Histone H3 serine-57 is a CHK1 substrate whose phosphorylation affects DNA repair Parisis N, Dans PD, Jbara M, Singh B, Schausi-Tiffoche D, Molina-Serrano D, Brun-Heath I, Hendrychová D, Maity SK, Buitrago D, Lema R, Nait Achour T, Giunta S, Girardot M, Talarek N, Rofidal V, Danezi K, Coudreuse D, Prioleau MN, Feil R, Orozco M, Brik A, Wu PYJ, Krasinska L, Fisher D. Nature Communications 14, 5104 (2023).



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Histone post-translational modifications promote a chromatin environment that controls transcription, DNA replication and repair, but surprisingly few phosphorylations have been documented. We report the discovery of histone H3 serine-57 phosphorylation (H3S57ph) and show that it is implicated in different DNA repair pathways from fungi to vertebrates. We identified CHK1 as a major human H3S57 kinase, and disrupting or constitutively mimicking H3S57ph had opposing effects on rate of recovery from replication stress, 53BP1 chromatin binding, and dependency on RAD52. In fission yeast, mutation of all H3 alleles to S57A abrogated DNA repair by both non-homologous end-joining and homologous recombination, while cells with phospho-mimicking S57D alleles were partly compromised for both repair pathways, presented aberrant Rad52 foci and were strongly sensitised to replication stress. Mechanistically, H3S57ph loosens DNA-histone contacts, increasing nucleosome mobility, and interacts with H3K56. Our results suggest that dynamic phosphorylation of H3S57 is required for DNA repair and recovery from replication stress, opening avenues for investigating the role of this modification in other DNA-related processes.

Impact of Chromosomal Context on Origin Selection and the Replication Program Lanteri L, Perrot A, Schausi-Tiffoche D, Wu PYJ Genes 13(7):1244. doi: 10.3390/genes13071244



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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.

Linking the organization of DNA replication with genome maintenance Singh B and Wu PY Current Genetics 65(3):677-683. doi: 10.1007/s00294-018-0923-8



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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.

Insights into the Link between the Organization of DNA Replication and the Mutational Landscape Gaboriaud J and Wu PY Genes 10(4). pii: E252. doi: 10.3390/genes10040252



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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.

The organization of genome duplication is a critical determinant of the landscape of genome maintenance Gómez-Escoda B and Wu PY Genome Research doi/10.1101/gr.224527.117 2018-06-22



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Genome duplication is essential for cell proliferation, and the mechanisms regulating its execution are highly conserved. These processes give rise to a spatiotemporal organization of replication initiation across the genome, referred to as the replication program. Despite the identification of such programs in diverse eukaryotic organisms, their biological importance for cellular physiology remains largely unexplored. We address this fundamental question in the context of genome maintenance, taking advantage of the inappropriate origin firing that occurs when fission yeast cells lacking the Rad3/ATR checkpoint kinase are subjected to replication stress. Using this model, we demonstrate that the replication program quantitatively dictates the extent of origin de-regulation and the clustered localization of these events. Furthermore, our results uncover an accumulation of abnormal levels of single-stranded DNA (ssDNA) and the Rad52 repair protein at de-regulated origins. We show that these loci constitute a defining source of the overall ssDNA and Rad52 hotspots in the genome, generating a signature pattern of instability along the chromosomes. We then induce a genome-wide reprogramming of origin usage and evaluate its consequences in our experimental system. This leads to a complete redistribution of the sites of both inappropriate initiation and associated Rad52 recruitment. We therefore conclude that the organization of genome duplication governs the checkpoint control of origin-associated hotspots of instability and plays an integral role in shaping the landscape of genome maintenance.

Regulation of the program of DNA replication by CDK: new findings and perspectives Singh B and Wu PY Current Genetics doi.org/10.1007/s00294-018-0860-6



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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.

CDK activity provides temporal and quantitative cues for organizing genome duplication Perrot A, Millington CL, Gómez-Escoda B, Schausi-Tiffoche D, and Wu PY PLOS Genetics 14(2):e1007214;doi.org/10.1371/journal. pgen.1007214



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In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs), which associate with various cyclin partners to drive cell proliferation. Surprisingly, although we possess detailed knowledge of the CDK regulators and targets that are crucial for origin activation, little is known about whether CDKs play a critical role in establishing the genome-wide pattern of origin selection. We have addressed this question in the fission yeast, taking advantage of a simplified cell cycle network in which cell proliferation is driven by a single cyclin-CDK module. This system allows us to precisely control CDK activity in vivo using chemical genetics. First, in contrast to previous reports, our results clearly show that distinct cyclin-CDK pairs are not essential for regulating specific subsets of origins and for establishing a normal replication program. Importantly, we then demonstrate that the timing at which CDK activity reaches the S phase threshold is critical for the organization of replication in distinct efficiency domains, while the level of CDK activity at the onset of S phase is a dose-dependent modulator of overall origin efficiencies. Our study therefore implicates these different aspects of CDK regulation as versatile mechanisms for shaping the architecture of DNA replication across the genome.

Thermoplastic elastomer with advanced hydrophilization and bonding performances for rapid (30s) and easy molding of microfluidic devices Julie Lachaux, Clara Alcaine, Blanca Gómez-Escoda, Cécile M. Perrault, David Olea Duplan, Pei-Yun Jenny Wu, Iñaki Ochoa, Luis Fernandez, Olaf Mercier, Damien Coudreuse and Emmanuel Roy Lab on a Chip 2017,17, 2581-2594; doi:10.1039/C7LC00488E



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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 (∼22° 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.

Roles of CDK and DDK in Genome Duplication and Maintenance: Meiotic Singularities Gómez-Escoda B and Wu PY Genes 8(3), 105; doi:10.3390/genes8030105



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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.

A drug-compatible and temperature-controlled microfluidic device for live-cell imaging Chen T, Gomez-Escoda B, Munoz-Garcia J, Babic J, Griscom L, Wu PY, and Coudreuse D Open Biology 6:160156



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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.

Replication origin selection regulates the distribution of meiotic recombination Wu PY and Nurse P Molecular Cell 53:655-662



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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.

The programme of DNA replication: beyond genome duplication. Gomez-Escoda B and Wu PY Biochemical Society Transactions 41:1720-1725



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The accurate duplication and transmission of genetic information is critical for cell growth and proliferation, and this is ensured in part by the multilayered regulation of DNA synthesis. One of the key steps in this process is the selection and activation of the sites of replication initiation, or origins, across the genome. Interestingly, origin usage changes during development and in different pathologies, suggesting an integral interplay between the establishment of replication initiation along the chromosomes and cellular function. The present review discusses how the spatiotemporal organization of replication origin activation may play crucial roles in the control of biological events.

Quantitative Control of Protein S-Palmitoylation Regulates Meiotic Entry in Fission Yeast Zhang MM, Wu PY, Kelly FD, Nurse P and Hang HC PLOS Biology 11:e1001597



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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.

Insights from a new tool for meiotic induction in fission yeast Wu PY Cell Cycle 11:2050



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Meiosis is the differentiation pathway by which diploid cells produce haploid progeny for sexual reproduction. This specialized process involves one round of DNA replication, exchange of genetic material through recombination and haploidization achieved by two consecutive nuclear divisions without an intervening S phase. Meiosis is a tightly regulated process, and the major steps in meiotic progression, including the duplication, recombination and segregation of the genome, are conserved among eukaryotes. The molecular mechanisms underlying these events have been the subject of extensive and fruitful investigations, in particular in two model systems, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In a previous issue of Cell Cycle, Guerra-Moreno and colleagues presented a novel method for the synchronous induction of meiosis in fission yeast and this approach to uncover new aspects of the meiosis-specific transcriptional program.

Establishing the Program of Origin Firing during S Phase in Fission Yeast Wu PY and Nurse P Cell 136:852-864



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Initiation 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.

Regulation of an intergenic transcript controls adjacent gene transcription in Saccharomyces cerevisiae Martens JA, Wu PY, and Winston F Genes and Development 19:2695-2704



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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.

Molecular Architecture of the S. cerevisiae SAGA Complex Wu PY, Ruhlmann C, Winston F, and Schultz P Molecular Cell 15:199-208



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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.

Analysis of Spt7 Function in the Saccharomyces cerevisiae SAGA Coactivator Complex Wu PY and Winson F Molecular and Cellular Biology 22:5367-6379



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The Saccharomyces cerevisiae SAGA complex is required for the normal transcription of a large number of genes. Complex integrity depends on three core subunits, Spt7, Spt20, and Ada1. We have investigated the role of Spt7 in the assembly and function of SAGA. Our results show that Spt7 is important in controlling the levels of the other core subunits and therefore of SAGA. In addition, partial SAGA complexes containing Spt7 can be assembled in the absence of both Spt20 and Ada1. Through biochemical and genetic analyses of a series of spt7 deletion mutants, we have identified a region of Spt7 required for interaction with the SAGA component Spt8. An adjacent Spt7 domain was found to be required for a processed form of Spt7 that is present in a previously identified altered form of SAGA called SLIK, SAGAalt, or SALSA. Analysis of an spt7 mutant with greatly reduced levels of SLIK/SAGAalt/SALSA suggests a subtle role for this complex in transcription that may be redundant with a subset of SAGA functions.