La réplication de l’ADN est un processus extrêmement complexe, impliquant des milliers de fourches de réplication progressant le long des chromosomes. Ces fourches sont fréquemment ralenties ou arrêtées par différents obstacles, tels que des structures secondaires de l’ADN, des protéines agissant sur la chromatine ou encore un manque de nucléotides. Ce ralentissement, qualifié de stress réplicatif, joue un rôle central dans le développement tumoral. Des processus complexes, qui ne sont pas encore totalement connus, sont mis en place pour répondre à ce stress. Certaines nucléases, comme MRE11 et DNA2, dégradent l’ADN néorépliqué au niveau des fourches bloquées, ce qui permet le redémarrage des réplisomes.La voie interféron est un mécanisme de défense contre les agents pathogènes qui détecte la présence d’acides nucléiques étrangers dans le cytoplasme et active la réponse immunitaire innée. Des fragments d’ADN issus du métabolisme de l’ADN génomique (réparation, rétrotransposition) peuvent diffuser dans le cytoplasme et activer cette voie. Une manifestation pathologique de ce processus est le syndrome d’Aicardi-Goutières, une maladie rare caractérisée par une inflammation chronique générant des problèmes neurodégénératifs et développementaux. Dans le cadre de cette encéphalopathie, il a été suggéré que la réplication de l’ADN pouvait générer des fragments d’ADN cytosoliques, mais les mécanismes impliqués n'avaient pas été caractérisés.SAMHD1 est fréquemment muté dans le syndrome d’Aicardi-Goutières ainsi que dans certains cancers, mais son rôle dans l’étiologie de ces maladies était jusqu’à présent largement inconnu. Ce facteur de restriction du VIH possède une activité dNTPase impliquée dans la régulation des pools de nucléotides en G1, ainsi qu’une activité 3’-5’ exonucléase qui est, encore aujourd’hui, controversée.Le but de mon projet de thèse était de comprendre les mécanismes moléculaires responsables de l’inflammation dans les cellules déficientes pour SAMHD1.Nous montrons que de l’ADN cytosolique s’accumule dans les cellules déficientes pour SAMHD1, particulièrement en présence de stress réplicatif, activant la réponse interféron. Par ailleurs, SAMHD1 est important pour la réplication de l’ADN en conditions normales et pour le processing des fourches arrêtées, indépendamment de son activité dNTPase. De plus, SAMHD1 stimule l’activité exonucléase de MRE11 in vitro. Lorsque SAMHD1 est absent, la dégradation de l’ADN néosynthétisé est inhibée, ce qui empêche l’activation du checkpoint de réplication et entraine un défaut de redémarrage des fourches de réplication. De plus, la résection des fourches de réplication est réalisée par un mécanisme alternatif qui libère des fragments d’ADN dans le cytosol, activant la réponse interféron.Les résultats obtenus pendant ma thèse montrent, pour la première fois, un lien direct entre la réponse au stress réplicatif et la production d’interférons. Ces résultats ont des conséquences importantes dans notre compréhension du syndrome d’Aicardi Goutières et des cancers liés à SAMHD1. Par exemple, nous avons démontré que MRE11 et RECQ1 sont responsables de la production des fragments d’ADN qui déclenchent la réponse inflammatoire dans les cellules déficientes pour SAMHD1. Nous pouvons donc imaginer que bloquer l’activité de ces enzymes pourrait diminuer la production des fragments d’ADN et, in fine, l’activation de l’immunité innée dans ces cellules. Par ailleurs, la voie interférons joue un rôle essentiel dans l’efficacité thérapeutique de l’irradiation et de certains agents chimiothérapiques comme l’oxaliplatine. Moduler cette réponse pourrait donc avoir un intérêt beaucoup plus large en thérapie anti-tumorale. / DNA replication is an utterly complex process, implicating thousands of replication forks that progress along chromosomes. These forks frequently slow-down or stall when they encounter obstacles such as DNA secondary structures, proteins acting on chromatin or a lack of dNTPs. Such impediment on the progression of replication forks, termed replication stress, plays a major role in tumorigenesis. Intricate processes, still not entirely understood, are established to respond to this stress. For instance, nucleases such as DNA2 and MRE11 degrade nascent DNA at arrested forks to allow their restart.The interferon pathway is a defense mechanism against pathogens that detects non-self-nucleic acids in the cytosol to activate the innate immune response. However, DNA fragments originating from the metabolism of genomic DNA, such as DNA repair and retrotransposition, may also diffuse into the cytosol to activate this pathway. The Aicardi-Goutières Syndrome (AGS), a rare genetic disorder characterized by neurological and developmental defects is caused by chronic inflammation due to the over-production of type I IFNs. It has been proposed that DNA replication may generate cytosolic DNA fragments triggering this encephalopathy. However, the mechanisms involved have remained unexplored.SAMHD1 is frequently mutated in the Aicardi-Goutières Syndrome as well as in some cancers. However, its role in the etiology of these diseases remains poorly understood. This HIV-1 restriction factor has a dNTPase activity involved in the regulation of dNTP pools and a putative 3’-5’ exonuclease activity.The goal of my PhD thesis was to understand the molecular mechanisms responsible for inflammation induced by SAMHD1 deficiency.We show that cytosolic DNA accumulates in SAMHD1-deficient cells, especially in conditions of replication stress, activating the interferon response. In addition, we demonstrate that SAMHD1 is necessary for DNA replication and for the processing of arrested forks, independently of its dNTPase activity. SAMHD1 stimulates the exonuclease activity of MRE11 in vitro. In the absence of SAMHD1, nascent DNA degradation is inhibited, preventing replication checkpoint activation and fork restart. Besides, forks are aberrantly processed by an alternative pathway that generates cytosolic DNA fragments, thereby activating the interferon response.Altogether, we demonstrate for the first time a direct link between the DNA replication stress response and interferon production. This result has important consequences regarding our understanding of the Aicardi-Goutières Syndrome and cancers caused by SAMHD1 mutations, which could be translated into new therapeutic opportunities. For instance, we have shown that MRE11 and RECQ1 are responsible for the production of DNA fragments triggering the pro-inflammatory response in SAMHD1-deficient cells. Inhibiting these enzymes decreases the production of cytosolic nucleic acids and, consequently, reduces the activation of cell-autonomous innate immunity in SAMHD1-depleted cells. Accordingly, inhibiting these enzymes may as well decrease IFN production in AGS in vivo models caused by SAMHD1 mutations. The interferon pathway also plays a major role in tumorigenesis as well as in the clinical efficacy of irradiation and some chemotherapeutic agents such as oxaliplatin. Modulating this response may therefore have much broader implications in anti-cancer therapy.

Aicardi-Goutières Syndrome (AGS) is a single gene, autoimmune disorder, with variable onset in the first year of life. Its clinical features exhibit similarities to several autoimmune diseases and congenital viral infections. AGS can result from mutations in ADAR1, TREX1 and SAMHD1 as well as any of the three genes that encode the protein subunits of the RNase H2 enzyme. It is hypothesised that impairment of nucleic acid metabolism results in abnormal nucleic acid species within the cell. This in turn is thought to cause the aberrant immune response that leads to AGS. The RNase H2 complex contains the catalytic RNASEH2A subunit and the auxiliary RNASEH2B and RNASEH2C subunits, which are thought to provide structural support and facilitate interactions with additional cellular proteins. RNase H2 can cleave the RNA strand of an RNA:DNA hybrid as well as 5’ of a single ribonucleotide embedded in dsDNA. Therefore, RNase H2 may have roles in several cellular processes, including DNA replication and repair, transcription, and viral infection. The aim of this PhD project was to investigate the physiological functions of RNase H2. The localisation of the RNase H2 proteins was investigated using EGFP-tagging and fluorescent microscopy. The interaction between the PIP-box of RNASEH2B and PCNA was found to localise RNase H2 and not RNase H1 to nuclear replication foci during S-phase. This suggests that RNase H2 is the dominant RNase H activity during DNA replication. Stable cell lines expressing EGFP-RNASEH2B and an alternative isoform, EGFP-RNASEH2Balt, were generated and used to perform a protein-protein interaction screen by GFP-Trap and mass spectrometry. The results indicate putative physical interactions between RNASEH2B and other factors involved in DNA replication and repair. Further evidence for a role in DNA repair was revealed when mammalian RNase H2 null cells were treated with hydroxyurea. Low doses of hydroxyurea increased ribonucleotide incorporation into genomic DNA and impaired S-phase progression. In contrast to wild-type cells, RNase H2 null cell proliferation also failed to recover from this replicative stress after HU withdrawal. However, the ribonucleotide content of genomic DNA from these cells did return to pre-hydroxyurea treatment levels. This suggests that an alternative repair pathway exists in mammalian cells, which can remove ribonucleotides from DNA in the absence of RNase H2, but that this pathway is also harmful to the cells. There is evidence that TREX1 facilitates viral infection while SAMHD1 has been shown to restrict viral infection. Therefore, experiments were performed to investigate if RNase H2 could be a viral facilitator or restriction factor. Ribonucleotides can be incorporated into viral DNA, so RNase H2 could act as a restriction factor by nicking and damaging the pre-integration complex. However, RNase H2 could also function as a facilitator of infection by processing viral RNA:DNA hybrid by-products and thus prevent the host immune response. The data obtained during this PhD project provides further evidence that RNase H2 is involved in DNA replication and repair and has contributed to the understanding of the function of RNase H2 in the cell. However, it is still unknown how mutations in RNase H2 lead to the pathology of AGS.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive encephalopathy with low incidence. The disease is caused by mutations in the genes encoding for TREX1, SAMHD1, ADAR, IFIH1 and the three genes encoding for the heterotrimeric RNase H2 enzyme. Biallelic mutations in any of the genes cause elevated type I interferon levels in the cerebrospinal fluid (CSF), the hallmark of AGS. In AGS patients, increased type I interferon levels cause massive inflammation in the brain that leads to mental and physical retardation that likely cause death in early childhood. AGS shows significant overlap with the prototypic autoimmune disease systemic lupus erythematosus (SLE). Like AGS patients, SLE patients are also characterized by increased type I interferon levels, anti-nuclear autoantibodies (ANAs) and arthritis. Moreover, heterozygous mutations in TREX1, SAMHD1 and RNase H2 are also found in a small fraction of SLE patients. Due to the genetic, molecular and clinical overlap, AGS is regarded as a monogenic variant of SLE. This overlap allows for the investigation of SLE pathomechanisms using genetically engineered mouse models with AGS-related mutations. In order to generate a mouse model that allows for the identification of pathomechanisms in AGS patients with mutations in the genes encoding for the RNase H2 enzyme, we generated mice with deficiency for the RNase H2 enzyme. Mice with complete deficiency for the RNase H2 enzyme (Rnaseh2c-/- or Rnaseh2bKOF/KOF) died perinatally or were stillborn. Mouse embryonic fibroblasts (MEFs) from E14.5 Rnaseh2bKOF/KOF embryos displayed impaired proliferation that was caused by the accumulation of MEF cells in G2/M of the cell cycle which increased with cultivation time or if MEF cells were isolated from E18.5 Rnaseh2bKOF/KOF embryos. Gene expression analysis of E14.5 fetal liver cells revealed a robust p53-mediated DNA damage response with the cell cycle inhibitor cyclin- dependent kinase inhibitor 1a (Cdkn1a, p21) being the most up-regulated gene. We found increased numbers of phosphorylated histone H2AX (γH2AX) in fetal liver and thymus cells from E18.5 Rnaseh2bKOF/KOF embryos, indicative of DNA double-strand breaks. Finally, we could show increased ribonucleotide loads in genomic DNA from embryos that were completely deficient for the RNase H2 enzyme. Collectively, we have demonstrated that complete RNase H2 deficiency causes persistent genomic ribonucleotide loads that render the DNA instable and prone to DNA strand breaks. DNA damage leads to the activation of p53 that in turn activates the cell cycle inhibitor p21 that inhibits cell cycle progression and likely causes accumulation of RNase H2-deficient cells in G2/M. To bypass early lethality we also generated bone marrow chimera and cell type-specific knockouts of the Rnaseh2b gene. While fetal liver cells of E14.5 Rnaseh2bKOF/KOF embryos could maintain hematopoiesis of irradiated recipient mice for almost one year, bone marrow cells from these primary recipients failed to reconstitute lethally irradiated mice in a secondary transfer. In line with this observation, VavCre-mediated deletion of the Rnaseh2b gene caused a more than hundred fold reduction of peripheral blood B cells, while B cell numbers remained unaltered upon CD19Cre-mediated deletion that occurs much later in B cell development. These data suggested that RNase H2 deficiency leads to the accumulation of genomic ribonucleotides that might cause the accumulation of a so far uncharacterized DNA damage species with increasing cell cycle passages. The data further supported our hypothesis that the impact of RNase H2 deficiency is determined by the number of cell proliferation. Finally, an epidermis-specific knockout of the Rnaseh2b gene displayed the most dramatic phenotype. Knockout mice were characterized by hyperpigmentation, hair loss and spontaneous ulcerations of the skin. Microscopically, these mice displayed moderate thickening of the epidermis and dermal fibrosis as indicated by increased collagen deposition. Macroscopic skin phenotypes were completely dependent on p53 expression, since concomitant deletion of the p53 gene rescued mice from hyperpigmentation, hair loss and ulcerations. This data demonstrated that Rnaseh2b deficiency in the epidermis may also lead to DNA damage and subsequent p53 activation as shown for fetal liver from E14.5 RNase H2-deficient embryos. Preliminary data also indicate an increased incidence of cancer formation in RNase H2/p53 double knockouts, identifying the RNase H2 enzyme as an important tumor suppressor.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive encephalopathy with low incidence. The disease is caused by mutations in the genes encoding for TREX1, SAMHD1, ADAR, IFIH1 and the three genes encoding for the heterotrimeric RNase H2 enzyme. Biallelic mutations in any of the genes cause elevated type I interferon levels in the cerebrospinal fluid (CSF), the hallmark of AGS. In AGS patients, increased type I interferon levels cause massive inflammation in the brain that leads to mental and physical retardation that likely cause death in early childhood. AGS shows significant overlap with the prototypic autoimmune disease systemic lupus erythematosus (SLE). Like AGS patients, SLE patients are also characterized by increased type I interferon levels, anti-nuclear autoantibodies (ANAs) and arthritis. Moreover, heterozygous mutations in TREX1, SAMHD1 and RNase H2 are also found in a small fraction of SLE patients. Due to the genetic, molecular and clinical overlap, AGS is regarded as a monogenic variant of SLE. This overlap allows for the investigation of SLE pathomechanisms using genetically engineered mouse models with AGS-related mutations. In order to generate a mouse model that allows for the identification of pathomechanisms in AGS patients with mutations in the genes encoding for the RNase H2 enzyme, we generated mice with deficiency for the RNase H2 enzyme. Mice with complete deficiency for the RNase H2 enzyme (Rnaseh2c-/- or Rnaseh2bKOF/KOF) died perinatally or were stillborn. Mouse embryonic fibroblasts (MEFs) from E14.5 Rnaseh2bKOF/KOF embryos displayed impaired proliferation that was caused by the accumulation of MEF cells in G2/M of the cell cycle which increased with cultivation time or if MEF cells were isolated from E18.5 Rnaseh2bKOF/KOF embryos. Gene expression analysis of E14.5 fetal liver cells revealed a robust p53-mediated DNA damage response with the cell cycle inhibitor cyclin- dependent kinase inhibitor 1a (Cdkn1a, p21) being the most up-regulated gene. We found increased numbers of phosphorylated histone H2AX (γH2AX) in fetal liver and thymus cells from E18.5 Rnaseh2bKOF/KOF embryos, indicative of DNA double-strand breaks. Finally, we could show increased ribonucleotide loads in genomic DNA from embryos that were completely deficient for the RNase H2 enzyme. Collectively, we have demonstrated that complete RNase H2 deficiency causes persistent genomic ribonucleotide loads that render the DNA instable and prone to DNA strand breaks. DNA damage leads to the activation of p53 that in turn activates the cell cycle inhibitor p21 that inhibits cell cycle progression and likely causes accumulation of RNase H2-deficient cells in G2/M. To bypass early lethality we also generated bone marrow chimera and cell type-specific knockouts of the Rnaseh2b gene. While fetal liver cells of E14.5 Rnaseh2bKOF/KOF embryos could maintain hematopoiesis of irradiated recipient mice for almost one year, bone marrow cells from these primary recipients failed to reconstitute lethally irradiated mice in a secondary transfer. In line with this observation, VavCre-mediated deletion of the Rnaseh2b gene caused a more than hundred fold reduction of peripheral blood B cells, while B cell numbers remained unaltered upon CD19Cre-mediated deletion that occurs much later in B cell development. These data suggested that RNase H2 deficiency leads to the accumulation of genomic ribonucleotides that might cause the accumulation of a so far uncharacterized DNA damage species with increasing cell cycle passages. The data further supported our hypothesis that the impact of RNase H2 deficiency is determined by the number of cell proliferation. Finally, an epidermis-specific knockout of the Rnaseh2b gene displayed the most dramatic phenotype. Knockout mice were characterized by hyperpigmentation, hair loss and spontaneous ulcerations of the skin. Microscopically, these mice displayed moderate thickening of the epidermis and dermal fibrosis as indicated by increased collagen deposition. Macroscopic skin phenotypes were completely dependent on p53 expression, since concomitant deletion of the p53 gene rescued mice from hyperpigmentation, hair loss and ulcerations. This data demonstrated that Rnaseh2b deficiency in the epidermis may also lead to DNA damage and subsequent p53 activation as shown for fetal liver from E14.5 RNase H2-deficient embryos. Preliminary data also indicate an increased incidence of cancer formation in RNase H2/p53 double knockouts, identifying the RNase H2 enzyme as an important tumor suppressor.

Mutations in TREX1, encoding three prime repair exonuclease 1, cause Aicardi-Goutières syndrome (AGS) 1, an autoinflammatory disease characterized by neurodegeneration and constitutive activation of the antiviral cytokine type I interferon. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts from two AGS patients with biallelic TREX1 mutations. These cell lines offer a unique resource to investigate disease processes in a cell-type specific manner.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18135号 / 医博第3855号 / 新制||医||1001(附属図書館) / 30993 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 亮, 教授 三森 経世, 教授 中畑 龍俊 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19625号 / 医博第4132号 / 新制||医||1015(附属図書館) / 32661 / 京都大学大学院医学研究科医学専攻 / (主査)教授 高田 穣, 教授 松田 文彦, 教授 小泉 昭夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Mutations in SAMHD1, encoding SAM and HD domain-containing protein 1, cause Aicardi-Goutières syndrome (AGS) 5, an infancy-onset autoinflammatory disease characterized by neurodegeneration and chronic activation of type I interferon. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts and peripheral blood mononuclear cells from three AGS patients with biallelic SAMHD1 mutations. These cell lines provide a valuable source to study disease mechanisms and to assess therapeutic molecules.

Aicardi-Goutières syndrome (AGS) is a rare inflammatory encephalopathy mimicking in utero acquired viral infection. Cardinal findings comprise leukodystrophy, basal ganglia calcifications and cerebral atrophy along with cerebrospinal fluid lymphocytosis and elevated interferon-α. In the majority of cases AGS is inherited as an autosomal recessive trait and caused by mutations in six genes including RNASEH2A, RNASEH2B, RNASEH2C, TREX1, SAMHD1 and ADAR1, all of which encode enzymes acting on nucleic acid species. Most patients present with first neurological signs in early infancy and experience severe global developmental delay. Here, we report on the unusual divergent phenotype of two siblings who both carry the most frequent AGS causing p.A177T (c.529G > A) RNASEH2B mutation in the homozygous state. While one sibling showed a typical AGS presentation with early onset and severe statomotor and mental impairment, the older sibling was intellectually completely normal. She was only diagnosed because of mild spasticity of the legs and serological signs of autoimmunity. These findings highlight the phenotypic variability of AGS and suggest that AGS may be underdiagnosed among children with mild cerebral palsy.

Aicardi-Goutières syndrome (AGS) is a rare inflammatory encephalopathy mimicking in utero acquired viral infection. Cardinal findings comprise leukodystrophy, basal ganglia calcifications and cerebral atrophy along with cerebrospinal fluid lymphocytosis and elevated interferon-α. In the majority of cases AGS is inherited as an autosomal recessive trait and caused by mutations in six genes including RNASEH2A, RNASEH2B, RNASEH2C, TREX1, SAMHD1 and ADAR1, all of which encode enzymes acting on nucleic acid species. Most patients present with first neurological signs in early infancy and experience severe global developmental delay. Here, we report on the unusual divergent phenotype of two siblings who both carry the most frequent AGS causing p.A177T (c.529G > A) RNASEH2B mutation in the homozygous state. While one sibling showed a typical AGS presentation with early onset and severe statomotor and mental impairment, the older sibling was intellectually completely normal. She was only diagnosed because of mild spasticity of the legs and serological signs of autoimmunity. These findings highlight the phenotypic variability of AGS and suggest that AGS may be underdiagnosed among children with mild cerebral palsy.