RESUMEN
Repair of DNA double-strand breaks (DSBs) by non-homologous end joining is critical for neural development, and brain cells frequently contain somatic genomic variations that might involve DSB intermediates. We now use an unbiased, high-throughput approach to identify genomic regions harboring recurrent DSBs in primary neural stem/progenitor cells (NSPCs). We identify 27 recurrent DSB clusters (RDCs), and remarkably, all occur within gene bodies. Most of these NSPC RDCs were detected only upon mild, aphidicolin-induced replication stress, providing a nucleotide-resolution view of replication-associated genomic fragile sites. The vast majority of RDCs occur in long, transcribed, and late-replicating genes. Moreover, almost 90% of identified RDC-containing genes are involved in synapse function and/or neural cell adhesion, with a substantial fraction also implicated in tumor suppression and/or mental disorders. Our characterization of NSPC RDCs reveals a basis of gene fragility and suggests potential impacts of DNA breaks on neurodevelopment and neural functions.
Asunto(s)
Roturas del ADN , Células-Madre Neurales/metabolismo , Animales , Afidicolina/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Encéfalo/citología , Adhesión Celular , Moléculas de Adhesión Celular Neuronal/metabolismo , Roturas del ADN/efectos de los fármacos , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Proteínas Ligadas a GPI/metabolismo , Genoma , Humanos , Ratones , Proteínas del Tejido Nervioso/metabolismo , Sinapsis , Factores de Transcripción/metabolismo , Translocación GenéticaRESUMEN
DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN Polimerasa Dirigida por ADN , ADN , Mutagénesis , Mutación , Animales , Humanos , Ratones , Alquilación/efectos de la radiación , Línea Celular , ADN/química , ADN/genética , ADN/metabolismo , ADN/efectos de la radiación , Aductos de ADN/química , Aductos de ADN/genética , Aductos de ADN/metabolismo , Aductos de ADN/efectos de la radiación , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Reparación del ADN/genética , Reparación del ADN/fisiología , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Mutagénesis/genética , Mutagénesis/efectos de la radiación , Mutación/genética , Mutación/efectos de la radiación , Neoplasias/genética , Transcripción Genética , Rayos Ultravioleta/efectos adversosRESUMEN
Mild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes ("RDC-genes") that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome-wide DSB identification approach that captures DSBs via their ability to join to specific genomic Cas9/single-guide RNA-generated bait DSBs. In XRCC4/p53-deficient ESCs, we detected seven RDCs, all of which were in genes and two of which were robust. In contrast, in NPCs derived from these ESC lines we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ESCs provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias de Ratones/citología , Células-Madre Neurales/metabolismo , Animales , Línea Celular , Células Cultivadas , Roturas del ADN , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Genes p53/genética , Genoma , Humanos , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Familia de Multigenes/genética , Neurogénesis , Neuronas/citologíaRESUMEN
Ig heavy chain (IgH) class switch recombination (CSR) in B lymphocytes switches IgH constant regions to change antibody functions. CSR is initiated by DNA double-strand breaks (DSBs) within a donor IgH switch (S) region and a downstream acceptor S region. CSR is completed by fusing donor and acceptor S region DSB ends by classical nonhomologous end-joining (C-NHEJ) and, in its absence, by alternative end-joining that is more biased to use longer junctional microhomologies (MHs). Deficiency for DSB response (DSBR) factors, including ataxia telangiectasia-mutated (ATM) and 53BP1, variably impair CSR end-joining, with 53BP1 deficiency having the greatest impact. However, studies of potential impact of DSBR factor deficiencies on MH-mediated CSR end-joining have been technically limited. We now use a robust DSB joining assay to elucidate impacts of deficiencies for DSBR factors on CSR and chromosomal translocation junctions in primary mouse B cells and CH12F3 B-lymphoma cells. Compared with wild-type, CSR and c-myc to S region translocation junctions in the absence of 53BP1, and, to a lesser extent, other DSBR factors, have increased MH utilization; indeed, 53BP1-deficient MH profiles resemble those associated with C-NHEJ deficiency. However, translocation junctions between c-myc DSB and general DSBs genome-wide are not MH-biased in ATM-deficient versus wild-type CH12F3 cells and are less biased in 53BP1- and C-NHEJ-deficient cells than CSR junctions or c-myc to S region translocation junctions. We discuss potential roles of DSBR factors in suppressing increased MH-mediated DSB end-joining and features of S regions that may render their DSBs prone to MH-biased end-joining in the absence of DSBR factors.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Cambio de Clase de Inmunoglobulina , Translocación Genética , Animales , Línea Celular , Secuenciación de Nucleótidos de Alto Rendimiento , RatonesRESUMEN
We recently discovered 27 recurrent DNA double-strand break (DSB) clusters (RDCs) in mouse neural stem/progenitor cells (NSPCs). Most RDCs occurred across long, late-replicating RDC genes and were found only after mild inhibition of DNA replication. RDC genes share intriguing characteristics, including encoding surface proteins that organize brain architecture and neuronal junctions, and are genetically implicated in neuropsychiatric disorders and/or cancers. RDC identification relies on high-throughput genome-wide translocation sequencing (HTGTS), which maps recurrent DSBs based on their translocation to "bait" DSBs in specific chromosomal locations. Cellular heterogeneity in 3D genome organization allowed unequivocal identification of RDCs on 14 different chromosomes using HTGTS baits on three mouse chromosomes. Additional candidate RDCs were also implicated, however, suggesting that some RDCs were missed. To more completely identify RDCs, we exploited our finding that joining of two DSBs occurs more frequently if they lie on the same cis chromosome. Thus, we used CRISPR/Cas9 to introduce specific DSBs into each mouse chromosome in NSPCs that were used as bait for HTGTS libraries. This analysis confirmed all 27 previously identified RDCs and identified many new ones. NSPC RDCs fall into three groups based on length, organization, transcription level, and replication timing of genes within them. While mostly less robust, the largest group of newly defined RDCs share many intriguing characteristics with the original 27. Our findings also revealed RDCs in NSPCs in the absence of induced replication stress, and support the idea that the latter treatment augments an already active endogenous process.
Asunto(s)
Roturas del ADN de Doble Cadena , Animales , Encéfalo , Reparación del ADN , Eliminación de Gen , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Células-Madre Neurales/metabolismo , Interferencia de ARN , Translocación GenéticaRESUMEN
The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD ) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD , but not DNA-PKcs-/- , B cells. Meanwhile, the residual joints from DNA-PKcsKD/KD cells and the efficient Sµ-Sγ1 junctions from DNA-PKcs-/- B cells both display similar preferences for small (2-6 nt) microhomologies (MH). In DNA-PKcs-/- cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Sε and Sµ-Sµ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.
Asunto(s)
Linfocitos B/enzimología , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Cambio de Clase de Inmunoglobulina/fisiología , Inmunoglobulina G/biosíntesis , Proteínas Nucleares/metabolismo , Recombinación Genética/fisiología , Animales , Linfocitos B/citología , Línea Celular , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/genética , Inmunoglobulina G/genética , Ratones , Ratones Noqueados , Proteínas Nucleares/genéticaRESUMEN
NPGPx is a member of the glutathione peroxidase (GPx) family; however, it lacks GPx enzymatic activity due to the absence of a critical selenocysteine residue, rendering its function an enigma. Here, we show that NPGPx is a newly identified stress sensor that transmits oxidative stress signals by forming the disulfide bond between its Cys57 and Cys86 residues. This oxidized form of NPGPx binds to glucose-regulated protein (GRP)78 and forms covalent bonding intermediates between Cys86 of NPGPx and Cys41/Cys420 of GRP78. Subsequently, the formation of the disulfide bond between Cys41 and Cys420 of GRP78 enhances its chaperone activity. NPGPx-deficient cells display increased reactive oxygen species, accumulated misfolded proteins, and impaired GRP78 chaperone activity. Complete loss of NPGPx in animals causes systemic oxidative stress, increases carcinogenesis, and shortens life span. These results suggest that NPGPx is essential for releasing excessive ER stress by enhancing GRP78 chaperone activity to maintain physiological homeostasis.
Asunto(s)
Proteínas Portadoras/metabolismo , Estrés del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Estrés Oxidativo , Peroxidasas/metabolismo , Deficiencias en la Proteostasis/enzimología , Transducción de Señal , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Cisteína , Daño del ADN , Disulfuros/metabolismo , Relación Dosis-Respuesta a Droga , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Fibroblastos/enzimología , Fibroblastos/patología , Glutatión Peroxidasa , Proteínas de Choque Térmico/genética , Homeostasis , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Mutación , Oxidantes/farmacología , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Peroxidasas/genética , Unión Proteica , Pliegue de Proteína , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/patología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Tiempo , TransfecciónRESUMEN
High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs. Comparative analyses of transcription profiles in NSPCs and B cells revealed that the great majority of TSS-proximal junctions occurred in genes commonly expressed in both cell types, possibly because this common set has higher transcription levels on average than genes transcribed in only one or the other cell type. In the latter context, among all actively transcribed genes containing translocation junctions in NSPCs, those with junctions located within 2 kb of the TSS show a significantly higher transcription rate on average than genes with junctions in the gene body located at distances greater than 2 kb from the TSS. Finally, analysis of repair junction signatures of TSS-associated translocations in wild-type versus classical nonhomologous end-joining (C-NHEJ)-deficient NSPCs reveals that both C-NHEJ and alternative end-joining pathways can generate translocations by joining TSS-proximal DSBs to DSBs on other chromosomes. Our studies show that the generation of transcription-associated DSBs is conserved across divergent cell types.
Asunto(s)
Roturas del ADN de Doble Cadena , Células-Madre Neurales/metabolismo , Transcripción Genética , Translocación Genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Linfocitos B/metabolismo , Células Cultivadas , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes myc , Genes p53 , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-myc/deficiencia , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Sitio de Iniciación de la Transcripción , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
We reported that non-targeting siRNA (NT-siRNA) stress induces non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx) expression to cooperate with exoribonuclease XRN2 for releasing the stress [Wei,P.C., Lo,W.T., Su,M.I., Shew,J.Y. and Lee,W.H. (2011) Non-targeting siRNA induces NPGPx expression to cooperate with exoribonuclease XRN2 for releasing the stress. Nucleic Acids Res., 40, 323-332]. However, how NT-siRNA stress inducing NPGPx expression remains elusive. In this communication, we showed that the proximal promoter of NPGPx contained a mixed G-quadruplex (G4) structure, and disrupting the structure diminished NT-siRNA induced NPGPx promoter activity. We also demonstrated that nucleolin (NCL) specifically bonded to the G4-containing sequences to replace the originally bound Sp1 at the NPGPx promoter on NT-siRNA stress. Consistently, overexpression of NCL further increased NPGPx promoter activity, whereas depletion of NCL desensitized NPGPx promoter to NT-siRNA stress. These results suggest that the cis-element with mixed G4 structure at the NPGPx promoter plays an essential role for its transactivation mediated by NCL to release cells from NT-siRNA stress.
Asunto(s)
G-Cuádruplex , Peroxidasas/genética , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , ARN Interferente Pequeño , Proteínas de Unión al ARN/metabolismo , Estrés Fisiológico/genética , Activación Transcripcional , Sitios de Unión , Línea Celular , Secuencia Rica en GC , Humanos , Peroxidasas/metabolismo , Factor de Transcripción Sp1/metabolismo , Regulación hacia Arriba , NucleolinaRESUMEN
Short interfering RNAs (siRNAs) target specific mRNAs for their degradation mediated by RNA-induced silencing complex (RISC). Persistent activation of siRNA-RISC frequently leads to non-targeting toxicity. However, how cells mediate this stress remains elusive. In this communication, we found that the presence of non-targeting siRNA selectively induced the expression of an endoplasmic reticulum (ER)-resident protein, non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), but not other ER-stress proteins including GRP78, Calnexin and XBP1. Cells suffering from constant non-targeting siRNA stress grew slower and prolonged G1 phase, while NPGPx-depleted cells accumulated mature non-targeting siRNA and underwent apoptosis. Upon the stress, NPGPx covalently bound to exoribonuclease XRN2, facilitating XRN2 to remove accumulated non-targeting siRNA. These results suggest that NPGPx serves as a novel responder to non-targeting siRNA-induced stress in facilitating XRN2 to release the non-targeting siRNA accumulation.
Asunto(s)
Exorribonucleasas/metabolismo , Glutatión Peroxidasa/metabolismo , Peroxidasas/metabolismo , ARN Interferente Pequeño/metabolismo , Estrés Fisiológico , Animales , Apoptosis , Daño del ADN , Chaperón BiP del Retículo Endoplásmico , Exorribonucleasas/fisiología , Fase G1 , Glutatión Peroxidasa/biosíntesis , Glutatión Peroxidasa/genética , Humanos , Ratones , Peroxidasas/biosíntesis , Peroxidasas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico/genéticaRESUMEN
Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unraveled the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA revealed co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity revealed that head-to-head interactions between replication and transcription machinery resulted in 60% DSB contribution to the head-on compared to 40% for co-directional. Our findings revealed TTR as a novel fragile class and highlighted how the linear interaction between transcription and replication impacts genome stability.
RESUMEN
Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unravel the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA reveals co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity uncovers that head-to-head interactions between replication and transcription machinery result in 60% DSB contribution to the head-on compared to 40% for co-directional. Taken together we reveal TTR as a fragile class and show how the linear interaction between transcription and replication impacts genome stability.
Asunto(s)
Roturas del ADN de Doble Cadena , Replicación del ADN , Inestabilidad Genómica , Transcripción Genética , Animales , Ratones , Células-Madre Neurales/metabolismo , ADN/metabolismo , ADN/genética , Origen de Réplica , Telómero/metabolismo , Telómero/genética , Centrómero/metabolismo , Centrómero/genéticaRESUMEN
BACKGROUND: Medulloblastomas with chromothripsis developing in children with Li-Fraumeni Syndrome (germline TP53 mutations) are highly aggressive brain tumors with dismal prognosis. Conventional photon radiotherapy and DNA-damaging chemotherapy are not successful for these patients and raise the risk of secondary malignancies. We hypothesized that the pronounced homologous recombination deficiency in these tumors might offer vulnerabilities that can be therapeutically utilized in combination with high linear energy transfer carbon ion radiotherapy. METHODS: We tested high-precision particle therapy with carbon ions and protons as well as topotecan with or without PARP inhibitor in orthotopic primary and matched relapsed patient-derived xenograft models. Tumor and normal tissue underwent longitudinal morphological MRI, cellular (markers of neurogenesis and DNA damage-repair), and molecular characterization (whole-genome sequencing). RESULTS: In the primary medulloblastoma model, carbon ions led to complete response in 79% of animals irrespective of PARP inhibitor within a follow-up period of 300 days postirradiation, as detected by MRI and histology. No sign of neurologic symptoms, impairment of neurogenesis or in-field carcinogenesis was detected in repair-deficient host mice. PARP inhibitors further enhanced the effect of proton irradiation. In the postradiotherapy relapsed tumor model, median survival was significantly increased after carbon ions (96 days) versus control (43 days, P < .0001). No major change in the clonal composition was detected in the relapsed model. CONCLUSION: The high efficacy and favorable toxicity profile of carbon ions warrants further investigation in primary medulloblastomas with chromothripsis. Postradiotherapy relapsed medulloblastomas exhibit relative resistance compared to treatment-naïve tumors, calling for exploration of multimodal strategies.
Asunto(s)
Neoplasias Cerebelosas , Cromotripsis , Radioterapia de Iones Pesados , Síndrome de Li-Fraumeni , Meduloblastoma , Animales , Carbono , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/radioterapia , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/radioterapia , RatonesRESUMEN
Emerging evidences implicate the contribution of ROS to T cell activation and signaling. The tyrosine kinase, ζ-chain-associated protein of 70â¯kDa (ZAP70), is essential for T cell development and activation. However, it remains elusive whether a direct redox regulation affects ZAP70 activity upon TCR stimulation. Here, we show that deficiency of non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), a redox sensor, results in T cell hyperproliferation and elevated cytokine productions. T cell-specific NPGPx-knockout mice reveal enhanced T-dependent humoral responses and are susceptible to experimental autoimmune encephalomyelitis (EAE). Through proteomic approaches, ZAP70 is identified as the key interacting protein of NPGPx through disulfide bonding. NPGPx is activated by ROS generated from TCR stimulation, and modulates ZAP70 activity through redox switching to reduce ZAP70 recruitment to TCR/CD3 complex in membrane lipid raft, therefore subduing TCR responses. These results reveal a delicate redox mechanism that NPGPx serves as a modulator to curb ZAP70 functions in maintaining T cell homeostasis.
Asunto(s)
Proteómica , Linfocitos T , Animales , Homeostasis , Ratones , Oxidación-Reducción , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/metabolismoRESUMEN
The association between macrocephaly and autism spectrum disorder (ASD) suggests that the mechanisms underlying excessive neural growth could contribute to ASD pathogenesis. Consistently, neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) of ASD individuals with early developmental brain enlargement are inherently more proliferative than control NPCs. Here, we show that hiPSC-derived NPCs from ASD individuals with macrocephaly display an altered DNA replication program and increased DNA damage. When compared with the control NPCs, high-throughput genome-wide translocation sequencing (HTGTS) demonstrates that ASD-derived NPCs harbored elevated DNA double-strand breaks in replication stress-susceptible genes, some of which are associated with ASD pathogenesis. Our results provide a mechanism linking hyperproliferation of NPCs with the pathogenesis of ASD by disrupting long neural genes involved in cell-cell adhesion and migration.
Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Células Madre Pluripotentes Inducidas , Trastorno del Espectro Autista/genética , Proliferación Celular , Inestabilidad Genómica , HumanosRESUMEN
Interferons (IFNs) are key regulators for both innate and adaptive immune responses. By screening ENU-mutagenized mice, we identified a pedigree- P117 which displayed impaired response to type I, but not type II, IFNs. Through inheritance test, genetic mapping and sequencing, we found a T to A point mutation in the 5' splice site of STAT2 intron 4-5, leading to cryptic splicing and frame shifting. As a result, the expression of STAT2 protein was greatly diminished in the mutant mice. Nonetheless, a trace amount of functional STAT2 protein was still detectable and was capable of inducing, though to a lesser extent, IFNalpha-downstream gene expressions, suggesting that P117 is a STAT2 hypomorphic mutant. The restoration of mouse or human STAT2 gene in P117 MEFs rescued the response to IFNalpha, suggesting that the mutation in STAT2 is most likely the cause of the phenotypes seen in the pedigree. Development of different subsets of lymphocytes appeared to be normal in the mutant mice except that the percentage and basal expression of CD86 in splenic pDC and cDC were reduced. In addition, in vitro Flt3L-dependent DC development and TLR ligand-mediated DC differentiation were also defective in mutant cells. These results suggest that STAT2 positively regulates DC development and differentiation. Interestingly, a severe impairment of antiviral state and increased susceptibility to EMCV infection were observed in the mutant MEFs and mice, respectively, suggesting that the remaining STAT2 is not sufficient to confer antiviral response. In sum, the new allele of STAT2 mutant reported here reveals a role of STAT2 for DC development and a threshold requirement for full functions of type I IFNs.
Asunto(s)
Células Dendríticas/fisiología , Fenómenos del Sistema Inmunológico/fisiología , Interferón-alfa/inmunología , Interferón gamma/inmunología , Mutación , Factor de Transcripción STAT2 , Virus/metabolismo , Animales , Células Cultivadas , Análisis Mutacional de ADN , Células Dendríticas/citología , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Intrones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linaje , Empalme del ARN , Factor de Transcripción STAT2/genética , Factor de Transcripción STAT2/metabolismo , Virus/patogenicidadRESUMEN
Alzheimer's disease (AD), probably caused by abnormal accumulation of ß-amyloid (Aß) and aberrant phosphorylation of tau, is the most common cause of dementia among older people. Generation of patient-specific neurons by induced pluripotent stem cell (iPSC) technology facilitates exploration of the disease features in live human neurons from AD patients. In this study, we generated iPSCs from two familial AD patients carrying a heterozygous D678H mutation in the APP gene (AD-iPSCs). The neurons derived from our AD-iPSCs demonstrated aberrant accumulation of intracellular and secreted Aß42 and Aß40, reduction of serine 9 phosphorylation in glycogen synthase kinase 3ß (GSK3ß) hyperphosphorylation of threonine 181 and serine 396 in tau protein, impaired neurite outgrowth, downregulation of synaptophysin, and increased caspase 1 activity. The comparison between neurons derived from a sibling pair of wild-type and mutated iPSCs successfully recapitulated these AD phenotypes. Treatment with indole compound NC009-1 (3-((1H-Indole-3-yl)methyl)-4-(2-nitrophenyl)but-3-en-2-one), a potential Aß aggregation reducer, normalized the Aß levels and GSK3ß and tau phosphorylation, attenuated caspase 1 activity, and improved neurite outgrowth in AD-iPSC-derived neurons. Thus, APP D678H iPSCs-derived neurons recapitulate the cellular characteristics relevant to AD and enable exploration of the underlying pathogenesis and therapeutic strategies for AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Mutación/genética , Adulto , Animales , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Femenino , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Indoles/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Masculino , Ratones , Persona de Mediana Edad , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Linaje , Fenotipo , Fosforilación/efectos de los fármacos , Proteínas tau/metabolismoRESUMEN
Chromothripsis and chromoanasynthesis are catastrophic events leading to clustered genomic rearrangements. Whole-genome sequencing revealed frequent complex genomic rearrangements (n = 16/26) in brain tumors developing in mice deficient for factors involved in homologous-recombination-repair or non-homologous-end-joining. Catastrophic events were tightly linked to Myc/Mycn amplification, with increased DNA damage and inefficient apoptotic response already observable at early postnatal stages. Inhibition of repair processes and comparison of the mouse tumors with human medulloblastomas (n = 68) and glioblastomas (n = 32) identified chromothripsis as associated with MYC/MYCN gains and with DNA repair deficiencies, pointing towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Daño del ADN/genética , Reparación del ADN/genética , Genoma , Animales , Apoptosis/genética , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades/genética , Proteínas de Unión al ADN/metabolismo , Amplificación de Genes , Reordenamiento Génico/genética , Recombinación Homóloga/genética , Humanos , Cariotipificación , Ratones , Proteína Proto-Oncogénica N-Myc/genética , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Proteínas Proto-Oncogénicas c-myc/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Lymph node (LN) metastasis is commonly associated with systemic distant organ metastasis in human breast cancer and is an important prognostic predictor for survival of breast cancer patients. However, whether tumor-draining LNs (TDLNs) play a significant role in modulating the malignancy of cancer cells for distant metastasis remains controversial. Using a syngeneic mouse mammary tumor model, we found that breast tumor cells derived from TDLN have higher malignancy and removal of TDLNs significantly reduced distant metastasis. Up-regulation of oncogenic Il-17rb in cancer cells derived from TDLNs contributes to their malignancy. TGF-ß1 secreted from regulatory T cells (Tregs) in the TDLNs mediated the up-regulation of Il-17rb through downstream Smad2/3/4 signaling. These phenotypes can be abolished by TGF-ß1 neutralization or depletion of Tregs. Consistently, clinical data showed that the up-regulation of IL-17RB in cancer cells from LN metastases correlated with the increased prevalence of Tregs as well as the aggressive growth of tumors in mouse xenograft assay. Together, these results indicate that Tregs in TDLNs play an important role in modulating the malignancy of breast cancer cells for distant metastasis. Blocking IL-17RB expression could therefore be a potential approach to curb the process.
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Neoplasias de la Mama/patología , Ganglios Linfáticos/patología , Receptores de Interleucina-17/metabolismo , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/radioterapia , Femenino , Humanos , Ganglios Linfáticos/inmunología , Metástasis Linfática , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , ARN Interferente Pequeño/metabolismo , Receptores de Interleucina-17/antagonistas & inhibidores , Receptores de Interleucina-17/genética , Transducción de Señal , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Trasplante Homólogo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
NPGPx (GPx7) is a member of the glutathione peroxidase (GPx) family without any GPx activity. GPx7 displays a unique function which serves as a stress sensor/transmitter to transfer the signal to its interacting proteins by shuttling disulfide bonds in response to various stresses. In this review, we focus on the exceptional structural and biochemical features of GPx7 compared to other 7 family members and described how GPx7 regulates the diverse signaling targets including GRP78, PDI, CPEB2, and XRN2, and their different roles in unfolded protein response, oxidative stress, and non-targeting siRNA stress response, respectively. The phenotypes associated with GPx7 deficiency in mouse or human including ROS accumulations, highly elevated cancer incidences, auto-immune disorders, and obesity are also revealed in this paper. Finally, we compare GPx8 with GPx7, which shares the highest structural similarity but different biological roles in stress response. These insights have thus provided a more comprehensive understanding of the role of GPx7 in the maintenance of redox homeostasis.