RESUMEN
Macrophages produce genotoxic agents, such as reactive oxygen and nitrogen species, that kill invading pathogens. Here we show that these agents activate the DNA damage response (DDR) kinases ATM and DNA-PKcs through the generation of double stranded breaks (DSBs) in murine macrophage genomic DNA. In contrast to other cell types, initiation of this DDR depends on signaling from the type I interferon receptor. Once activated, ATM and DNA-PKcs regulate a genetic program with diverse immune functions and promote inflammasome activation and the production of IL-1ß and IL-18. Indeed, following infection with Listeria monocytogenes, DNA-PKcs-deficient murine macrophages produce reduced levels of IL-18 and are unable to optimally stimulate IFN-γ production by NK cells. Thus, genomic DNA DSBs act as signaling intermediates in murine macrophages, regulating innate immune responses through the initiation of a type I IFN-dependent DDR.
Asunto(s)
Regulación de la Expresión Génica , Inmunidad Innata , Inflamasomas/metabolismo , Interferón Tipo I/metabolismo , Listeria monocytogenes/inmunología , Macrófagos/inmunología , Animales , Roturas del ADN de Doble Cadena , Daño del ADN , Ratones , Proteínas Quinasas/metabolismoRESUMEN
Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib has been approved for treatment of advanced ovarian cancer associated with BRCA1 and BRCA2 mutations. BRCA1- and BRCA2-mutated cells, which are homologous recombination (HR) deficient, are hypersensitive to PARPi through the mechanism of synthetic lethality. Here we examine the effect of PARPi on HR-proficient cells. Olaparib pretreatment, PARP1 knockdown or Parp1 heterozygosity of Brca2(cko/ko) mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2(ko/ko) cells. PARP1 deficiency does not restore HR in Brca2(ko/ko) cells, but protects stalled replication forks from MRE11-mediated degradation through its impaired recruitment. The functional consequence of Parp1 heterozygosity on BRCA2 loss is demonstrated by a significant increase in tumorigenesis in Brca2(cko/cko) mice. Thus, while olaparib efficiently kills BRCA2-deficient cells, we demonstrate that it can also contribute to the synthetic viability if PARP is inhibited before BRCA2 loss.
Asunto(s)
Proteína BRCA2/deficiencia , Poli(ADP-Ribosa) Polimerasa-1/deficiencia , Animales , Proteína BRCA2/metabolismo , Supervivencia Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Recombinación Homóloga/efectos de los fármacos , Humanos , Integrasas/metabolismo , Proteína Homóloga de MRE11/metabolismo , Ratones , Modelos Biológicos , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacologíaRESUMEN
The structure of broken DNA ends is a critical determinant of the pathway used for DNA double-strand break (DSB) repair. Here, we develop an approach involving the hairpin capture of DNA end structures (HCoDES), which elucidates chromosomal DNA end structures at single-nucleotide resolution. HCoDES defines structures of physiologic DSBs generated by the RAG endonuclease, as well as those generated by nucleases widely used for genome editing. Analysis of G1 phase cells deficient in H2AX or 53BP1 reveals DNA ends that are frequently resected to form long single-stranded overhangs that can be repaired by mutagenic pathways. In addition to 3' overhangs, many of these DNA ends unexpectedly form long 5' single-stranded overhangs. The divergence in DNA end structures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in end protection. Thus, the high-resolution end structures obtained by HCoDES identify features of DNA end processing during DSB repair.
Asunto(s)
Cromosomas Humanos/genética , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Células Cultivadas , Roturas del ADN de Doble Cadena , HumanosAsunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B/metabolismo , Dineínas Citoplasmáticas/metabolismo , Proteínas Nucleares/genética , Animales , HumanosRESUMEN
The resection of broken DNA ends is required for DNA double-strand break (DSB) repair by homologous recombination (HR) but can inhibit normal repair by nonhomologous end joining (NHEJ), the main DSB repair pathway in G1-phase cells. Antigen receptor gene assembly proceeds through DNA DSB intermediates generated in G1-phase lymphocytes by the RAG endonuclease. These DSBs activate ATM, which phosphorylates H2AX, forming γ-H2AX in flanking chromatin. γ-H2AX prevents CtIP from initiating resection of RAG DSBs. Whether there are additional proteins required to promote resection of these DNA ends is not known. KRAB-associated protein 1 (KAP-1) (TRIM28) is a transcriptional repressor that modulates chromatin structure and has been implicated in the repair of DNA DSBs in heterochromatin. Here, we show that in murine G1-phase lymphocytes, KAP-1 promotes resection of DSBs that are not protected by H2AX and its downstream effector 53BP1. In these murine cells, KAP-1 activity in DNA end resection is attenuated by a single-amino-acid change that reflects a KAP-1 polymorphism between primates and other mammalian species. These findings establish KAP-1 as a component of the machinery that can resect DNA ends in G1-phase cells and suggest that there may be species-specific features to this activity.
Asunto(s)
Reparación del ADN por Unión de Extremidades/genética , Proteínas de Unión al ADN/metabolismo , Fase G1/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Linfocitos/metabolismo , Animales , Células Cultivadas , ADN/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos C57BL , Fosforilación/genéticaRESUMEN
The carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) is known to function in 5' strand resection during homologous recombination, similar to the budding yeast Sae2 protein, but its role in this process is unclear. Here, we characterize recombinant human CtIP and find that it exhibits 5' flap endonuclease activity on branched DNA structures, independent of the MRN complex. Phosphorylation of CtIP at known damage-dependent sites and other sites is essential for its catalytic activity, although the S327 and T847 phosphorylation sites are dispensable. A catalytic mutant of CtIP that is deficient in endonuclease activity exhibits wild-type levels of homologous recombination at restriction enzyme-generated breaks but is deficient in processing topoisomerase adducts and radiation-induced breaks in human cells, suggesting that the nuclease activity of CtIP is specifically required for the removal of DNA adducts at sites of DNA breaks.
Asunto(s)
Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Endonucleasas/metabolismo , Proteínas Nucleares/metabolismo , Reparación del ADN por Recombinación/genética , Sitios de Unión/genética , Proteínas Portadoras/genética , Catálisis , Línea Celular , Supervivencia Celular/genética , ADN/genética , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas , Endonucleasas/genética , Humanos , Proteínas Nucleares/genética , Fosforilación/genética , Procesamiento Proteico-Postraduccional/genética , Radiación Ionizante , Recombinación GenéticaRESUMEN
Allelic exclusion is enforced through the ability of antigen receptor chains expressed from one allele to signal feedback inhibition of V-to-(D)J recombination on the other allele. To achieve allelic exclusion by such means, only one allele can initiate V-to-(D)J recombination within the time required to signal feedback inhibition. DNA double-strand breaks (DSBs) induced by the RAG endonuclease during V(D)J recombination activate the Ataxia Telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) kinases. We demonstrate that ATM enforces Igκ allelic exclusion, and that RAG DSBs induced during Igκ recombination in primary pre-B cells signal through ATM, but not DNA-PK, to suppress initiation of additional Igκ rearrangements. ATM promotes high-density histone H2AX phosphorylation to create binding sites for MDC1, which functions with H2AX to amplify a subset of ATM-dependent signals. However, neither H2AX nor MDC1 is required for ATM to enforce Igκ allelic exclusion and suppress Igκ rearrangements. Upon activation in response to RAG Igκ cleavage, ATM signals down-regulation of Gadd45α with concomitant repression of the Gadd45α targets Rag1 and Rag2. Our data indicate that ATM kinases activated by RAG DSBs during Igκ recombination transduce transient H2AX/MDC1-independent signals that suppress initiation of further Igκ rearrangements to control Igκ allelic exclusion.
Asunto(s)
Proteínas de Ciclo Celular/inmunología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Reordenamiento Génico de Cadena Ligera de Linfocito B , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/inmunología , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Alelos , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Linfocitos B/inmunología , Linfocitos B/metabolismo , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Histonas/deficiencia , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Modelos Biológicos , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/genéticaRESUMEN
DNA double-strand breaks (DSBs) are generated by the recombination activating gene (RAG) endonuclease in all developing lymphocytes as they assemble antigen receptor genes. DNA cleavage by RAG occurs only at the G1 phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening before their repair by classical non-homologous end-joining (NHEJ). Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently. Here, in vivo, we show that in murine cells the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1 (mediator of DNA damage checkpoint 1), which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ataxia telangiectasia mutated (ATM) kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle, in which it is essential for homology-mediated repair. In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and show significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes, thereby preventing these DNA ends from accessing repair pathways that promote genomic instability.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Fase G1 , Reordenamiento Génico de Linfocito B , Histonas/metabolismo , Linfocitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Línea Celular Transformada , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas , Reordenamiento Génico de Linfocito B/genética , Inestabilidad Genómica , Histonas/deficiencia , Histonas/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Linfocitos/citología , Ratones , Proteínas Nucleares , Células Precursoras de Linfocitos B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinación Genética/genética , Especificidad por Sustrato , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Men1 is a tumor suppressor gene mutated in endocrine neoplasms. Besides its endocrine role, the Men1 gene product menin interacts with the mixed lineage leukemia (MLL) protein, a histone H3 lysine 4 methyltransferase. Although menin and MLL fusion proteins cooperate to activate Homeobox (Hox) gene expression during transformation, little is known about the normal hematopoietic functions of menin. Here, we studied hematopoiesis after Men1 ablation. Menin loss modestly impaired blood neutrophil, lymphocyte, and platelet counts. Without hematopoietic stress, multilineage and myelo-erythroid bone marrow progenitor numbers were preserved, while B lymphoid progenitors were decreased. In contrast, competitive transplantation revealed a marked functional defect of long-term hematopoietic stem cells (HSC) in the absence of menin, despite normal initial homing of progenitors to the bone marrow. HoxA9 gene expression was only modestly decreased in menin-deficient HSCs. These observations reveal a novel and essential role for menin in HSC homeostasis that was most apparent during situations of hematopoietic recovery, suggesting that menin regulates molecular pathways that are essential during the adaptive HSC response to stress.
Asunto(s)
Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Leucemia/patología , Linfocitos/citología , Neoplasia Endocrina Múltiple Tipo 1/patología , Proteínas Proto-Oncogénicas/metabolismo , Animales , Trasplante de Médula Ósea , Citometría de Flujo , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/fisiología , Leucemia/genética , Leucemia/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasia Endocrina Múltiple Tipo 1/genética , Neoplasia Endocrina Múltiple Tipo 1/fisiopatología , Neutrófilos/citología , Recuento de Plaquetas , Proteínas Proto-Oncogénicas/genética , Receptores Mensajeros de Linfocitos/metabolismoRESUMEN
Menin is the product of the tumor suppressor gene Men1 that is mutated in the inherited tumor syndrome multiple endocrine neoplasia type 1 (MEN1). Menin has been shown to interact with SET-1 domain-containing histone 3 lysine 4 (H3K4) methyltransferases including mixed lineage leukemia proteins to regulate homeobox (Hox) gene expression in vitro. Using conditional Men1 knockout mice, we have investigated the requirement for menin in hematopoiesis and myeloid transformation. Men1 excision causes reduction of Hoxa9 expression, colony formation by hematopoietic progenitors, and the peripheral white blood cell count. Menin directly activates Hoxa9 expression, at least in part, by binding to the Hoxa9 locus, facilitating methylation of H3K4, and recruiting the methylated H3K4 binding protein chd1 to the locus. Consistent with signaling downstream of menin, ectopic expression of both Hoxa9 and Meis1 rescues colony formation defects in Men1-excised bone marrow. Moreover, Men1 excision also suppresses proliferation of leukemogenic mixed lineage leukemia-AF9 fusion-protein-transformed myeloid cells and Hoxa9 expression. These studies uncover an important role for menin in both normal hematopoiesis and myeloid transformation and provide a mechanistic understanding of menin's function in these processes that may be used for therapy.