RESUMO
Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.
Assuntos
Álcool Desidrogenase/genética , Aldeído-Desidrogenase Mitocondrial/genética , Formaldeído/sangue , Leucemia/genética , Adolescente , Aldeídos/sangue , Animais , Criança , Pré-Escolar , Adutos de DNA/genética , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Feminino , Formaldeído/toxicidade , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lactente , Leucemia/sangue , Leucemia/patologia , Masculino , Camundongos , Mutação/genética , Especificidade por SubstratoRESUMO
Haematopoietic stem cells renew blood. Accumulation of DNA damage in these cells promotes their decline, while misrepair of this damage initiates malignancies. Here we describe the features and mutational landscape of DNA damage caused by acetaldehyde, an endogenous and alcohol-derived metabolite. This damage results in DNA double-stranded breaks that, despite stimulating recombination repair, also cause chromosome rearrangements. We combined transplantation of single haematopoietic stem cells with whole-genome sequencing to show that this damage occurs in stem cells, leading to deletions and rearrangements that are indicative of microhomology-mediated end-joining repair. Moreover, deletion of p53 completely rescues the survival of aldehyde-stressed and mutated haematopoietic stem cells, but does not change the pattern or the intensity of genome instability within individual stem cells. These findings characterize the mutation of the stem-cell genome by an alcohol-derived and endogenous source of DNA damage. Furthermore, we identify how the choice of DNA-repair pathway and a stringent p53 response limit the transmission of aldehyde-induced mutations in stem cells.
Assuntos
Acetaldeído/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Etanol/metabolismo , Etanol/farmacologia , Instabilidade Genômica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Mutação , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades , Etanol/administração & dosagem , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Feminino , Deleção de Genes , Genes p53/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Autoantígeno Ku/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reparo de DNA por Recombinação/efeitos dos fármacos , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sequenciamento Completo do GenomaRESUMO
Maternal metabolism provides essential nutrients to enable embryonic development. However, both mother and embryo produce reactive metabolites that can damage DNA. Here we discover how the embryo is protected from these genotoxins. Pregnant mice lacking Aldh2, a key enzyme that detoxifies reactive aldehydes, cannot support the development of embryos lacking the Fanconi anemia DNA repair pathway gene Fanca. Remarkably, transferring Aldh2(-/-)Fanca(-/-) embryos into wild-type mothers suppresses developmental defects and rescues embryonic lethality. These rescued neonates have severely depleted hematopoietic stem and progenitor cells, indicating that despite intact maternal aldehyde catabolism, fetal Aldh2 is essential for hematopoiesis. Hence, maternal and fetal aldehyde detoxification protects the developing embryo from DNA damage. Failure of this genome preservation mechanism might explain why birth defects and bone marrow failure occur in Fanconi anemia, and may have implications for fetal well-being in the many women in Southeast Asia that are genetically deficient in ALDH2.
Assuntos
Acetaldeído/metabolismo , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Embrião de Mamíferos/metabolismo , Etanol/toxicidade , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Acetaldeído/toxicidade , Família Aldeído Desidrogenase 1 , Aldeído-Desidrogenase Mitocondrial , Animais , Animais Recém-Nascidos , Dano ao DNA , Modelos Animais de Doenças , Embrião de Mamíferos/embriologia , Feminino , Genoma , Células-Tronco Hematopoéticas/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismoRESUMO
Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.
Assuntos
Proteínas Aviárias/metabolismo , Galinhas/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Ubiquitinação , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Linhagem Celular , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Haematopoietic stem cells (HSCs) regenerate blood cells throughout the lifespan of an organism. With age, the functional quality of HSCs declines, partly owing to the accumulation of damaged DNA. However, the factors that damage DNA and the protective mechanisms that operate in these cells are poorly understood. We have recently shown that the Fanconi anaemia DNA-repair pathway counteracts the genotoxic effects of reactive aldehydes. Mice with combined inactivation of aldehyde catabolism (through Aldh2 knockout) and the Fanconi anaemia DNA-repair pathway (Fancd2 knockout) display developmental defects, a predisposition to leukaemia, and are susceptible to the toxic effects of ethanol-an exogenous source of acetaldehyde. Here we report that aged Aldh2(-/-) Fancd2(-/-) mutant mice that do not develop leukaemia spontaneously develop aplastic anaemia, with the concomitant accumulation of damaged DNA within the haematopoietic stem and progenitor cell (HSPC) pool. Unexpectedly, we find that only HSPCs, and not more mature blood precursors, require Aldh2 for protection against acetaldehyde toxicity. Additionally, the aldehyde-oxidizing activity of HSPCs, as measured by Aldefluor stain, is due to Aldh2 and correlates with this protection. Finally, there is more than a 600-fold reduction in the HSC pool of mice deficient in both Fanconi anaemia pathway-mediated DNA repair and acetaldehyde detoxification. Therefore, the emergence of bone marrow failure in Fanconi anaemia is probably due to aldehyde-mediated genotoxicity restricted to the HSPC pool. These findings identify a new link between endogenous reactive metabolites and DNA damage in HSCs, and define the protective mechanisms that counteract this threat.
Assuntos
Aldeídos/toxicidade , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Mutagênicos/toxicidade , Acetaldeído/metabolismo , Acetaldeído/toxicidade , Envelhecimento , Aldeído Desidrogenase/deficiência , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Aldeído-Desidrogenase Mitocondrial , Aldeídos/metabolismo , Animais , Medula Óssea/patologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA , Etanol/toxicidade , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Feminino , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/metabolismo , Estimativa de Kaplan-Meier , Leucemia/metabolismo , Leucemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
RING (Really Interesting New Gene)-in-between-RING (RBR) enzymes are a distinct class of E3 ubiquitin ligases possessing a cluster of three zinc-binding domains that cooperate to catalyse ubiquitin transfer. The regulation and biological function for most members of the RBR ligases is not known, and all RBR E3s characterized to date are auto-inhibited for in vitro ubiquitylation. Here, we show that TRIAD1 and HHARI, two members of the Ariadne subfamily ligases, associate with distinct neddylated Cullin-RING ligase (CRL) complexes. In comparison to the modest E3 ligase activity displayed by isolated TRIAD1 or HHARI, binding of the cognate neddylated CRL to TRIAD1 or HHARI greatly stimulates RBR ligase activity in vitro, as determined by auto-ubiquitylation, their ability to stimulate dissociation of a thioester-linked UBCH7â¼ubiquitin intermediate, and reactivity with ubiquitin-vinyl methyl ester. Moreover, genetic evidence shows that RBR ligase activity impacts both the levels and activities of neddylated CRLs in vivo. Cumulatively, our work proposes a conserved mechanism of CRL-induced Ariadne RBR ligase activation and further suggests a reciprocal role of this special class of RBRs as regulators of distinct CRLs.
Assuntos
Proteínas de Transporte/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Culina/metabolismo , Ciclopentanos/farmacologia , Células HEK293 , Humanos , Proteína NEDD8 , Pirimidinas/farmacologia , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/metabolismoRESUMO
Reactive aldehydes are common carcinogens. They are also by-products of several metabolic pathways and, without enzymatic catabolism, may accumulate and cause DNA damage. Ethanol, which is metabolised to acetaldehyde, is both carcinogenic and teratogenic in humans. Here we find that the Fanconi anaemia DNA repair pathway counteracts acetaldehyde-induced genotoxicity in mice. Our results show that the acetaldehyde-catabolising enzyme Aldh2 is essential for the development of Fancd2(-/-) embryos. Nevertheless, acetaldehyde-catabolism-competent mothers (Aldh2(+/-)) can support the development of double-mutant (Aldh2(-/-)Fancd2(-/-)) mice. However, these embryos are unusually sensitive to ethanol exposure in utero, and ethanol consumption by postnatal double-deficient mice rapidly precipitates bone marrow failure. Lastly, Aldh2(-/-)Fancd2(-/-) mice spontaneously develop acute leukaemia. Acetaldehyde-mediated DNA damage may critically contribute to the genesis of fetal alcohol syndrome in fetuses, as well as to abnormal development, haematopoietic failure and cancer predisposition in Fanconi anaemia patients.
Assuntos
Aldeídos/antagonistas & inibidores , Aldeídos/toxicidade , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Acetaldeído/metabolismo , Acetaldeído/toxicidade , Aldeído Desidrogenase/deficiência , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Aldeído-Desidrogenase Mitocondrial , Aldeídos/metabolismo , Alelos , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Medula Óssea/fisiopatologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Células Clonais/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/genética , Perda do Embrião/induzido quimicamente , Perda do Embrião/etiologia , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/embriologia , Etanol/metabolismo , Etanol/toxicidade , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Feminino , Transtornos do Espectro Alcoólico Fetal/etiologia , Deleção de Genes , Genes Essenciais , Hematopoese/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras/induzido quimicamente , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiologia , Gravidez , Teratogênicos/metabolismo , Teratogênicos/toxicidade , DesmameRESUMO
The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak, and their priming toward primitive streak fates. How these different events are coordinated remains unknown. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that captures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this system in combination with microfabrication and in vivo experiments, we found that proliferation-induced crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that the right cell types become specified at the right place during embryonic development.
Assuntos
Diferenciação Celular , Gastrulação , Camadas Germinativas , Animais , Camundongos , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas com Domínio T/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/citologia , Linha Primitiva/metabolismo , Proteínas Fetais/metabolismo , Proteínas Fetais/genética , Via de Sinalização Wnt , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismoRESUMO
The helicase-associated endonuclease for fork-structured DNA (Hef) is an archaeabacterial protein that processes blocked replication forks. Here we have isolated the vertebrate Hef ortholog and investigated its molecular function. Disruption of this gene in chicken DT40 cells results in genomic instability and sensitivity to DNA cross-links. The similarity of this phenotype to that of cells lacking the Fanconi anemia-related (FA) tumor-suppressor genes led us to investigate whether Hef functions in this pathway. Indeed, we found a genetic interaction between the FANCC and Hef genes. In addition, Hef is a component of the FA nuclear protein complex that facilitates its DNA damage-inducible chromatin localization and the monoubiquitination of the FA protein FANCD2. Notably, Hef interacts directly with DNA structures that are intermediates in DNA replication. This discovery sheds light on the origins, regulation and molecular function of the FA tumor-suppressor pathway in the maintenance of genome stability.
Assuntos
Proteínas Aviárias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Galinhas , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Anemia de Fanconi/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas Aviárias/química , Proteínas Aviárias/deficiência , Proteínas Aviárias/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Galinhas/genética , Galinhas/metabolismo , DNA/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases/metabolismo , Evolução Molecular , Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi , Proteínas de Grupos de Complementação da Anemia de Fanconi , Instabilidade Genômica , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Metabolism is predicted to generate formaldehyde, a toxic, simple, reactive aldehyde that can damage DNA. Here we report a synthetic lethal interaction in avian cells between ADH5, encoding the main formaldehyde-detoxifying enzyme, and the Fanconi anemia (FA) DNA-repair pathway. These results define a fundamental role for the combined action of formaldehyde catabolism and DNA cross-link repair in vertebrate cell survival.
Assuntos
Reparo do DNA , Anemia de Fanconi/metabolismo , Formaldeído/metabolismo , Aldeído Oxirredutases/genética , Animais , Linhagem Celular , Galinhas/genética , Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/fisiologia , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/fisiologia , Técnicas de Inativação de Genes , Redes e Vias MetabólicasRESUMO
The evolutionarily conserved SLX4 protein, a key regulator of nucleases, is critical for DNA damage response. SLX4 nuclease complexes mediate repair during replication and can also resolve Holliday junctions formed during homologous recombination. Here we describe the phenotype of the Btbd12 knockout mouse, the mouse ortholog of SLX4, which recapitulates many key features of the human genetic illness Fanconi anemia. Btbd12-deficient animals are born at sub-Mendelian ratios, have greatly reduced fertility, are developmentally compromised and are prone to blood cytopenias. Btbd12(-/-) cells prematurely senesce, spontaneously accumulate damaged chromosomes and are particularly sensitive to DNA crosslinking agents. Genetic complementation reveals a crucial requirement for Btbd12 (also known as Slx4) to interact with the structure-specific endonuclease Xpf-Ercc1 to promote crosslink repair. The Btbd12 knockout mouse therefore establishes a disease model for Fanconi anemia and genetically links a regulator of nuclease incision complexes to the Fanconi anemia DNA crosslink repair pathway.
Assuntos
Anemia de Fanconi/genética , Recombinases/genética , Recombinases/fisiologia , Animais , Senescência Celular , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA , Feminino , Fibroblastos/metabolismo , Teste de Complementação Genética , Células-Tronco Hematopoéticas , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos KnockoutRESUMO
The Fanconi anemia (FA) nuclear core complex and the E2 ubiquitin-conjugating enzyme UBE2T are required for the S phase and DNA damage-restricted monoubiquitination of FANCD2. This constitutes a key step in the FA tumor suppressor pathway, and much attention has been focused on the regulation at this point. Here, we address the importance of the assembly of the FA core complex and the subcellular localization of UBE2T in the regulation of FANCD2 monoubiquitination. We establish three points. First, the stable assembly of the FA core complex can be dissociated of its ability to function as an E3 ubiquitin ligase. Second, the actual E3 ligase activity is not determined by the assembly of the FA core complex but rather by its DNA damage-induced localization to chromatin. Finally, UBE2T and FANCD2 access this subcellular fraction independently of the FA core complex. FANCD2 monoubiquitination is therefore not regulated by multiprotein complex assembly but by the formation of an active E2/E3 holoenzyme on chromatin.
Assuntos
Cromatina/enzimologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Animais , Domínio Catalítico , Ciclo Celular , Linhagem Celular , Galinhas , Dano ao DNA , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Humanos , Ligação ProteicaRESUMO
Monoubiquitination of FANCD2 and PCNA promotes DNA repair. It causes chromatin accumulation of FANCD2 and facilitates PCNA's recruitment of translesion polymerases to stalled replication. USP1, a protease that removes monoubiquitin from FANCD2 and PCNA, was thought to reverse the DNA damage response of these substrates. We disrupted USP1 in chicken cells to dissect its role in a stable genetic system. USP1 ablation increases FANCD2 and PCNA monoubiquitination but unexpectedly results in DNA crosslinker sensitivity. This defective DNA repair is associated with constitutively chromatin-bound, monoubiquitinated FANCD2. In contrast, persistent PCNA monoubiquitination has negligible impact on DNA repair or mutagenesis. USP1 was previously shown to autocleave after DNA damage. In DT40, USP1 autocleavage is not stimulated by DNA damage, and expressing a noncleavable mutant in the USP1 knockout strain partially rescues crosslinker sensitivity. We conclude that efficient DNA crosslink repair requires FANCD2 deubiquitination, whereas FANCD2 monoubiquitination is not dependent on USP1 autocleavage.