Philippe Pasero

• PhD
• Institute of Human Genetics

In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetyl...

The cohesin complex holds together newly replicated chromatids and is involved in diverse pathways that preserve genome integrity. We show that in budding yeast, cohesin is transiently recruited to active replication origins, and it spreads along DNA as forks progress. When DNA synthesis is impeded, cohesin accumulates at replication sites and is c...

Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here...

The over-expression of RNase H1 enables the progression of replication forks under stress and safeguards genome stability. The most common interpretation of these observations is that RNase H1 over-expression removes R-loops, whose formation would hinder replication fork progression. Here we challenge this model using an innovative strategy to rapi...

Background In order to better understand why about 15% of people living with Human Immunodeficiency Virus-1 (PWH) on highly active antiretroviral therapy do not restore their CD4 count, we explored the link previously reported between glutamate plasma level and CD4 count. Methods We recruited fourty-four adults living with HIV-1 aviremic under ant...

Cancer cells display high levels of oncogene-induced replication stress (RS) and rely on DNA damage checkpoint for viability. This feature is exploited by cancer therapies to either increase RS to unbearable levels or inhibit checkpoint kinases involved in the DNA damage response. Thus far, treatments that combine these two strategies have shown pr...

Cancer cells display high levels of oncogene-induced replication stress (RS) and rely on DNA damage checkpoint for viability. This feature is exploited by cancer therapies to either increase RS to unbearable levels or to inhibit checkpoint kinases involved in the DNA damage response (DDR). Thus far, treatments that combine these two strategies have...

Cancer cells display high levels of oncogene-induced replication stress (RS) and rely on DNA damage checkpoint for viability. This feature is exploited by cancer therapies to either increase RS to unbearable levels or to inhibit checkpoint kinases involved in the DNA damage response (DDR). Thus far, treatments that combine these two strategies have...

Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown....

Cancer cells display high levels of oncogene-induced replication stress (RS) and rely on DNA damage checkpoint for viability. This feature is exploited by cancer therapies to either increase RS to unbearable levels or to inhibit checkpoint kinases involved in the DNA damage response (DDR). Thus far, treatments that combine these two strategies have...

Cancer cells display high levels of oncogene-induced replication stress (RS) and rely on DNA damage checkpoint for viability. This feature is exploited by cancer therapies to either increase RS to unbearable levels or to inhibit checkpoint kinases involved in the DNA damage response (DDR). Thus far, treatments that combine these two strategies have...

Members of the conserved Pif1 family of 5′-3′ DNA helicases can unwind G4s and mitigate their negative impact on genome stability. In Saccharomyces cerevisiae, two Pif1 family members, Pif1 and Rrm3, contribute to the suppression of genomic instability at diverse regions including telomeres, centromeres and tRNA genes. While Pif1 can resolve laggin...

Multiple myeloma (MM) is a hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents that have significantly improved clinical outcome, most patients relapse and develop drug resistance. MM is characterized by genomic instability and a high level of replicative str...

Although ethanol is a class I carcinogen and is linked to more than 700,000 cancer incidences, a clear understanding of the molecular mechanisms underlying ethanol-related carcinogenesis is still lacking. Further understanding of ethanol-related cell damage can contribute to reducing or treating alcohol-related cancers. Here, we investigated the ef...

R‐loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest, and restart in Saccharomyces cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that whil...

Replication stress is an alteration in the progression of replication forks caused by a variety of events of endogenous or exogenous origin. In precancerous lesions, this stress is exacerbated by the deregulation of oncogenic pathways, which notably disrupts the coordination between replication and transcription, and leads to genetic instability an...

Upon DNA damage, cells activate the DNA damage response (DDR) to coordinate proliferation and DNA repair. Dietary, metabolic, and environmental inputs are emerging as modulators of how DNA surveillance and repair take place. Lipids hold potential to convey these cues, although little is known about how. We observed that lipid droplet (LD) number sp...

Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown....

Oncogene-induced senescence (OIS) is a tumor-suppressive mechanism that arrests cell proliferation in response to oncogene-induced replication stress (RS). OIS also depends on the cGAS-STING pathway, which detects cytosolic DNA and promotes the expression of type I interferons (IFN) and pro-inflammatory cytokines. Whether and how the RS and IFN res...

Multiple myeloma (MM) is a hematologic cancer characterized by accumulation of malignant plasma cells in the bone marrow. To date, no definitive cure exists for MM and resistance to current treatments is one of the major challenges of this disease. The DNA helicase BLM, whose depletion or mutation causes the cancer-prone Bloom’s syndrome (BS), is a...

In brief Aiello et al. show that the helicase Sen1 functions broadly for limiting genome-wide conflicts of RNA polymerase II with replication and other RNA polymerases and collaborates with RNases H to maintaingenome stability. A new approach (H-CRAC) allows for sensitive and high-resolution mapping of R-loops genome wide. SUMMARY Cellular homeosta...

Upon DNA damage, cells activate the DNA Damage Response (DDR) to coordinate proliferation and DNA repair. Dietary, metabolic, and environmental inputs are emerging as modulators of how DNA surveillance and repair take place. Lipids hold potential to convey these cues, although little is known about how. We observed that lipid droplet (LD) number sp...

Background Lymphopenia is predictive of survival in Coronavirus disease 2019 (COVID-19) patients. Objective The aim of this study was to understand the cause of a lymphocyte count drop in severe forms of SARS-CoV-2 infection. Methods Monocytic production of reactive oxygen species (ROS) and T cell apoptosis were measured by flow cytometry, DNA da...

The current methods for measuring the DNA damage response (DDR) are relatively labor-intensive and usually based on Western blotting, flow cytometry, and/or confocal immunofluorescence analyses. They require many cells and are often limited to the assessment of a single or few proteins. Here, we used the Celigo® image cytometer to evaluate the cell...

Cellular homeostasis requires the coordination of several machineries concurrently engaged on the DNA. Wide-spread transcription can interfere with other processes and transcription-replication conflicts (TRCs) threaten genome stability. The conserved Sen1 helicase terminates non-coding transcription, but also interacts with the replisome and repor...

Diffuse large B-cell lymphoma (DLBCL) is the most common hematological malignancy. Although more than half of patients with DLBCL achieve long-term remission, the majority of remaining patients succumb to the disease. As abnormal iron homeostasis is implicated in carcinogenesis and the progression of many tumors, we searched for alterations in iron...

Camptothecin (CPT) is a specific inhibitor of the DNA topoisomerase I (Top1p), currently used in cancer therapy, which induces DNA damage and cell death. Top1p is highly active at the repeated ribosomal DNA locus (rDNA) to relax DNA supercoiling caused by elevated transcription and replication occurring in opposite directions. Fob1p interacts with,...

Multiple myeloma (MM) is the second most frequent hematological malignancy, characterized by the accumulation of malignant plasma cells (PCs) within the bone marrow. To date, there is no definitive treatment for this pathology and a majority of patients will invariably relapse. Antibody secretion, the key biological function of PCs, is maintained i...

Replication-associated single-ended DNA double-strand breaks (seDSBs) are repaired predominantly through RAD51-mediated homologous recombination (HR). Removal of the non-homologous end-joining (NHEJ) factor Ku from resected seDSB ends is crucial for HR. The coordinated actions of MRE11-CtIP nuclease activities orchestrated by ATM define one pathway...

Replication stress (RS) is a hallmark of cancer cells that is associated with increased genomic instability. RS occurs when replication forks encounter obstacles along the DNA. Stalled forks are signaled by checkpoint kinases that prevent fork collapse and coordinate fork repair pathways. Fork restart also depends on chromatin remodelers to increas...

Immunotherapy has revolutionized cancer treatment and substantially improved patient outcome with regard to multiple tumour types. However, most patients still do not benefit from such therapies, notably because of the absence of pre-existing T cell infiltration. DNA damage response (DDR) deficiency has recently emerged as an important determinant...

Transcription-replication conflicts (TRCs) represent a potential source of endogenous replication stress (RS) and genomic instability in eukaryotic cells but the mechanisms that underlie this instability remain poorly understood. Part of the problem could come from non-B DNA structures called R-loops, which are formed of a RNA:DNA hybrid and a disp...

Plasma cells (PCs) have an essential role in humoral immune response by secretion of antibodies, and represent the final stage of B lymphocytes differentiation. During this differentiation, the pre-plasmablastic stage is characterized by highly proliferative cells that start to secrete immunoglobulins (Igs). Thus, replication and transcription must...

This protocol describes how to culture, image, and determine the nuclear position of a fluorescently tagged DNA locus in the 3D nucleoplasm of fixed Saccharomyces cerevisiae cells. Here, we propose a manual scoring method based on widefield images and an automated method based on 3D-SIM images. Yeast culture conditions have to be followed meticulou...

The genome of eukaryotic cells is particularly at risk during the S phase of the cell cycle, when megabases of chromosomal DNA are unwound to generate two identical copies of the genome. This daunting task is executed by thousands of micro-machines called replisomes, acting at fragile structures called replication forks. The correct execution of th...

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 gen...

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 d...

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 gen...

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 noncodin...

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...

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...

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...

DNA replication is an extremely complex process, involving thousands of replication forks progressing along chromosomes. These forks are frequently slowed down or stopped by various obstacles, such as secondary DNA structures, chromatin-acting proteins or a lack of nucleotides. This slowing down, known as replicative stress, plays a central role in...

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...

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 isolated from HS patients and more precisely the Outer Root Sheath Cells (ORS). We show that hair follicles from HS patients have an incr...

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 be...

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 compo...

Introduction Une anomalie du follicule pileux serait impliquée dans l’hidradénite suppurée (HS). Nous avons étudié les cellules de la gaine externe du follicule pileux (ORS). Nos résultats montrent que les ORS des patients HS (ORS-HS) sécrètent spontanément IP-10 et RANTES qui recrutent les cellules immunitaires. Notre étude actuelle se focalise su...

Diffuse large B cell lymphoma (DLBCL) is the most common hematologic malignancy. Although more than half of these patients may achieve long-term remission, the majority of the remaining patients succumb to DLBCL.These patients relapse after conventional Rituximab (R)-based chemotherapy regimen, such as CHOP (cyclophosphamide, doxorubicin, vincristi...

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 chro...

Impediments to DNA replication threaten genome stability. The homologous recombination (HR) pathway is involved in the restart of blocked replication forks. Here, we used a new method to study at the molecular level the restart of replication in response to DNA topoisomerase I poisoning by camptothecin (CPT). We show that HR-mediated restart at the...

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 investiga...

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 t...

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 pro...

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 mann...

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 scr...

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...

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 check...

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...

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...

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 hav...

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 sta...

Supplementary Figure 1: DDK activity is required for CHK1 activation and RPA2 accumulation in response to various replication stresses. (A) HCC1954 cells were pre-treated with DMSO or DDKi for the indicated times, then exposed to HU for 2 hours, followed by blotting for pCHK1 S317 or total CHK1. (B, C) HCC1954 cells were transfected with scrambled...

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...

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 th...

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 th...

Despite the recent advances achieved in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. The aim of this project is thus to identify new targets involved in resistance to oxaliplatin using a phenotypic high-throughput screening. We established oxaliplatin-resistant cellular clones from the colon carcinoma...

In a lifetime, a human being synthesizes approximately 2×10¹⁶ 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...

CDC7-DBF4 kinase (DDK) is required to initiate DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has also been reported to have diverse and sometimes conflicting roles in the replication checkpoint response in various organisms but the underlying mechanisms are far from settled. Here we show that human DDK promotes limit...

Emerging genome-wide methods for mapping DNA double-strand breaks (DSBs) by sequencing (e.g. BLESS) are limited to measuring relative frequencies of breaks between loci. Knowing the absolute DSB frequency per cell, however, is key to understanding their physiological relevance. Here, we propose quantitative DSB sequencing (qDSB-Seq), a method to in...

DNA double-strand breaks (DSBs) can be detected by label-based sequencing or pulsed-field gel electrophoresis (PFGE). Sequencing yields population-average DSB frequencies genome-wide, while PFGE reveals percentages of broken chromosomes. We constructed a mathematical framework to combine advantages of both: high-resolution DSB locations and their p...

Interference between DNA replication and transcription represents a major source of genomic instability. In this issue of Cell, Lang et al. and Hamperl et al. show that head-on collisions, but not codirectional collisions, impede fork progression in bacteria and in human cells by promoting the formation of RNA-DNA hybrids known as R-loops.

The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical...

Multiple myeloma (MM) is a plasma cell cancer with poor survival, characterized by the expansion of multiple myeloma cells (MMCs) in the bone marrow. Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the most downregulated genes. RecQ helicases are DNA...

Leishmaniasis is a common tropical disease that affects mainly poor people in underdeveloped and developing countries. This largely neglected infection is caused by Leishmania spp, a parasite from the Trypanosomatidae family. This parasitic disease has different clinical manifestations, ranging from localized cutaneous to more harmful visceral form...