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International audience; Eukaryotic DNA replication is regulated by conserved mechanisms that bring about a spatial and temporal organization in which distinct genomic domains are copied at characteristic times during S phase. Although this replication program has been closely linked with genome architecture, we still do not understand key aspects of how chromosomal context modulates the activity of replication origins. To address this question, we have exploited models that combine engineered genomic rearrangements with the unique replication programs of post-quiescence and pre-meiotic S phases. Our results demonstrate that large-scale inversions surprisingly do not affect cell proliferation and meiotic progression, despite inducing a restructuring of replication domains on each rearranged chromosome. Remarkably, these alterations in the organization of DNA replication are entirely due to changes in the positions of existing origins along the chromosome, as their efficiencies remain virtually unaffected genome wide. However, we identified striking alterations in origin firing proximal to the fusion points of each inversion, suggesting that the immediate chromosomal neighborhood of an origin is a crucial determinant of its activity. Interestingly, the impact of genome reorganization on replication initiation is highly comparable in the post-quiescent and pre-meiotic S phases, despite the differences in DNA metabolism in these two physiological states. Our findings therefore shed new light on how origin selection and the replication program are governed by chromosomal architecture.

International audience; The generation of a complete and accurate copy of the genetic material during each cell cycle is integral to cell growth and proliferation. However, genetic diversity is essential for adaptation and evolution, and the process of DNA replication is a fundamental source of mutations. Genome alterations do not accumulate randomly, with variations in the types and frequencies of mutations that arise in different genomic regions. Intriguingly, recent studies revealed a striking link between the mutational landscape of a genome and the spatial and temporal organization of DNA replication, referred to as the replication program. In our review, we discuss how this program may contribute to shaping the profile and spectrum of genetic alterations, with implications for genome dynamics and organismal evolution in natural and pathological contexts.

International audience; Progression through the cell cycle is driven by the activities of the cyclin-dependent kinase (CDK) family of enzymes, which establish an ordered passage through the cell cycle phases. CDK activity is crucial for the cellular transitions from G1 to S and G2 to M, which are highly controlled to promote the faithful duplication of the genetic material and the transmission of the genome into daughter cells, respectively. While oscillations in CDK activity are essential for cell division, how its specific dynamics may shape cellular processes remains an open question. Recently, we have investigated the potential role of CDK in establishing the profile of replication initiation along the chromosomes, also referred to as the replication program. Our results demonstrated that the timing and level of CDK activity at G1/S provide two critical and independent inputs that modulate the pattern of origin usage. In this review, we will present the conclusions of our study and discuss the implications of our findings for cellular function and physiology.

International audience; The spatial and temporal organization of genome duplication, also referred to as the replication program, is defined by the distribution and the activities of the sites of replication initiation across the genome. Alterations to the replication profile are associated with cell fate changes during development and in pathologies, but the importance of undergoing S phase with distinct and specific programs remains largely unexplored. We have recently addressed this question, focusing on the interplay between the replication program and genome maintenance. In particular, we demonstrated that when cells encounter challenges to DNA synthesis, the organization of DNA replication drives the response to replication stress that is mediated by the ATR/Rad3 checkpoint pathway, thus shaping the pattern of genome instability along the chromosomes. In this review, we present the major findings of our study and discuss how they may bring new perspectives to our understanding of the biological importance of the replication program.

International audience; Cells reproduce using two types of divisions: mitosis, which generates two daughter cells each with the same genomic content as the mother cell, and meiosis, which reduces the number of chromosomes of the parent cell by half and gives rise to four gametes. The mechanisms that promote the proper progression of the mitotic and meiotic cycles are highly conserved and controlled. They require the activities of two types of serine-threonine kinases, the cyclin-dependent kinases (CDKs) and the Dbf4-dependent kinase (DDK). CDK and DDK are essential for genome duplication and maintenance in both mitotic and meiotic divisions. In this review, we aim to highlight how these kinases cooperate to orchestrate diverse processes during cellular reproduction, focusing on meiosis-specific adaptions of their regulation and functions in DNA metabolism.

International audience; One of the most important areas of research on microfluidic technologies focuses on the identification and characterisation of novel materials with enhanced properties and versatility. Here we present a fast, easy and inexpensive microstructuration method for the fabrication of novel, flexible, transparent and biocompatible microfluidic devices. Using a simple hot press, we demonstrate the rapid (30 s) production of various microfluidic prototypes embossed in a commercially available soft thermoplastic elastomer (sTPE). This styrenic block copolymer (BCP) material is as flexible as PDMS and as thermoformable as classical thermoplastics. It exhibits high fidelity of replication using SU-8 and epoxy master molds in a highly convenient low-isobar (0.4 bar) and iso-thermal process. Microfluidic devices can then be easily sealed using either a simple hot plate or even a room-temperature assembly, allowing them to sustain liquid pressures of 2 and 0.6 bar, respectively. The excellent sorption and biocompatibility properties of the microchips were validated via a standard rhodamine dye assay as well as a sensitive yeast cell-based assay. The morphology and composition of the surface area after plasma treatment for hydrophilization purposes are stable and show constant and homogenous distribution of block nanodomains (similar to 22 degrees after 4 days). These domains, which are evenly distributed on the nanoscale, therefore account for the uniform and convenient surface of a "microfluidic scale device". To our knowledge, this is the first thermoplastic elastomer material that can be used for fast and reliable fabrication and assembly of microdevices while maintaining a high and stable hydrophilicity.

International audience; Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging.

The Saccharomyces cerevisiae SAGA complex is a multifunctional coactivator that regulates transcription by RNA polymerase II. The 3D structure of SAGA, revealed by electron microscopy, is formed by five modular domains and shows a high degree of structural conservation to human TFTC, reflecting their related subunit composition. The positions of several SAGA subunits were mapped by immunolabeling and by analysis of mutant complexes. The Taf (TBP-associated factor) subunits, shared with TFIID, occupy a central region in SAGA and form a similar structure in both complexes. The locations of two histone fold-containing core subunits, Spt7 and Ada1, are consistent with their role in providing a SAGA-specific interface with the Tafs. Three components that perform distinct regulatory functions, Spt3, Gcn5, and Tra1, are spatially separated, underscoring the modular nature of the complex. Our data provide insights into the molecular architecture of SAGA and imply a functional organization to the complex.

Summary The program of DNA replication, defined by the temporal and spatial pattern of origin activation, is altered during development and in cancers. However, whether changes in origin usage play a role in regulating specific biological processes remains unknown. We investigated the consequences of modifying origin selection on meiosis in fission yeast. Genome-wide changes in the replication program of premeiotic S phase do not affect meiotic progression, indicating that meiosis neither activates nor requires a particular origin pattern. In contrast, local changes in origin efficiencies between different replication programs lead to changes in Rad51 recombination factor binding and recombination frequencies in these domains. We observed similar results for Rad51 when changes in efficiencies were generated by directly targeting expression of the Cdc45 replication factor. We conclude that origin selection is a key determinant for organizing meiotic recombination, providing evidence that genome-wide modifications in replication program can modulate cellular physiology., Graphical Abstract, Highlights • Premeiotic S phase neither activates nor requires a specific origin usage program • Origin selection is a key regulator of the distribution of meiotic recombination • Organization of genome duplication has a functional impact on cellular physiology, Changes in the pattern of DNA replication are observed during development and in various pathologies, but it is unclear whether they contribute to these processes. Wu and Nurse show, by altering replication origin usage in fission yeast, that origin selection is critical for the organization of meiotic recombination.

Transcriptional control of DHHC palmitoyltransferase activity levels shapes cellular palmitoylation patterns and influences meiotic entry in fission yeast., Protein S-palmitoylation, a lipid modification mediated by members of the palmitoyltransferase family, serves as an important membrane-targeting mechanism in eukaryotes. Although changes in palmitoyltransferase expression are associated with various physiological and disease states, how these changes affect global protein palmitoylation and cellular function remains unknown. Using a bioorthogonal chemical reporter and labeling strategy to identify and analyze multiple cognate substrates of a single Erf2 palmitoyltransferase, we demonstrate that control of Erf2 activity levels underlies the differential modification of key substrates such as the Rho3 GTPase in vegetative and meiotic cells. We show further that modulation of Erf2 activity levels drives changes in the palmitoylome as cells enter meiosis and affects meiotic entry. Disruption of Erf2 function delays meiotic entry, while increasing Erf2 palmitoyltransferase activity triggers aberrant meiosis in sensitized cells. Erf2-induced meiosis requires the function of the Rho3 GTPase, which is regulated by its palmitoylation state. We propose that control of palmitoyltransferase activity levels provides a fundamental mechanism for modulating palmitoylomes and cellular functions., Author Summary Palmitoylation is a fatty acid modification, which increases the hydrophobicity of proteins, known to control the membrane trafficking and function of many proteins important for cellular homeostasis and disease. Being a reversible process, palmitoylation is proposed to be a major cellular regulator. While changes in global protein palmitoylation patterns are often associated with different physiological states, the mechanisms that regulate this posttranslational modification and their functional consequences have been elusive. Using improved methods for protein palmitoylation analysis in the genetically tractable fission yeast Schizosaccharomyces pombe, we discovered that the global protein palmitoylation pattern is significantly altered and correlates with entry into meiosis, a major developmental transition. We further showed that the precise level of a palmitoylating enzyme is important to shape the subset of lipid-modified proteins and influence the start of this cellular transition. As palmitoylated proteins are involved in eukaryotic cellular pathways, our results provide a crucial insight into how the quantitative control of palmitoylating enzymes may impact human physiology and disease.

International audience; Comment on: Guerra-Moreno A, et al. Cell Cycle 2012; 1621-5.

SummaryInitiation of eukaryotic DNA synthesis occurs at origins of replication that are utilized with characteristic times and frequencies during S phase. We have investigated origin usage by evaluating the kinetics of replication factor binding in fission yeast and show that similar to metazoa, ORC binding is periodic during the cell cycle, increasing during mitosis and peaking at M/G1. At an origin, the timing of ORC binding in M and pre-RC assembly in G1 correlates with the timing of firing during S, and the level of pre-IC formation reflects origin efficiency. Extending mitosis allows ORC to become more equally associated with origins and leads to genome-wide changes in origin usage, while overproduction of pre-IC factors increases replication of both efficient and inefficient origins. We propose that differential recruitment of ORC to origins during mitosis followed by competition among origins for limiting replication factors establishes the timing and efficiency of origin firing.

Recent studies have revealed that transcription of noncoding, intergenic DNA is abundant among eukaryotes. However, the functions of this transcription are poorly understood. We have previously shown that in Saccharomyces cerevisiae, expression of an intergenic transcript, SRG1, represses the transcription of the adjacent gene, SER3, by transcription interference. We now show that SRG1 transcription is regulated by serine, thereby conferring regulation of SER3, a serine biosynthetic gene. This regulation requires Cha4, a serine-dependent activator that binds to the SRG1 promoter and is required for SRG1 induction in the presence of serine. Furthermore, two coactivator complexes, SAGA and Swi/Snf, are also directly required for activation of SRG1 and transcription interference of SER3. Taken together, our results elucidate a physiological role for intergenic transcription in the regulation of SER3. Moreover, our results demonstrate a mechanism by which intergenic transcription allows activators to act indirectly as repressors.

The Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a multisubunit coactivator complex that is important for transcription in vivo (27, 32). Whole-genome mRNA analysis of SAGA mutants has shown that the expression of approximately 10% of S. cerevisiae genes are affected by the loss of the SAGA complex (23). Both in vivo and in vitro experiments have shown that SAGA activates transcription after its recruitment by transcriptional activators (5, 22, 42, 43). Other results have suggested that SAGA also represses transcription at particular promoters (4, 23). In addition, several studies have shown that SAGA often acts coordinately with other coactivator complexes at a promoter to achieve normal levels of transcription (27). The SAGA complex is conserved between yeast and humans, strongly suggesting that this type of coactivator is also important in mammalian transcription (6, 25, 26, 28, 46). The subunits in SAGA can be grouped functionally based on a large body of in vivo and in vitro experiments (27). Three classes of SAGA proteins are involved in distinct aspects of transcriptional control. First, Gcn5 contains the catalytic activity for the histone acetyltransferase (HAT) activity of SAGA (8), and Gcn5's HAT activity is modulated by the Ada2 and Ada3 proteins (2, 16, 39). Second, Spt3 and Spt8 of SAGA have been shown to control the TATA box binding protein (TBP)-TATA interaction at particular promoters (4, 5, 9, 11, 22). Third, Tra1 has been shown to interact with several transcriptional activators in vitro, suggesting that SAGA is recruited to promoters via this subunit and subsequently activates or represses transcription through its different activities (7). SAGA also contains two additional classes of proteins that are not known to participate directly in regulation and therefore may serve structural roles. In the first class are three proteins, Spt7, Spt20, and Ada1, that function as SAGA core components based on their requirement for integrity of the complex. SAGA is absent from spt7Δ, spt20Δ, and ada1Δ mutants, as assayed by Western analysis and HAT assays (16, 38). Moreover, these mutations cause a broad variety of severe phenotypes, consistent with a complete loss of SAGA function (21, 30, 38). The second class contains a subset of the Tafs (Tafs 5, 6, 9, 10, and 12), proteins initially identified as components of the TFIID complex (17). In referring to the Tafs, we are using the recently revised uniform Taf nomenclature (41). At least one of the Tafs, Taf12, appears to be important for SAGA structure, as its removal via a taf12 temperature sensitivity mutation affects both SAGA integrity as well as its nucleosomal HAT activity (17). While the control of transcription by SAGA has been extensively studied, less is known about the control of its assembly and the protein-protein associations within the complex. Previous studies have shown that both Spt7 and Ada1 have histone fold motifs that interact with Taf components of SAGA: Ada1 with Taf12 and Spt7 with Taf10 (13, 14). However, it is not known how the core subunits contribute toward the structural integrity of the complex. To learn more about the roles of one of the SAGA core components, we have chosen Spt7 as the focus of our studies. Spt7 is a 1,332-amino-acid, highly negatively charged protein whose sequence contains two motifs of note. First, as mentioned previously, Spt7 contains a histone fold (amino acids 979 to 1045) required for interaction with the Taf10 subunit of SAGA (13). Second, Spt7 contains a bromodomain (amino acids 463 to 523), a motif found in many transcription factors (20; reviewed in reference 44). A deletion that removes the Spt7 bromodomain does not cause any detectable phenotypes, suggesting that this domain is either redundant or not crucial for Spt7 function (15, 38). Previous studies suggest that Spt7 may act dynamically to regulate SAGA function. These studies demonstrated that SAGA exists in at least one alternate form that has been named SLIK (SAGA-like), SAGAalt, or SALSA (4, 18, 34; D. Sterner and S. Berger, personal communication). This complex is referred to hereafter as SLIK/SALSA. The SLIK/SALSA complex has two identified differences from SAGA: a smaller form of Spt7 and the absence of Spt8 (4; Patrick Grant, personal communication). Although little is known about the formation of SLIK/SALSA, evidence suggests involvement of the general amino acid control pathway, because SLIK/SALSA levels increase in cells grown in amino acid starvation conditions (4). However, the requirements for SLIK/SALSA formation, as well as its functions in vivo, remain unknown. In this study, we examine several aspects of Spt7 protein structure and function. We demonstrate that Spt7 is involved in regulating the levels of Spt20 and Ada1, the two other SAGA core components, suggesting that Spt7 levels control the amount of SAGA present in vivo. Additional experiments reveal that partial Spt7-containing complexes form in the absence of the other core components. To determine regions of function and interaction with other subunits, we generated and analyzed a set of spt7 partial deletion mutants. This analysis allowed us to delineate a region of minimal function in Spt7 as well as a domain for Spt8 interaction. Moreover, we have defined a region of Spt7 that is required for its processing and, hence, for the formation of SLIK/SALSA. Analysis of the spt7 mutant that impairs processing suggests that SLIK/SALSA is not strongly required for transcriptional activation.

Synthetic biology is one of the most exciting strategies for the investigation of living organisms and lies at the intersection of biology and engineering. Originally developed in prokaryotes, the idea of deciphering biological phenomena through building artificial genetic circuits and studying their behaviours has rapidly demonstrated its potential in a broad range of fields in the life sciences. From the assembly of synthetic genomes to the generation of novel biological functions, yeast cells have imposed themselves as the most powerful eukaryotic model for this approach. However, we are only beginning to explore the possibilities of synthetic biology, and the perspectives it offers in a genetically amenable system such as yeasts are endless. Copyright © 2016 John WileySons, Ltd.

DNA replication is a highly conserved and regulated step of the cell cycle. Defects in genome duplication have severe consequences for cell proliferation and have been linked to different pathologies, including cancer. Replication initiates at discrete sites along the chromosomes known as origins. For a population of cells, the program of replication is defined by the distribution and activation of origins across the genome. However, cells within the same population display plasticity in origin usage, and the subset of origins activated during S-phase varies from one cell cycle to the next. While changes in the replication program have been observed during development and differentiation as well as in cancer, the functional importance of these alterations remained unknown. Our laboratory previously demonstrated in the fission yeast that the program of origin selection during pre-meiotic S phase regulates the sites of double-strand break (DSB) formation during meiosis, providing the first evidence for the functional consequences of genome-wide changes in origin usage. Building on this work, my thesis takes two approaches to investigate the crosstalk between genome duplication and meiotic recombination, using the fission yeast Schizosaccharomyces pombe as a model system. First, we explored the impact of chromosomal organization on the program of DNA replication and meiotic recombination. To this end, we engineered chromosomal rearrangements that exchange the positions of replication domains with different efficiency and timing characteristics. Our results demonstrated that this induced local changes in origin efficiency near the endpoints of the rearranged region during both mitotic and pre-meiotic S phase. Interestingly, while genome-wide analysis of the DSB profile showed alterations near the rearranged ends, these differences did not reflect the changes in origin usage. This unexpected finding suggests a complex regulation of DSB formation during meiosis that hints at a potential role for chromosomal context in this process. In addition, we aimed to investigate the steps in DNA replication that are important for promoting DSB formation. Specifically, we focused on evaluating whether the replication machinery must progress through a DSB site before breaks are formed or whether origin activation is sufficient to induce nearby DSBs. For these studies, we constructed and characterized a system for inducing a replication fork barrier during pre-meiotic S phase. Taken together, my thesis work provides new directions for investigating the interplay between chromosomal organization, DNA replication and meiotic recombination., La réplication de l’ADN est une étape essentielle du cycle cellulaire et les étapes qui la composent sont fortement conservées et régulées. Des erreurs dans la duplication du génome peuvent avoir de graves conséquences sur la proliferation cellulaire et ont été liées à différentes pathologies telles que le cancer. La duplication du génome commence à partir de sites distribués tout au long du génome appelés origines de réplication. Cette duplication est réalisé suivant un programme de réplication précis qui est défini, pour une population de cellules, par la distribution et l’activation de ses origines le long du génome. Toutefois, les cellules au sein d’une même population font preuve d’une certaine plasticité quant à l’utilisation de ces origines. Par exemple, l’ensemble des origines activées varie d’une phase S à une autre ainsi que d’une cellule à une autre. Bien que ces changements du programme de réplication aient été observés durant le développement, la différenciation cellulaire ainsi que dans les cancers, l’importance de ces changements sur les fonctions cellulaires reste peu caractérisée. En utilisant la levure à fission Schizosaccharomyces pombe comme modèle d’étude, notre laboratoire a montré que l’utilisation d’un programme de réplication particulier durant la phase S de méiose avait des conséquences sur la formation de cassures doubles brins de l’ADN. Ces résultats montrent pour la première fois qu’un changement du programme de réplication à l’échelle du génome a des conséquences sur les fonctions cellulaires. Basés sur ces résultats, mes travaux de thèse visent à comprendre le lien existant entre la replication du génome et la recombinaison méiotique, via deux approches. Dans un premier temps, nous avons exploré l’impact de l’organisation chromosomique sur la réplication de l’ADN et la recombinaison méiotique. Pour celanous avons généré des réarrangements chromosomiques qui nous ont permis d’échanger la position de domaines de réplication ayant des efficacités et des timings différents. Nos résultats ont montré que ces réarrangements induisaient des changements de l’efficacité des origines localisées spécifiquement de part et d’autre des extrémités des régions réarrangées, et ce durant les phases S mitotique et méiotique. Alors que l’analyse des cassures doubles brins de l’ADN sur l’ensemble du génome montre également des changements aux extrémités de la region réarrangée, ces changements ne reflètent pas les changements du programme de réplication. Ces résultats inattendus suggèrent que le contrôle des cassures doubles brins de l’ADN durant la méiose est régulé de manière complexe et que le context chromosomique pourrait jouer un rôle dans ce procédé. En parallèle, nous nous sommes intéressés à déterminer quelles étaient les étapes de la réplication de l’ADN importantes pour la formation des cassures doubles brins. Nous nous sommes spécialement concentrés à déterminer si la machinerie de réplication doit passer par un site de cassure avant que celle-ci soit faite ou si l’initiation de la réplication est suffisante pour induire les cassures doubles brins adjacentes. Pour cela nous avons construit et caractérisé un système dans lequel nous pouvons induire une barrière de réplication durant la phase S de méiose. Mes travaux de thèse ouvrent ainsi de nouvelles pistes de recherche pour comprendre le lien entre l’organisation chromosomique, la réplication de l’ADN et la recombinaison méiotique.