RESUMEN
Deficiency of FANCD2/FANCI-associated nuclease 1 (FAN1) in humans leads to karyomegalic interstitial nephritis (KIN), a rare hereditary kidney disease characterized by chronic renal fibrosis, tubular degeneration, and characteristic polyploid nuclei in multiple tissues. The mechanism of how FAN1 protects cells is largely unknown but is thought to involve FAN1's function in DNA interstrand cross-link (ICL) repair. Here, we describe a Fan1-deficient mouse and show that FAN1 is required for cellular and organismal resistance to ICLs. We show that the ubiquitin-binding zinc finger (UBZ) domain of FAN1, which is needed for interaction with FANCD2, is not required for the initial rapid recruitment of FAN1 to ICLs or for its role in DNA ICL resistance. Epistasis analyses reveal that FAN1 has cross-link repair activities that are independent of the Fanconi anemia proteins and that this activity is redundant with the 5'-3' exonuclease SNM1A. Karyomegaly becomes prominent in kidneys and livers of Fan1-deficient mice with age, and mice develop liver dysfunction. Treatment of Fan1-deficient mice with ICL-inducing agents results in pronounced thymic and bone marrow hypocellularity and the disappearance of c-kit(+) cells. Our results provide insight into the mechanism of FAN1 in ICL repair and demonstrate that the Fan1 mouse model effectively recapitulates the pathological features of human FAN1 deficiency.
Asunto(s)
Endodesoxirribonucleasas/deficiencia , Endodesoxirribonucleasas/genética , Riñón/patología , Hepatopatías/genética , Animales , Médula Ósea/efectos de los fármacos , Reactivos de Enlaces Cruzados/farmacología , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Epistasis Genética , Exodesoxirribonucleasas/metabolismo , Hígado/patología , Ratones , Enzimas Multifuncionales , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
Current methods for detecting mutations in Fanconi anemia (FA)-suspected patients are inefficient and often miss mutations. We have applied recent advances in DNA sequencing and genomic capture to the diagnosis of FA. Specifically, we used custom molecular inversion probes or TruSeq-enrichment oligos to capture and sequence FA and related genes, including introns, from 27 samples from the International Fanconi Anemia Registry at The Rockefeller University. DNA sequencing was complemented with custom array comparative genomic hybridization (aCGH) and RNA sequencing (RNA-seq) analysis. aCGH identified deletions/duplications in 4 different FA genes. RNA-seq analysis revealed lack of allele specific expression associated with a deletion and splicing defects caused by missense, synonymous, and deep-in-intron variants. The combination of TruSeq-targeted capture, aCGH, and RNA-seq enabled us to identify the complementation group and biallelic germline mutations in all 27 families: FANCA (7), FANCB (3), FANCC (3), FANCD1 (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (2), and FANCL (3). FANCC mutations are often the cause of FA in patients of Ashkenazi Jewish (AJ) ancestry, and we identified 2 novel FANCC mutations in 2 patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of FA.
Asunto(s)
Hibridación Genómica Comparativa/métodos , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Judíos/genética , Análisis de Secuencia de ARN/métodos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Salud de la Familia , Anemia de Fanconi/etnología , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación L de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Eliminación de Gen , Duplicación de Gen , Humanos , MutaciónRESUMEN
The Alternative Lengthening of Telomeres (ALT) pathway is a telomerase-independent pathway for telomere maintenance that is active in a significant subset of human cancers and in vitro immortalized cell lines. ALT is thought to involve templated extension of telomeres through homologous recombination, but the genetic or epigenetic changes that unleash ALT are not known. Recently, mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 were found to correlate with features of ALT in pancreatic neuroendocrine cancers, pediatric glioblastomas, and other tumors of the central nervous system, suggesting that these mutations might contribute to the activation of the ALT pathway in these cancers. We have taken a comprehensive approach to deciphering ALT by applying genomic, molecular biological, and cell biological approaches to a panel of 22 ALT cell lines, including cell lines derived in vitro. Here we show that loss of ATRX protein and mutations in the ATRX gene are hallmarks of ALT-immortalized cell lines. In addition, ALT is associated with extensive genome rearrangements, marked micronucleation, defects in the G2/M checkpoint, and altered double-strand break (DSB) repair. These attributes will facilitate the diagnosis and treatment of ALT positive human cancers.
Asunto(s)
ADN Helicasas/genética , Histonas , Proteínas Nucleares/genética , Homeostasis del Telómero/genética , Telómero/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas Co-Represoras , Roturas del ADN de Doble Cadena , Daño del ADN/genética , ADN Helicasas/metabolismo , Reparación del ADN/genética , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Inestabilidad Genómica , Células HeLa , Histonas/genética , Histonas/metabolismo , Recombinación Homóloga , Humanos , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Transducción de Señal , Telomerasa/genética , Telómero/metabolismo , Proteína Nuclear Ligada al Cromosoma XRESUMEN
Hematopoietic stem cell transplantation (HSCT) is a curative treatment for patients with many different blood and immune diseases; however, current treatment regimens contain non-specific chemotherapy and/or irradiation conditioning, which carry both short-term and long-term toxicities. The use of such agents may be particularly harmful for patients with Fanconi anemia (FA), who have genetic mutations resulting in deficiencies in DNA repair, leading to increased sensitivity to genotoxic agents. mAb-based conditioning has been proposed as an alternative conditioning strategy for HSCT that minimizes these toxicities by eliminating collateral tissue damage. Given the high need for improved treatments for FA patients, we aimed to evaluate the efficacy of different αCD117 mAb agents and immunosuppression on hematopoietic stem cell (HSC) depletion and explored their ability to safely establish therapeutic donor hematopoiesis post-HSCT in FA disease models. We evaluated the effects of different concentrations of αCD117 mAbs in vitro and in vivo on HSC growth and depletion. To further assess the efficacy of mAb-based conditioning, Fancd2-/- animals were treated with αCD117 mAb and combination agents with αCD47 mAb and antibody-drug-conjugates (ADCs) for syngeneic HSCT. Immunosuppression αCD4 mAb was added to all in vivo experiments due to a slightly mismatched background between the donor grafts and recipients. Immunosuppressant cocktails were also given to Fancd2-/- animals to evaluate the efficacy of mAb-based conditioning in the haploidentical setting. Statistical analyses were done using the unpaired t-test. We found that antagonistic αCD117 mAbs alone do not deplete host HSCs or enhance HSCT effectively in FA mouse models; however, the potency of αCD117 mAbs can be safely augmented through combination with αCD47 mAbs and with ADCs, both of which lead to profound HSC depletion and establishment of long-term donor engraftment post-syngeneic HSCT. This is the first time these approaches have been tested in parallel in any disease setting, with the greatest donor engraftment observed after CD117-ADC conditioning. Interestingly, our data also suggest that HSC-targeted conditioning is not necessary in HSCT for FA, as high donor HSC engraftment was observed with mAb-based immune suppression alone with immunologically matched and mismatched haploidentical grafts. These results demonstrate the safety and efficacy of several different non-genotoxic mAb-based conditioning strategies in the FA setting. In addition, they show that if sufficient immunosuppression is given to obtain initial donor HSC engraftment, turnover of a majority of the hematolymphoid system can result, likely owing to the survival advantage of wild-type HSCs over FA HSCs. Such non-toxic all-mAb-based conditioning strategies could be transformative for FA patients and those with other hematolymphoid diseases.
Asunto(s)
Anemia de Fanconi , Trasplante de Células Madre Hematopoyéticas , Animales , Ratones , Anemia de Fanconi/etiología , Anemia de Fanconi/terapia , Acondicionamiento Pretrasplante/métodos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Inmunosupresores , Terapia de Inmunosupresión/métodos , Anticuerpos MonoclonalesRESUMEN
Recent studies revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes that may give rise to somatic brain mosaicism. The mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. Here, we elucidated the genome-wide landscape of RNA:DNA hybrid structures called "R-loops" in primary neural stem/progenitor cells undergoing aphidicolin-induced, mild replication stress to assess the potential contribution of R-loops to the different, recurrent classes of DNA break "hotspots". We find that R-loops in neural stem/progenitor cells undergoing mild replication stress are present primarily in early-replicating, transcribed regions and in genes with promoter GC skew that are associated with cell lineage-specific processes. Surprisingly, most long, neural genes that form recurrent DSB clusters do not show R-loop formation under conditions of mild replication stress. Our findings are consistent with a role of R-loop-associated processes in promoter-proximal DNA break formation in highly transcribed, early replicating regions but suggest that R-loops do not drive replication stress-induced, recurrent DSB cluster formation in most long, neural genes.
Asunto(s)
Células-Madre Neurales , Estructuras R-Loop , ADN/genética , Roturas del ADN de Doble Cadena , Reparación del ADNRESUMEN
Severe cellular sensitivity and aberrant chromosomal rearrangements in response to DNA interstrand crosslink (ICL) inducing agents are hallmarks of Fanconi anemia (FA) deficient cells. These phenotypes have previously been ascribed to inappropriate activity of non-homologous end joining (NHEJ) rather than a direct consequence of DNA ICL repair defects. Here we used chemical inhibitors, RNAi, and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 to inactivate various components of NHEJ in cells from FA patients. We show that suppression of DNA-PKcs, DNA Ligase IV, and 53BP1 is not capable of rescuing ICL-induced proliferation defects and only 53BP1 knockout partially suppresses the chromosomal abnormalities of FA patient cells.
Asunto(s)
Daño del ADN , Reparación del ADN por Unión de Extremidades , Anemia de Fanconi/metabolismo , Fibroblastos/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular Transformada , Proliferación Celular , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/patología , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Células HCT116 , Humanos , Mutación , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
Chronic kidney disease (CKD) represents a major health burden. Its central feature of renal fibrosis is not well understood. By exome sequencing, we identified mutations in FAN1 as a cause of karyomegalic interstitial nephritis (KIN), a disorder that serves as a model for renal fibrosis. Renal histology in KIN is indistinguishable from that of nephronophthisis, except for the presence of karyomegaly. The FAN1 protein has nuclease activity and acts in DNA interstrand cross-link (ICL) repair within the Fanconi anemia DNA damage response (DDR) pathway. We show that cells from individuals with FAN1 mutations have sensitivity to the ICL-inducing agent mitomycin C but do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from individuals with Fanconi anemia. We complemented ICL sensitivity with wild-type FAN1 but not with cDNA having mutations found in individuals with KIN. Depletion of fan1 in zebrafish caused increased DDR, apoptosis and kidney cysts. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms contributing to renal fibrosis and CKD.