From the nuclear pore to DNA damage : the ATR-mediated shuttling of Ddx19 to resolve transcription-replication conflicts

Les cellules sont constamment exposées à des agents endommageant de l'ADN d'origine exogène, notamment les rayons ultraviolets, les irradiations γ, et l'exposition aux agents chimiques génotoxiques, mais également d'origine endogène générés par le métabolisme cellulaire. De plus en plus d'évidences montrent que la transcription est un processus biologique qui peut mettre en péril l'intégrité du génome. Un mécanisme actuellement très étudié qui lie la transcription à l'instabilité génomique est la formation des boucles R (R-loops), des structures hybrides ARN:ADN qui exposent un ADN simple brin déplacé. Ces structures aberrantes se présentent en tant que sous-produits de la transcription et/ou lors de l'interférence entre la réplication et la transcription, et plus récemment ils ont été montrées s'accumuler lorsque la biogénèse de l'ARNm est perturbée. La persistance des boucles R est une source importante d'instabilité génomique car elle peut générer des cassures double brin de l'ADN et favoriser la recombinaison. Pour faire face aux conséquences néfastes des endommagements de l'ADN, les cellules activent une cascade élaborée de voies de signalisation qui permet de coordonner la prolifération cellulaire avec la réparation de l'ADN. L'ensemble de ces acteurs moléculaires constitue un réseau de réponse aux dommages de l'ADN qui est indispensable pour la stabilité génomique. Récemment chez la levure, l'activation transitoire de ce réseau a été également proposée être important dans la coordination de la transcription et de la réplication, afin d'éviter d'une part des contraintes topologiques et d'autre part la formation de structures aberrantes générées lors de conflits entre ces deux processus cellulaires essentiels. Dans la perspective d'identifier des nouveaux gènes impliqués dans ce réseau de signalisation, un crible fonctionnel in vitro précédemment établi au laboratoire a conduit à l'identification de Ddx19, une hélicase à motif DEAD-box, en tant que nouvel élément répondant à l'endommagement de l'ADN. Ddx19 interagit avec le pore nucléaire via CAN/Nup214, et il est impliqué dans l'export des ARNm grâce à son activité hélicase et ATPase, stimulé par les facteurs IP6 et Gle1. Le présent travail de thèse dévoile une nouvelle fonction de Ddx19 distincte de son rôle connu dans l'export de l'ARNm. Je pu montrer que, lors de l'induction des dommages à l'ADN par les rayons UV, Ddx19 se relocalise transitoirement de la face cytoplasmique du nucléopore vers le noyau de façon dépendant d'ATR. L'inactivation de Ddx19 entraîne des endommagements spontanées dépendant de la prolifération, démontré par l'activation de la voie de signalisation d'ATM-Chk2 et la formation de foyers nucléaires de γH2AX et 53BP1. Ces phénotypes sont concomitants avec le ralentissement des fourches de réplication qui ne peuvent plus redémarrer après leur blocage par la camptothécine. En outre, les cellules déplétées de Ddx19 présentent une forte accumulation des boucles R nucléaires, enrichi dans le compartiment nucléolaire, et aussi autour de la périphérie nucléaire. Par ailleurs, ces cellules présentent une viabilité réduite et une létalité synthétique lorsque la déplétion de Ddx19 est combinée avec l'inhibition de l'expression de la topoisomérase I. Je propose Ddx19 comme deuxième hélicase nécessaire pour la résolution des boucles R, et qui fonctionne à côté mais de façon indépendante de la Senataxin, l'hélicase précédemment connue pour résoudre ces structures in vivo chez les cellules de mammifères. Je démontre que cette nouvelle fonction de Ddx19 ne dépend pas de son interaction avec le pore nucléaire, mais plutôt de son activité hélicase et d'un résidu de sérine phosphorylée par Chk1 qui stimule sa relocalisation vers le noyau. Ces données proposent Ddx19 en tant que nouvelle ARN hélicase qui facilite la coordination de la réplication et la transcription, médiée par ATR à travers de la résolution des boucles R, préservant ainsi l'intégrité du génome.Cells are continuously challenged by DNA damage resulting from external cues as UV light, γ-irradiation and exposure to genotoxic chemicals, as well as from endogenous stress caused by cellular metabolism. Growing evidence points to transcription as a biological process that could adversely affect genome integrity. One currently highly investigated mechanism by which transcription can induce genome instability is through the formation of R-loops, RNA:DNA hybrid structures exposing a displaced single-stranded DNA tract. These aberrant structures occur as byproducts of transcription and/or upon interference between replication and transcription, and more recently were also shown to accumulate upon disruption of mRNA biogenesis and processing. Persistent unresolved R-loops are a potent source of genomic instability as they ultimately generate double strand breaks and promote recombination events. To deal with the deleterious consequences of DNA damage, cells activate elaborate DNA damage response (DDR) pathways to delay cell division and stimulate repair of lesions, thus preserving genome stability. Recently in yeast transient DDR activation has also been proposed to be important in the coordination of transcription and replication, in order to avoid topological constraints and the formation of aberrant structures generated upon collision of their machineries. By means of an in vitro screen aimed at identifying new DDR genes, we isolated Ddx19, a DEAD-Box helicase known to be involved in mRNA export, as a novel DNA damage responsive gene. Ddx19 interacts with the nucleopore complex via nucleoporin CAN/Nup214, and is involved in mRNA remodelling and export through its ATPase and helicase activities, stimulated by IP6 and the Gle1 factor. My present thesis work unravels a novel function of Ddx19 in preserving genome stability in mammalian cells, distinct from its known role in mRNA export. I show that upon UV-induced damage, Ddx19 transiently relocalizes from the cytoplasmic face of the nucleopore to the nucleus in an ATR-dependent manner. Downregulation of Ddx19 gives rise to spontaneous, proliferation-dependent DNA damage, as determined by the specific activation of the ATM-Chk2 pathway and formation of γH2AX and 53BP1 nuclear foci. This is concomitant with the slowing down of replication forks that are unable to restart after being stalled with camptothecin. In addition, cells depleted of Ddx19 display strong accumulation of nuclear R-loops, enriched in the nucleolar compartment, and around the nuclear periphery. Moreover, these cells show low viability and exhibited synthetic lethality when combined with inhibition of topoisomerase I expression. I propose Ddx19 as a second helicase required for R-loops resolution, functioning alongside but independently of Senataxin, the first known RNA helicase to resolve these structures in vivo in mammalian cells. I provide evidence that this new function of Ddx19 does not depend on its interaction with the nuclear pore, but rather on its helicase activity and on a serine residue phosphorylated by Chk1 which promotes its relocalization into the nucleus upon damage. These data put forward Ddx19 as a novel RNA helicase that facilitates ATR-dependent coordination of DNA replication and transcription through R-loops resolution, thus preserving genome integrity

Ddx19 links mRNA nuclear export with progression of transcription and replication and suppresses genomic instability upon DNA damage in proliferating cells

International audienceThe DEAD-box Helicase 19 (Ddx19) gene codes for an RNA helicase involved in both mRNA (mRNA) export from the nucleus into the cytoplasm and in mRNA translation. In unperturbed cells, Ddx19 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore. Here we review recent findings related to an additional Ddx19 function in the nucleus in resolving RNA:DNA hybrids (R-loops) generated during collision between transcription and replication, and upon DNA damage. Activation of a DNA damage response pathway dependent upon the ATR kinase, a major regulator of replication fork progression, stimulates translocation of the Ddx19 protein from the cytoplasm into the nucleus. Only nuclear Ddx19 is competent to resolve R-loops, and down regulation of Ddx19 expression induces DNA double strand breaks only in proliferating cells. Overall these observations put forward Ddx19 as an important novel mediator of the crosstalk between transcription and replication

Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites

International audienceImbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihy-dropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of di-hydropyrimidines induces the formation of DNA-protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication in vitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromo-somal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors

Combination of arsenic and interferon-α inhibits expression of KSHV latent transcripts and synergistically improves survival of mice with primary effusion lymphomas.

BACKGROUND: Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphomas (PEL). PEL cell lines infected with KSHV, but negative for Epstein-Barr virus have a tumorigenic potential in non-obese diabetic/severe combined immunodeficient mice and result in efficient engraftment and formation of malignant ascites with notable abdominal distension, consistent with the clinical manifestations of PEL in humans. METHODOLOGY/PRINCIPAL FINDINGS: Using this preclinical mouse model, we demonstrate that the combination of arsenic trioxide and interferon-alpha (IFN) inhibits proliferation, induces apoptosis and downregulates the latent viral transcripts LANA-1, v-FLIP and v-Cyc in PEL cells derived from malignant ascites. Furthermore, this combination decreases the peritoneal volume and synergistically increases survival of PEL mice. CONCLUSION/SIGNIFICANCE: These results provide a promising rationale for the therapeutic use of arsenic/IFN in PEL patients

MCM8-and MCM9 Deficiencies Cause Lifelong Increased Hematopoietic DNA Damage Driving p53- Dependent Myeloid Tumors

International audienceHematopoiesis is particularly sensitive to DNA damage. Myeloid tumor incidence increases in patients with DNA repair defects and after chemotherapy. It is not known why hematopoietic cells are highly vulnerable to DNA damage. Addressing this question is complicated by the paucity of mouse models of hematopoietic malignancies due to defective DNA repair. We show that DNA repair-deficient Mcm8- and Mcm9-knockout mice develop myeloid tumors, phenocopying prevalent myelodysplastic syndromes. We demonstrate that these tumors are preceded by a lifelong DNA damage burden in bone marrow and that they acquire proliferative capacity by suppressing signaling of the tumor suppressor and cell cycle controller RB, as often seen in patients. Finally, we found that absence of MCM9 and the tumor suppressor Tp53 switches tumorigenesis to lymphoid tumors without precedent myeloid malignancy. Our results demonstrate that MCM8/9 deficiency drives myeloid tumor development and establishes a DNA damage burdened mouse model for hematopoietic malignancies

Arsenic/IFN synergistically inhibited proliferation and induced apoptosis of ascites-derived BC3 (left) and BCBL-1 cells (right).

<p>(<b>A</b>) Cell Proliferation: Cells were plated in a 96 well format and treated with the single agent drugs or their combinations for 24, 48, and 72h. Results are expressed as percent of control, plotted as mean ± SD, and are representative of two independent experiments. (<b>B</b>) Annexin V staining: BC-3 and BCBL-1 ascites were treated for 48h. Histograms represent the proportion of apoptotic cells. Results are plotted as mean ± SD and are representative of at least 2 independent experiments. (<b>C</b>) TUNEL assay: BC-3 and BCBL-1 cells derived from PEL ascites were treated for 72h. Histograms represent apoptotic cells as percentage of the untreated controls and are plotted as mean ± SD.</p

Arsenic combined with IFN induced capase-dependent apoptosis and latent viral proteins downregulation in BC-3 cells.

<p>(<b>A</b>) Western blot analysis of BC-3 cells treated for 48h using PARP-specific antibody. (<b>B</b>) Western blot analysis of <i>ex-vivo</i> treated (48h) ascites derived BC-3 cells using LANA-1 and LANA-2 specific antibodies.</p

Arsenic and IFN synergistically prolonged survival in PEL mice.

<p>(<b>A</b>) Mice phenotype before and after treatment with arsenic/IFN. (<b>B</b>) Solid PEL tumors in untreated mice. (<b>C</b>) PCR for V-FLIP gene expression in different organs. (<b>D</b>) Kaplan–Meier analysis of overall survival curves of PEL NOD/SCID mice. Mice (<i>n</i> = 10 for each condition) were inoculated with 2x10⁶ of BC-3 (left) and BCBL-1 (right) cells, respectively. Treatment with the single agent drugs or their combinations was initiated at 2 days post-inoculation of PEL cells for a total of 21 days. The symbol * was used to compare treatment groups to control, while the symbol ‡ was used to compare combination treatments to single treatments. (*, ‡) indicates p< 0.05; (**, ‡‡) indicates p< 0.01; and (***, ‡‡‡) indicates p< 0.001.</p

Arsenic and IFN delayed ascites formation in PEL mice.

<p>Peritoneal volume on day 45 post-treatment. PEL NOD/SCID mice (n=10 per condition) were visually monitored. Peritoneal diameter (d) was measured with a caliber to assess ascites development. Peritoneal volume was calculated according to the formula: v=4/3π(d/2)³. The symbol * was used to compare treatment groups to control (*, **, and *** indicates p< 0.05, p< 0.01, and p< 0.001, respectively).</p

Arsenic/IFN synergistically inhibited expression of latent viral transcripts.

<p>Relative expression levels of different samples were calculated by standardization of the amount of target transcript for (A) LANA-1, (B) v-Cyclin or (C) v-FLIP, in a sample to the amount of housekeeping Glucose-6-phosphate dehydrogenase (G6PDH) RNA analyzed in the same sample. In addition, the averages of the normalized control values of Glucose-6-phosphate dehydrogenase (G6PDH) for each sample were used to determine the relative changes in gene expression of the KSHV latency protein LANA-1 by the comparative CT method (2^-ΔΔC_T) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079474#B12" target="_blank">12</a>]. Untreated BC3 and BCBL-1 ascites were used as a calibrator for viral gene expression. The Y-axis represents the relative quantities expressed as percent of the control. The symbol * was used to compare treatment groups to control, while the symbol ‡ was used to compare combination treatments to single treatments. (*, ‡) indicates p< 0.05; (**, ‡‡) indicates p< 0.01; and (***, ‡‡‡) indicates p< 0.001.</p