observations suggest that the non-random organization of the nucleus affects where repair occurs. The aim of this project is to reach a better understanding of the influence of the nuclear spatial architecture and organization at replication fork blocks.

Gene amplification has been observed in different organisms in response to environmental constraints, such as limited nutrients or exposure to a variety of toxic compounds, conferring them specific phenotypic adaptations by increasing expression levels. But the presence of multiple gene copies has generally not been found in natural genomes in absence of specific functional selection. Here we show that the massive amplification of a chromosomal locus (up to 880 copies per cell) occurs in absence of any direct selection, associated with low-order amplifications of flanking segments in complex chromosomal alterations. These results were obtained in the mutants with restored phenotypes that spontaneously appear from genetically engineered strains of the yeast Saccharomyces cerevisiae with severe fitness reduction. Grossly extended chromosomes (macrotene) were formed, with complex structural alterations but sufficient stability to propagate unchanged over successive generations. Their detailed molecular analysis, including complete genome sequencing, identification of sequence breakpoints and comparisons between mutants reveals novel mechanisms to their formation whose combined action underlies the astonishing dynamics of eukaryotic chromosomes and its consequences.

Project context and summary : Cryptococcus neoformans is a sugar-coated yeast that is able to interact closely with numerous organisms in the environment including amebae, paramecium of nematodes. The interaction with these organisms probably shaped its virulence. The ability to survive nutrient starvation, oxidative stress, desiccation, both in the environment and in humans, indicates a high level of physiological and metabolic plasticity of the yeast. In humans, after primary infection during childhood, the yeast is able to survive within the host for years before reactivation, leading to a deadly disseminated fungal infection. This phenomenon, called dormancy / quiescence is one of the main biological features of this fungus in relation with disease pathogenesis. It is known in bacteria, parasites and other fungi. There is no consensus on the definition of dormancy. Most often, dormant cells are characterized by a low metabolic activity sometimes undetectable under normal laboratory conditions and the ability to be resuscitated by adequate stimuli. In C. neoformans, dormancy has only been demonstrated epidemiologically in our laboratory but not experimentally so far. We developed an assay where yeasts cells exhibiting characteristics of potentially dormant cells were generated. Our current project aims at exploring the conditions leading to, the biology of the entry in and the mechanisms sustaining dormancy.

Our aim is to characterize the fine organization of chromatin on homologs during meiotic prophase, and how this organization is functionally related to recombination.

We are interested in the cytoplasmic quality control of gene expression and more especially into the behavior of aberrant peptides which could be generated from non-conform translation events. We are now investigating the role of a Saccharomyces cerevisiae RNA helicase protein that we named Tac4 (for Translation associated Component 4). We showed that this protein is involved in translation. We demonstrated, by sucrose gradient and affinity purification that Tac4 interacts with the ribosome. A first UV cross-linking and cDNA analysis (CRAC) experiment clearly revealed that Tac4 interacts with the 18S rRNA of the 40S ribosomal subunit and we precisely defined the crosslink point. These preliminary results also suggested an enrichment of the 3’-end regions of mRNAs. This implies that Tac4 could not only interact with the small ribosomal subunit but also directly with mRNA. Tac4 is conserved through the evolution and its mammalian homologue is involved in initiation of translation. Therefore, we thought that Tac4 could be associated with the 5’-end rather than with the 3’-end. However, a recent paper from the Rachel Green’s lab showed that translation reinitiation into the 3’-UTR region may occurs when translation termination is affected (Young et al., Cell 2015). The factors and molecular mechanisms implicated in these events are not known. Altogether, our preliminary results suggest that Tac4 is an excellent candidate participating to the unwinding of RNA structure or to the release of some RNA-binding proteins into the 3’-end mRNA. We now would like to 1) confirm that Tac4 preferentially interacts with the 3’-end of mRNA, 2) determine whether Tac4 interacts with a region upstream the Stop codon or in the 3’-UTR of the mRNA, 3) identify the mRNA targets to determine whether Tac4 could have a general role in translation or could only be involved in translation of some specific mRNA.

We are interested in the cytoplasmic quality control of gene expression and more especially into the behavior of aberrant peptides which could be generated from non-conform translation events. We are now investigating the role of a Saccharomyces cerevisiae RNA helicase protein that we named Tac4 (for Translation Associated Component 4). We showed that this protein is involved in translation. We demonstrated, by sucrose gradient and affinity purification that Tac4 interacts with the ribosome. A first UV cross-linking and cDNA analysis (CRAC) experiment clearly revealed that Tac4 interacts with the 18S rRNA of the 40S ribosomal subunit and we precisely defined the crosslink point. These preliminary results also suggested an enrichment of the 3’-end regions of mRNAs. This implies that Tac4 could not only interact with the small ribosomal subunit but also directly with mRNA. Tac4 is conserved through the evolution and its mammalian homologue is involved in initiation of translation. Therefore, we thought that Tac4 could be associated with the 5’-end rather than with the 3’-end. However, recent data showed that translation reinitiation into the 3’-UTR region may occurs when translation termination is affected. The factors and molecular mechanisms implicated in these events are not known. Altogether, our preliminary results suggest that Tac4 is an excellent candidate participating to the unwinding of RNA structure or to the release of some RNA-binding proteins into the 3’-end mRNA. We now would like to 1) confirm that Tac4 preferentially interacts with the 3’-end of mRNA, 2) determine whether Tac4 interacts with a region upstream the Stop codon or in the 3’-UTR of the mRNA, 3) determine whether Tac4 could also interact with other mRNA region, such as the 5'-UTR region, 4) identify the mRNA targets to determine whether Tac4 could have a general role in translation or could only be involved in tra

Analysing the transcriptome of exponentially grown or stationary phase yeasts in a genetic background that stabilises pervasive transcipts, we identified a first subset of ≈ 140 antisense transcripts anti-correlated with gene transcripts that are specifically expressed in quiescence. We are further investigating whether these genes are subject to a transcriptional interference and what are the mechanisms underlying this regulation. More in detail, we would like to analyse the loci where an antisense ncRNA are detected.

Many neurodegenerative disorders are caused by the large expansion of a repeated sequence, called a "trinucleotide repeat". Our laboratory is using the CRISPR-Cas family of endonucleases in order to shorten the repeat tract below the length that is known to be pathological in humans. Some of the nucleases tested are very efficient at cutting the repeat tract, but in order to make this approach a viable gene therapy strategy, we must ensure that the nuclease is not inducing mutations in other parts of the genome (so-called "off-targets"). The present project aims at analyzing all possible off-targets of these nucleases on the different repeated sequences involved in neurodegenerative disorders, in order to validate (or invalidate) this approach.