Publications by authors named "Philippe Pasero"

89 Publications

RNA-sequencing data-driven dissection of human plasma cell differentiation reveals new potential transcription regulators.

Leukemia 2021 Apr 6. Epub 2021 Apr 6.

Department of Biological Hematology, CHU Montpellier, Montpellier, France.

Plasma cells (PCs) play an important role in the adaptive immune system through a continuous production of antibodies. We have demonstrated that PC differentiation can be modeled in vitro using complex multistep culture systems reproducing sequential differentiation process occurring in vivo. Here we present a comprehensive, temporal program of gene expression data encompassing human PC differentiation (PCD) using RNA sequencing (RNA-seq). Our results reveal 6374 differentially expressed genes classified into four temporal gene expression patterns. A stringent pathway enrichment analysis of these gene clusters highlights known pathways but also pathways largely unknown in PCD, including the heme biosynthesis and the glutathione conjugation pathways. Additionally, our analysis revealed numerous novel transcriptional networks with significant stage-specific overexpression and potential importance in PCD, including BATF2, BHLHA15/MIST1, EZH2, WHSC1/MMSET, and BLM. We have experimentally validated a potent role for BLM in regulating cell survival and proliferation during human PCD. Taken together, this RNA-seq analysis of PCD temporal stages helped identify coexpressed gene modules with associated up/downregulated transcription regulator genes that could represent major regulatory nodes for human PC maturation. These data constitute a unique resource of human PCD gene expression programs in support of future studies for understanding the underlying mechanisms that control PCD.
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http://dx.doi.org/10.1038/s41375-021-01234-0DOI Listing
April 2021

Topoisomerase I prevents transcription-replication conflicts at transcription termination sites.

Mol Cell Oncol 2020 Dec 7;8(1):1843951. Epub 2020 Dec 7.

Institut Curie, PSL Research University, CNRS, UMR3244, Sorbonne Université, Paris, France.

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops, we mapped RNA:DNA hybrids, markers of replication fork stalling and DNA double-strand breaks along the human genome. This analysis indicates that transient replication fork pausing occurs at the transcription termination sites of highly expressed genes enriched in R-loops and prevents head-on conflicts with transcription, in a topoisomerase I-dependent manner.
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http://dx.doi.org/10.1080/23723556.2020.1843951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849740PMC
December 2020

High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS.

Nat Protoc 2021 02 21;16(2):1034-1061. Epub 2020 Dec 21.

Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Warsaw, Poland.

DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such as cancer. Therefore, there is a great need for a precise, sensitive, and universal method for DSB detection, to enable both the study of their mechanisms of formation and repair as well as to explore their therapeutic potential. We provide a detailed protocol for our recently developed ultrasensitive and genome-wide DSB detection method: immobilized direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (i-BLESS), which relies on the encapsulation of cells in agarose beads and labeling breaks directly and specifically with biotinylated linkers. i-BLESS labels DSBs with single-nucleotide resolution, allows detection of ultrarare breaks, takes 5 d to complete, and can be applied to samples from any organism, as long as a sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach to enable the measurement of absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Such normalization using qDSB-Seq is especially useful for the evaluation of spontaneous DSB levels and the estimation of DNA damage induced rather uniformly in the genome (e.g., by irradiation or radiomimetic chemotherapeutics).
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http://dx.doi.org/10.1038/s41596-020-00448-3DOI Listing
February 2021

A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization.

Mol Cell 2021 01 4;81(1):183-197.e6. Epub 2020 Dec 4.

Institut de Génétique Humaine, CNRS, Université de Montpellier, Equipe Labéllisée Ligue contre le Cancer, 34396 Montpellier, France; University of Basel and Friedrich Miescher Institute for Biomedical Research, Faculty of Natural Sciences, Klingelbergstrasse 50, 4056 Basel, Switzerland. Electronic address:

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.
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http://dx.doi.org/10.1016/j.molcel.2020.11.010DOI Listing
January 2021

TDP-43 dysfunction results in R-loop accumulation and DNA replication defects.

J Cell Sci 2020 10 30;133(20). Epub 2020 Oct 30.

Division of Oncology, Department of Medicine, Washington University in St. Louis, St. Louis, MO 63110, USA

TAR DNA-binding protein 43 (TDP-43; also known as TARDBP) is an RNA-binding protein whose aggregation is a hallmark of the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 loss increases DNA damage and compromises cell viability, but the actual function of TDP-43 in preventing genome instability remains unclear. Here, we show that loss of TDP-43 increases R-loop formation in a transcription-dependent manner and results in DNA replication stress. TDP-43 nucleic-acid-binding and self-assembly activities are important in inhibiting R-loop accumulation and preserving normal DNA replication. We also found that TDP-43 cytoplasmic aggregation impairs TDP-43 function in R-loop regulation. Furthermore, increased R-loop accumulation and DNA damage is observed in neurons upon loss of TDP-43. Together, our findings indicate that TDP-43 function and normal protein homeostasis are crucial in maintaining genomic stability through a co-transcriptional process that prevents aberrant R-loop accumulation. We propose that the increased R-loop formation and genomic instability associated with TDP-43 loss are linked to the pathogenesis of TDP-43 proteinopathies.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.244129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648616PMC
October 2020

Resolution of R-loops by INO80 promotes DNA replication and maintains cancer cell proliferation and viability.

Nat Commun 2020 09 10;11(1):4534. Epub 2020 Sep 10.

Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.
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http://dx.doi.org/10.1038/s41467-020-18306-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484789PMC
September 2020

Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites.

Nat Commun 2020 08 7;11(1):3940. Epub 2020 Aug 7.

Institut de Génétique Humaine, CNRS et Université de Montpellier, Equipe labélisée Ligue contre le Cancer, Montpellier, France.

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.
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http://dx.doi.org/10.1038/s41467-020-17858-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414224PMC
August 2020

Ethanol exposure increases mutation rate through error-prone polymerases.

Nat Commun 2020 07 21;11(1):3664. Epub 2020 Jul 21.

Laboratory of Systems Biology, VIB-KU Leuven Center for Microbiology, Leuven, Belgium.

Ethanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes.
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http://dx.doi.org/10.1038/s41467-020-17447-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374746PMC
July 2020

Sir2 takes affirmative action to ensure equal opportunity in replication origin licensing.

Proc Natl Acad Sci U S A 2020 07 30;117(29):16723-16725. Epub 2020 Jun 30.

Institut de Génétique Humaine, CNRS and Université de Montpellier, Equipe Labélisée Ligue contre le Cancer, 34396 Montpellier, France

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http://dx.doi.org/10.1073/pnas.2010001117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382207PMC
July 2020

Homologous recombination and Mus81 promote replication completion in response to replication fork blockage.

EMBO Rep 2020 07 17;21(7):e49367. Epub 2020 May 17.

Institut de Génétique Humaine, Université de Montpellier-CNRS, Montpellier, France.

Impediments to DNA replication threaten genome stability. The homologous recombination (HR) pathway has been involved in the restart of blocked replication forks. Here, we used a method to increase yeast cell permeability in order to study at the molecular level the fate of replication forks blocked by DNA topoisomerase I poisoning by camptothecin (CPT). Our results indicate that Rad52 and Rad51 HR factors are required to complete DNA replication in response to CPT. Recombination events occurring during S phase do not generally lead to the restart of DNA synthesis but rather protect blocked forks until they merge with convergent forks. This fusion generates structures requiring their resolution by the Mus81 endonuclease in G /M. At the global genome level, the multiplicity of replication origins in eukaryotic genomes and the fork protection mechanism provided by HR appear therefore to be essential to complete DNA replication in response to fork blockage.
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http://dx.doi.org/10.15252/embr.201949367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332989PMC
July 2020

Hair follicle stem cell replication stress drives IFI16/STING-dependent inflammation in hidradenitis suppurativa.

J Clin Invest 2020 07;130(7):3777-3790

INSERM U955, Equipe 16, Créteil, France.

Hidradenitis suppurativa (HS) is a chronic, relapsing, inflammatory skin disease. HS appears to be a primary abnormality in the pilosebaceous-apocrine unit. In this work, we characterized hair follicle stem cells (HFSCs) isolated from HS patients and more precisely the outer root sheath cells (ORSCs). We showed that hair follicle cells from HS patients had an increased number of proliferating progenitor cells and lost quiescent stem cells. Remarkably, we also showed that the progression of replication forks was altered in ORSCs from hair follicles of HS patients, leading to activation of the ATR/CHK1 pathway. These alterations were associated with an increased number of micronuclei and with the presence of cytoplasmic ssDNA, leading to the activation of the IFI16/STING pathway and the production of type I IFNs. This mechanistic analysis of the etiology of HS in the HFSC compartment establishes a formal link between genetic predisposition and skin inflammation observed in HS.
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http://dx.doi.org/10.1172/JCI131180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324185PMC
July 2020

Mec1 Is Activated at the Onset of Normal S Phase by Low-dNTP Pools Impeding DNA Replication.

Mol Cell 2020 05 12;78(3):396-410.e4. Epub 2020 Mar 12.

Institut de Génétique Humaine, CNRS, Université de Montpellier, Montpellier, France. Electronic address:

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.
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http://dx.doi.org/10.1016/j.molcel.2020.02.021DOI Listing
May 2020

Top1 and Top2 promote replication fork arrest at a programmed pause site.

Genes Dev 2020 01;34(1-2):1-3

Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Université de Montpellier, Montpellier 34396, France.

Programmed fork pausing is a complex process allowing cells to arrest replication forks at specific loci in a polar manner. Studies in budding yeast and other model organisms indicate that such replication fork barriers do not act as roadblocks passively impeding fork progression but rather elicit complex interactions between fork and barrier components. In this issue of , Shyian and colleagues (pp. 87-98) show that in budding yeast, the fork protection complex Tof1-Csm3 interacts physically with DNA topoisomerase I (Top1) at replication forks through the C-terminal domain of Tof1. Fork pausing at the ribosomal DNA (rDNA) replication fork barrier (RFB) is impaired in the absence of Top1 or in a mutant that does not bind Top1, but the function of Top1 can be partially compensated for by Top2. Together, these data indicate that topoisomerases play an unexpected role in the regulation of programmed fork pausing in .
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http://dx.doi.org/10.1101/gad.335463.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938665PMC
January 2020

MRX Increases Chromatin Accessibility at Stalled Replication Forks to Promote Nascent DNA Resection and Cohesin Loading.

Mol Cell 2020 01 20;77(2):395-410.e3. Epub 2019 Nov 20.

Institut de Génétique Humaine, CNRS, Université de Montpellier, Equipe Labellisée Ligue contre le Cancer, Montpellier, France. Electronic address:

The recovery of stalled replication forks depends on the controlled resection of nascent DNA and on the loading of cohesin. These processes operate in the context of nascent chromatin, but the impact of nucleosome structure on a fork restart remains poorly understood. Here, we show that the Mre11-Rad50-Xrs2 (MRX) complex acts together with the chromatin modifiers Gcn5 and Set1 and the histone remodelers RSC, Chd1, and Isw1 to promote chromatin remodeling at stalled forks. Increased chromatin accessibility facilitates the resection of nascent DNA by the Exo1 nuclease and the Sgs1 and Chl1 DNA helicases. Importantly, increased ssDNA promotes the recruitment of cohesin to arrested forks in a Scc2-Scc4-dependent manner. Altogether, these results indicate that MRX cooperates with chromatin modifiers to orchestrate the action of remodelers, nucleases, and DNA helicases, promoting the resection of nascent DNA and the loading of cohesin, two key processes involved in the recovery of arrested forks.
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http://dx.doi.org/10.1016/j.molcel.2019.10.029DOI Listing
January 2020

Kinome expression profiling to target new therapeutic avenues in multiple myeloma.

Haematologica 2020 03 9;105(3):784-795. Epub 2019 Jul 9.

IGH, CNRS, Université de Montpellier, Montpellier, France

Multiple myeloma (MM) account for approximately 10% of hematological malignancies and is the second most common hematological disorder. Kinases inhibitors are widely used and their efficiency for the treatment of cancers has been demonstrated. Here, in order to identify kinases of potential therapeutic interest for the treatment of MM, we investigated the prognostic impact of the kinome expression profile in large cohorts of patients. We identified 36 kinome-related genes significantly linked with a prognostic value to MM, and built a kinome index based on their expression. The Kinome Index (KI) is linked to prognosis, proliferation, differentiation, and relapse in MM. We then tested inhibitors targeting seven of the identified protein kinas-es (PBK, SRPK1, CDC7-DBF4, MELK, CHK1, PLK4, MPS1/TTK) in human myeloma cell lines. All tested inhibitors significantly reduced the viability of myeloma cell lines, and we confirmed the potential clinical interest of three of them on primary myeloma cells from patients. In addition, we demonstrated their ability to potentialize the toxicity of conventional treatments, including Melphalan and Lenalidomide. This highlights their potential beneficial effect in myeloma therapy. Three kinases inhibitors (CHK1i, MELKi and PBKi) overcome resistance to Lenalidomide, while CHK1, PBK and DBF4 inhibitors re-sensitize Melphalan resistant cell line to this conventional therapeutic agent. Altogether, we demonstrate that kinase inhibitors could be of therapeutic interest especially in high-risk myeloma patients defined by the KI. CHEK1, MELK, PLK4, SRPK1, CDC7-DBF4, MPS1/TTK and PBK inhibitors could represent new treatment options either alone or in combination with Melphalan or IMiD for refractory/relapsing myeloma patients.
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http://dx.doi.org/10.3324/haematol.2018.208306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049359PMC
March 2020

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2.

Mol Cell 2019 08 26;75(3):605-619.e6. Epub 2019 Jun 26.

The Institute of Cancer Research, London, UK. Electronic address:

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2 cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.
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http://dx.doi.org/10.1016/j.molcel.2019.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695479PMC
August 2019

Overexpression of the Fork Protection Complex: a strategy to tolerate oncogene-induced replication stress in cancer cells.

Mol Cell Oncol 2019 7;6(4):1607455. Epub 2019 May 7.

Equipe Labellisée Ligue contre le Cancer, Institut de Génétique Humaine, CNRS, Université de Montpellier, Montpellier, France.

Oncogene-induced replication stress (RS) plays an active role in tumorigenesis by promoting genomic instability but is also a challenge for cell proliferation. Recent evidence indicates that different types of cancer cells adapt to RS by overexpressing components of the ATR-CHK1 pathway that promote fork progression in a checkpoint-independent manner.
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http://dx.doi.org/10.1080/23723556.2019.1607455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548472PMC
May 2019

qDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing.

Nat Commun 2019 05 24;10(1):2313. Epub 2019 May 24.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, Texas, 77555, USA.

DNA double-strand breaks (DSBs) are among the most lethal types of DNA damage and frequently cause genome instability. Sequencing-based methods for mapping DSBs have been developed but they allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. Here we propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induce spike-in DSBs by a site-specific endonuclease and use them to quantify detected DSBs (labeled, e.g., using i-BLESS). Utilizing qDSB-Seq, we determine numbers of DSBs induced by a radiomimetic drug and replication stress, and reveal two orders of magnitude differences in DSB frequencies. We also measure absolute frequencies of Top1-dependent DSBs at natural replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.
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http://dx.doi.org/10.1038/s41467-019-10332-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534554PMC
May 2019

Inhibition of Ataxia-Telangiectasia Mutated and RAD3-Related () Overcomes Oxaliplatin Resistance and Promotes Antitumor Immunity in Colorectal Cancer.

Cancer Res 2019 06 15;79(11):2933-2946. Epub 2019 Apr 15.

Institut de Recherche en Cancérologie de Montpellier, INSERM U1194 Université Montpellier, CNRS, France.

Although many patients with colorectal cancer initially respond to the chemotherapeutic agent oxaliplatin, acquired resistance to this treatment remains a major challenge to the long-term management of this disease. To identify molecular targets of oxaliplatin resistance in colorectal cancer, we performed an shRNA-based loss-of-function genetic screen using a kinome library. We found that silencing of ataxia-telangiectasia mutated and RAD3-related (ATR), a serine/threonine protein kinase involved in the response to DNA stress, restored oxaliplatin sensitivity in a cellular model of oxaliplatin resistance. Combined application of the ATR inhibitor VE-822 and oxaliplatin resulted in strong synergistic effects in six different colorectal cancer cell lines and their oxaliplatin-resistant subclones, promoted DNA single- and double-strand break formation, growth arrest, and apoptosis. This treatment also increased replicative stress, cytoplasmic DNA, and signals related to immunogenic cell death such as calreticulin exposure and HMGB1 and ATP release. In a syngeneic colorectal cancer mouse model, combined administration of VE-822 and oxaliplatin significantly increased survival by promoting antitumor T-cell responses. Finally, a DNA repair gene signature discriminated sensitive from drug-resistant patients with colorectal cancer. Overall, our results highlight the potential of ATR inhibition combined with oxaliplatin to sensitize cells to chemotherapy as a therapeutic option for patients with colorectal cancer. SIGNIFICANCE: These findings demonstrate that resistance to oxaliplatin in colorectal cancer cells can be overcome with inhibitors of ATR and that combined treatment with both agents exerts synergistic antitumor effects. http://cancerres.aacrjournals.org/content/canres/79/11/2933/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-2807DOI Listing
June 2019

Overexpression of Claspin and Timeless protects cancer cells from replication stress in a checkpoint-independent manner.

Nat Commun 2019 02 22;10(1):910. Epub 2019 Feb 22.

Institut de Génétique Humaine, CNRS, Université de Montpellier, Equipe Labellisée Ligue Contre le Cancer, 34396, Montpellier, France.

Oncogene-induced replication stress (RS) promotes cancer development but also impedes tumor growth by activating anti-cancer barriers. To determine how cancer cells adapt to RS, we have monitored the expression of different components of the ATR-CHK1 pathway in primary tumor samples. We show that unlike upstream components of the pathway, the checkpoint mediators Claspin and Timeless are overexpressed in a coordinated manner. Remarkably, reducing the levels of Claspin and Timeless in HCT116 cells to pretumoral levels impeded fork progression without affecting checkpoint signaling. These data indicate that high level of Claspin and Timeless increase RS tolerance by protecting replication forks in cancer cells. Moreover, we report that primary fibroblasts adapt to oncogene-induced RS by spontaneously overexpressing Claspin and Timeless, independently of ATR signaling. Altogether, these data indicate that enhanced levels of Claspin and Timeless represent a gain of function that protects cancer cells from of oncogene-induced RS in a checkpoint-independent manner.
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http://dx.doi.org/10.1038/s41467-019-08886-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385232PMC
February 2019

DDR Inc., one business, two associates.

Curr Genet 2019 Apr 22;65(2):445-451. Epub 2018 Nov 22.

Equipe Labellisée Ligue contre le Cancer, Institut de Génétique Humaine, CNRS and Université de Montpellier, Montpellier, France.

Eukaryotic cells activate cell cycle checkpoints in response to DNA damage. In Saccharomyces cerevisiae, the DNA damage response is achieved by the activation of the sensor kinases Mec1 and Tel1 and transmitted to the effector kinase Rad53. Rad9 and Mrc1 are thought to differentially mediate the activation of Rad53 depending on the cell cycle phase. Rad9 can respond to DNA lesions throughout the cell cycle, whereas Mrc1 responds to replication impediments in S phase. It was not clear if Rad9 and Mrc1 were triggering the same response to DNA damage occurring in S phase. By carefully studying the kinetics of activation of Rad53 by different types of replication stresses, we recently showed that Rad9 and Mrc1 cooperate in time and space to trigger a unique response to DNA damage in S phase. This primarily includes the control of both DNA replication initiation and elongation. After showing that Rad9 plays a preponderant role during S phase, the data presented here provocatively suggest that Mrc1 could also mediate the activation of Rad53 outside of S phase.
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http://dx.doi.org/10.1007/s00294-018-0908-7DOI Listing
April 2019

i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks.

Commun Biol 2018 31;1:181. Epub 2018 Oct 31.

Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, 02-089, Warsaw, Poland.

Maintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.
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http://dx.doi.org/10.1038/s42003-018-0165-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6208412PMC
October 2018

SAMHD1 and the innate immune response to cytosolic DNA during DNA replication.

Curr Opin Immunol 2019 02 5;56:24-30. Epub 2018 Oct 5.

Institut de Génétique Humaine, CNRS, Université de Montpellier, Equipe Labellisée Ligue contre le Cancer, Montpellier France. Electronic address:

Cytosolic DNA of endogenous or exogenous origin is sensed by the cGAS-STING pathway to activate innate immune responses. Besides microbial DNA, this pathway detects self-DNA in the cytoplasm of damaged or abnormal cells and plays a central role in antitumor immunity. The mechanism by which cytosolic DNA accumulates under genotoxic stress conditions is currently unclear, but recent studies on factors mutated in the Aicardi-Goutières syndrome cells, such as SAMHD1, RNase H2 and TREX1, are shedding new light on this key process. In particular, these studies indicate that the rupture of micronuclei and the release of ssDNA fragments during the processing of stalled replication forks and chromosome breaks represent potent inducers of the cGAS-STING pathway.
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http://dx.doi.org/10.1016/j.coi.2018.09.017DOI Listing
February 2019

Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage.

EMBO J 2018 11 29;37(21). Epub 2018 Aug 29.

Institut de Génétique Humaine, CNRS, Equipe Labellisée Ligue contre le Cancer, Université de Montpellier, Montpellier, France

The S-phase checkpoint maintains the integrity of the genome in response to DNA replication stress. In budding yeast, this pathway is initiated by Mec1 and is amplified through the activation of Rad53 by two checkpoint mediators: Mrc1 promotes Rad53 activation at stalled forks, and Rad9 is a general mediator of the DNA damage response. Here, we have investigated the interplay between Mrc1 and Rad9 in response to DNA damage and found that they control DNA replication through two distinct but complementary mechanisms. Mrc1 rapidly activates Rad53 at stalled forks and represses late-firing origins but is unable to maintain this repression over time. Rad9 takes over Mrc1 to maintain a continuous checkpoint signaling. Importantly, the Rad9-mediated activation of Rad53 slows down fork progression, supporting the view that the S-phase checkpoint controls both the initiation and the elongation of DNA replication in response to DNA damage. Together, these data indicate that Mrc1 and Rad9 play distinct functions that are important to ensure an optimal completion of S phase under replication stress conditions.
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http://dx.doi.org/10.15252/embj.201899319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213276PMC
November 2018

DDK Has a Primary Role in Processing Stalled Replication Forks to Initiate Downstream Checkpoint Signaling.

Neoplasia 2018 10 26;20(10):985-995. Epub 2018 Aug 26.

Laboratory of Genome Integrity and Tumorigenesis, Van Andel Research Institute (VARI), Grand Rapids, MI 49503. Electronic address:

CDC7-DBF4 kinase (DDK) initiates DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has diverse and apparently conflicting roles in the replication checkpoint response in various organisms, but the underlying mechanisms are far from settled. We show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M cell cycle arrest. DDK exhibits genetic interactions with the ssDNA exonuclease EXO1 and phosphorylates EXO1 in vitro. EXO1 is also required for nascent strand degradation following exposure to HU, so DDK might regulate EXO1 directly. Lastly, sublethal DDK inhibition causes various mitotic abnormalities, which is consistent with a checkpoint deficiency. In summary, DDK has a primary and previously undescribed role in the replication checkpoint to promote ssDNA accumulation at stalled forks, which is required to initiate a robust checkpoint response and cell cycle arrest to maintain genome integrity.
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http://dx.doi.org/10.1016/j.neo.2018.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6111017PMC
October 2018

SAMHD1 acts at stalled replication forks to prevent interferon induction.

Nature 2018 05 18;557(7703):57-61. Epub 2018 Apr 18.

Institut de Génétique Humaine, CNRS, Université de Montpellier, Laboratoire Maintien de l'Intégrité du Génome au cours de la Réplication, Ligue Contre le Cancer, Montpellier, France.

SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.
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http://dx.doi.org/10.1038/s41586-018-0050-1DOI Listing
May 2018

Senataxin resolves RNA:DNA hybrids forming at DNA double-strand breaks to prevent translocations.

Nat Commun 2018 02 7;9(1):533. Epub 2018 Feb 7.

LBCMCP, Centre de Biologie Integrative (CBI), CNRS, Université de Toulouse, UT3, 118 Route de Narbonne, 31062, Toulouse, France.

Ataxia with oculomotor apraxia 2 (AOA-2) and amyotrophic lateral sclerosis (ALS4) are neurological disorders caused by mutations in the gene encoding for senataxin (SETX), a putative RNA:DNA helicase involved in transcription and in the maintenance of genome integrity. Here, using ChIP followed by high throughput sequencing (ChIP-seq), we report that senataxin is recruited at DNA double-strand breaks (DSBs) when they occur in transcriptionally active loci. Genome-wide mapping unveiled that RNA:DNA hybrids accumulate on DSB-flanking chromatin but display a narrow, DSB-induced, depletion near DNA ends coinciding with senataxin binding. Although neither required for resection nor for timely repair of DSBs, senataxin was found to promote Rad51 recruitment, to minimize illegitimate rejoining of distant DNA ends and to sustain cell viability following DSB production in active genes. Our data suggest that senataxin functions at DSBs in order to limit translocations and ensure cell viability, providing new insights on AOA2/ALS4 neuropathies.
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http://dx.doi.org/10.1038/s41467-018-02894-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803260PMC
February 2018

Dbf4 recruitment by forkhead transcription factors defines an upstream rate-limiting step in determining origin firing timing.

Genes Dev 2017 12 12;31(23-24):2405-2415. Epub 2018 Jan 12.

State Key Laboratory of Agro-Biotechnology, Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Biological Sciences, China Agricultural University, Beijing 100193, China.

Initiation of eukaryotic chromosome replication follows a spatiotemporal program. The current model suggests that replication origins compete for a limited pool of initiation factors. However, it remains to be answered how these limiting factors are preferentially recruited to early origins. Here, we report that Dbf4 is enriched at early origins through its interaction with forkhead transcription factors Fkh1 and Fkh2. This interaction is mediated by the Dbf4 C terminus and was successfully reconstituted in vitro. An interaction-defective mutant, , phenocopies alleles in terms of origin firing. Remarkably, genome-wide replication profiles reveal that the direct fusion of the DNA-binding domain (DBD) of Fkh1 to Dbf4 restores the Fkh-dependent origin firing but interferes specifically with the pericentromeric origin activation. Furthermore, Dbf4 interacts directly with Sld3 and promotes the recruitment of downstream limiting factors. These data suggest that Fkh1 targets Dbf4 to a subset of noncentromeric origins to promote early replication in a manner that is reminiscent of the recruitment of Dbf4 to pericentromeric origins by Ctf19.
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http://dx.doi.org/10.1101/gad.306571.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5795786PMC
December 2017

Nucleases Acting at Stalled Forks: How to Reboot the Replication Program with a Few Shortcuts.

Annu Rev Genet 2017 11;51:477-499

Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA; email:

In a lifetime, a human being synthesizes approximately 2×10 meters of DNA, a distance that corresponds to 130,000 times the distance between the Earth and the Sun. This daunting task is executed by thousands of replication forks, which progress along the chromosomes and frequently stall when they encounter DNA lesions, unusual DNA structures, RNA polymerases, or tightly-bound protein complexes. To complete DNA synthesis before the onset of mitosis, eukaryotic cells have evolved complex mechanisms to process and restart arrested forks through the coordinated action of multiple nucleases, topoisomerases, and helicases. In this review, we discuss recent advances in understanding the role and regulation of nucleases acting at stalled forks with a focus on the nucleolytic degradation of nascent DNA, a process commonly referred to as fork resection. We also discuss the effects of deregulated fork resection on genomic instability and on the unscheduled activation of the interferon response under replication stress conditions.
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http://dx.doi.org/10.1146/annurev-genet-120116-024745DOI Listing
November 2017