RESUMEN
ATRX gene mutations have been identified in syndromic and non-syndromic intellectual disabilities in humans. ATRX is known to maintain genomic stability in neuroprogenitor cells, but its function in differentiated neurons and memory processes remains largely unresolved. Here, we show that the deletion of neuronal Atrx in mice leads to distinct hippocampal structural defects, fewer presynaptic vesicles, and an enlarged postsynaptic area at CA1 apical dendrite-axon junctions. We identify male-specific impairments in long-term contextual memory and in synaptic gene expression, linked to altered miR-137 levels. We show that ATRX directly binds to the miR-137 locus and that the enrichment of the suppressive histone mark H3K27me3 is significantly reduced upon the loss of ATRX. We conclude that the ablation of ATRX in excitatory forebrain neurons leads to sexually dimorphic effects on miR-137 expression and on spatial memory, identifying a potential therapeutic target for neurological defects caused by ATRX dysfunction.
Asunto(s)
Eliminación de Gen , Regulación de la Expresión Génica , Trastornos de la Memoria/genética , Trastornos de la Memoria/fisiopatología , MicroARNs/genética , Caracteres Sexuales , Aprendizaje Espacial , Proteína Nuclear Ligada al Cromosoma X/deficiencia , Animales , Secuencia de Bases , Región CA1 Hipocampal/patología , Región CA1 Hipocampal/ultraestructura , Condicionamiento Operante , Dendritas/metabolismo , Dendritas/ultraestructura , Femenino , Genotipo , Histonas/metabolismo , Lisina/metabolismo , Imagen por Resonancia Magnética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/metabolismo , Neuronas , Especificidad de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Proteína Nuclear Ligada al Cromosoma X/metabolismoRESUMEN
α-Thalassemia X-linked intellectual disability (ATR-X) syndrome is a neurodevelopmental disorder caused by mutations in the ATRX gene that encodes a SNF2-type chromatin-remodeling protein. The ATRX protein regulates chromatin structure and gene expression in the developing mouse brain and early inactivation leads to DNA replication stress, extensive cell death, and microcephaly. However, the outcome of Atrx loss of function postnatally in neurons is less well understood. We recently reported that conditional inactivation of Atrx in postnatal forebrain excitatory neurons (ATRX-cKO) causes deficits in long-term hippocampus-dependent spatial memory. Thus, we hypothesized that ATRX-cKO mice will display impaired hippocampal synaptic transmission and plasticity. In the present study, evoked field potentials and current source density analysis were recorded from a multichannel electrode in male, urethane-anesthetized mice. Three major excitatory synapses, the Schaffer collaterals to basal dendrites and proximal apical dendrites, and the temporoammonic path to distal apical dendrites on hippocampal CA1 pyramidal cells were assessed by their baseline synaptic transmission, including paired-pulse facilitation (PPF) at 50-ms interpulse interval, and by their long-term potentiation (LTP) induced by theta-frequency burst stimulation. Baseline single-pulse excitatory response at each synapse did not differ between ATRX-cKO and control mice, but baseline PPF was reduced at the CA1 basal dendritic synapse in ATRX-cKO mice. While basal dendritic LTP of the first-pulse excitatory response was not affected in ATRX-cKO mice, proximal and distal apical dendritic LTP were marginally and significantly reduced, respectively. These results suggest that ATRX is required in excitatory neurons of the forebrain to achieve normal hippocampal LTP and PPF at the CA1 apical and basal dendritic synapses, respectively. Such alterations in hippocampal synaptic transmission and plasticity could explain the long-term spatial memory deficits in ATRX-cKO mice and provide insight into the physiological mechanisms underlying intellectual disability in ATR-X syndrome patients.
Asunto(s)
Hipocampo/metabolismo , Plasticidad Neuronal/fisiología , Prosencéfalo/metabolismo , Sinapsis/metabolismo , Proteína Nuclear Ligada al Cromosoma X/deficiencia , Animales , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/citología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Prosencéfalo/citología , Proteína Nuclear Ligada al Cromosoma X/genéticaRESUMEN
The SWI/SNF-family chromatin remodeling protein ATRX is a tumor suppressor in sarcomas, gliomas and other malignancies. Its loss of function facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells, while it also affects Polycomb repressive complex 2 (PRC2) silencing of its target genes. To further define the role of inactivating ATRX mutations in carcinogenesis, we knocked out atrx in our previously reported p53/nf1-deficient zebrafish line that develops malignant peripheral nerve sheath tumors and gliomas. Complete inactivation of atrx using CRISPR/Cas9 was lethal in developing fish and resulted in an alpha-thalassemia-like phenotype including reduced alpha-globin expression. In p53/nf1-deficient zebrafish neither peripheral nerve sheath tumors nor gliomas showed accelerated onset in atrx+/- fish, but these fish developed various tumors that were not observed in their atrx+/+ siblings, including epithelioid sarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma and rare types of carcinoma. These cancer types are included in the AACR Genie database of human tumors associated with mutant ATRX, indicating that our zebrafish model reliably mimics a role for ATRX-loss in the early pathogenesis of these human cancer types. RNA-seq of p53/nf1- and p53/nf1/atrx-deficient tumors revealed that down-regulation of telomerase accompanied ALT-mediated lengthening of the telomeres in atrx-mutant samples. Moreover, inactivating mutations in atrx disturbed PRC2-target gene silencing, indicating a connection between ATRX loss and PRC2 dysfunction in cancer development.
Asunto(s)
Sarcoma Experimental/etiología , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína Nuclear Ligada al Cromosoma X/deficiencia , Proteína Nuclear Ligada al Cromosoma X/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas , Carcinogénesis/genética , Carcinogénesis/metabolismo , Modelos Animales de Enfermedad , Eritropoyesis , Femenino , Técnicas de Inactivación de Genes , Globinas/genética , Humanos , Mutación con Pérdida de Función , Masculino , Neurofibromina 1/deficiencia , Neurofibromina 1/genética , Sarcoma Experimental/genética , Sarcoma Experimental/metabolismo , Homeostasis del Telómero/genética , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Cancers that utilize the alternative lengthening of telomeres (ALT) mechanism for telomere maintenance are often difficult to treat and have a poor prognosis. They are also commonly deficient for expression of ATRX protein, a repressor of ALT activity, and a component of promyelocytic leukemia nuclear bodies (PML NBs) that are required for intrinsic immunity to various viruses. Here, we asked whether ATRX deficiency creates a vulnerability in ALT cancer cells that could be exploited for therapeutic purposes. We showed in a range of cell types that a mutant herpes simplex virus type 1 (HSV-1) lacking ICP0, a protein that degrades PML NB components including ATRX, was ten- to one thousand-fold more effective in infecting ATRX-deficient cells than wild-type ATRX-expressing cells. Infection of co-cultured primary and ATRX-deficient cancer cells revealed that mutant HSV-1 selectively killed ATRX-deficient cells. Sensitivity to mutant HSV-1 infection also correlated inversely with PML protein levels, and we showed that ATRX upregulates PML expression at both the transcriptional and post-transcriptional levels. These data provide a basis for predicting, based on ATRX or PML levels, which tumors will respond to a selective oncolytic herpesvirus.
Asunto(s)
Herpes Simple/metabolismo , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Riñón/metabolismo , Proteína de la Leucemia Promielocítica/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Nuclear Ligada al Cromosoma X/deficiencia , Animales , Muerte Celular , Línea Celular Tumoral , Cricetinae , Herpes Simple/patología , Humanos , Proteínas Inmediatas-Precoces/genética , Inmunidad Innata/genética , Riñón/patología , Mutación/genética , Viroterapia Oncolítica , Proteína de la Leucemia Promielocítica/genética , Homeostasis del Telómero , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Mutational inactivation of ATRX (α-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. Yet the pathogenic consequences of ATRX deficiency remain unclear, as do tractable mechanisms for its therapeutic targeting. Here we report that ATRX loss in isogenic glioma model systems induces replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Moreover, these effects are associated with the acquisition of disease-relevant copy number alterations over time. We then demonstrate, both in vitro and in vivo, that ATRX deficiency selectively enhances DNA damage and cell death following chemical G4 stabilization. Finally, we show that G4 stabilization synergizes with other DNA-damaging therapies, including ionizing radiation, in the ATRX-deficient context. Our findings reveal novel pathogenic mechanisms driven by ATRX deficiency in glioma, while also pointing to tangible strategies for drug development.
Asunto(s)
Neoplasias Encefálicas/genética , G-Cuádruplex , Glioma/genética , Proteína Nuclear Ligada al Cromosoma X/deficiencia , Proteína Nuclear Ligada al Cromosoma X/genética , Animales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Daño del ADN , Replicación del ADN , ADN de Neoplasias/química , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Técnicas de Silenciamiento del Gen , Inestabilidad Genómica , Glioma/metabolismo , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , MutaciónRESUMEN
Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in North America, accounting for >30,000 deaths annually. Although somatic activating mutations in KRAS appear in 97% of PDAC patients, additional factors are required to initiate PDAC. Because mutations in genes encoding chromatin remodelling proteins have been implicated in KRAS-mediated PDAC, we investigated whether loss of chromatin remodeler É-thalassemia, mental-retardation, X-linked (ATRX) affects oncogenic KRAS's ability to promote PDAC. ATRX affects DNA replication, repair, and gene expression and is implicated in other cancers including glioblastomas and pancreatic neuroendocrine tumors. The hypothesis was that deletion of Atrx in pancreatic acinar cells will increase susceptibility to injury and oncogenic KRAS. Methods: Mice allowing conditional loss of Atrx within pancreatic acinar cells were examined after induction of recurrent cerulein-induced pancreatitis or oncogenic KRAS (KRASG12D ). Histologic, biochemical, and molecular analysis examined pancreatic pathologies up to 2 months after induction of Atrx deletion. Results: Mice lacking Atrx showed more progressive damage, inflammation, and acinar-to-duct cell metaplasia in response to injury relative to wild-type mice. In combination with KRASG12D, Atrx-deficient acinar cells showed increased fibrosis, inflammation, progression to acinar-to-duct cell metaplasia, and pre-cancerous lesions relative to mice expressing only KRASG12D. This sensitivity appears only in female mice, mimicking a significant prevalence of ATRX mutations in human female PDAC patients. Conclusions: Our results indicate the absence of ATRX increases sensitivity to injury and oncogenic KRAS only in female mice. This is an instance of a sex-specific mutation that enhances oncogenic KRAS's ability to promote pancreatic intraepithelial lesion formation.
Asunto(s)
Oncogenes , Páncreas/lesiones , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína Nuclear Ligada al Cromosoma X/deficiencia , Células Acinares/metabolismo , Células Acinares/patología , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Análisis Mutacional de ADN , Femenino , Eliminación de Gen , Masculino , Ratones , Páncreas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Proteína Nuclear Ligada al Cromosoma X/metabolismo , Neoplasias PancreáticasRESUMEN
Mutational inactivation of the SWI/SNF chromatin regulator ATRX occurs frequently in gliomas, the most common primary brain tumors. Whether and how ATRX deficiency promotes oncogenesis by epigenomic dysregulation remains unclear, despite its recent implication in both genomic instability and telomere dysfunction. Here we report that Atrx loss recapitulates characteristic disease phenotypes and molecular features in putative glioma cells of origin, inducing cellular motility although also shifting differentiation state and potential toward an astrocytic rather than neuronal histiogenic profile. Moreover, Atrx deficiency drives widespread shifts in chromatin accessibility, histone composition, and transcription in a distribution almost entirely restricted to genomic sites normally bound by the protein. Finally, direct gene targets of Atrx that mediate specific Atrx-deficient phenotypes in vitro exhibit similarly selective misexpression in ATRX-mutant human gliomas. These findings demonstrate that ATRX deficiency and its epigenomic sequelae are sufficient to induce disease-defining oncogenic phenotypes in appropriate cellular and molecular contexts.
Asunto(s)
Glioma/genética , Proteína Nuclear Ligada al Cromosoma X/deficiencia , Proteína Nuclear Ligada al Cromosoma X/genética , Animales , Diferenciación Celular , Línea Celular , Movimiento Celular , Ensamble y Desensamble de Cromatina , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Silenciador del Gen , Genes p53 , Humanos , Ratones Noqueados , Células-Madre Neurales/metabolismo , Células Neuroepiteliales/metabolismo , Fenotipo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG)n ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA-DNA hybrids (R-loops) whose abundance correlates with the recruitment of ATRX Here, we show loss of ATRX is also associated with increased R-loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology-directed repair previously observed upon loss of ATRX function.