RESUMEN
Accumulation of topological stress in the form of DNA supercoiling is inherent to the advance of RNA polymerase II (Pol II) and needs to be resolved by DNA topoisomerases to sustain productive transcriptional elongation. Topoisomerases are therefore considered positive facilitators of transcription. Here, we show that, in contrast to this general assumption, human topoisomerase IIα (TOP2A) activity at promoters represses transcription of immediate early genes such as c-FOS, maintaining them under basal repressed conditions. Thus, TOP2A inhibition creates a particular topological context that results in rapid release from promoter-proximal pausing and transcriptional upregulation, which mimics the typical bursting behavior of these genes in response to physiological stimulus. We therefore describe the control of promoter-proximal pausing by TOP2A as a layer for the regulation of gene expression, which can act as a molecular switch to rapidly activate transcription, possibly by regulating the accumulation of DNA supercoiling at promoter regions.
Asunto(s)
ADN-Topoisomerasas de Tipo II/genética , ADN Superhelicoidal/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas Proto-Oncogénicas c-fos/genética , ARN Polimerasa II/genética , Transcripción Genética , Línea Celular Transformada , ADN-Topoisomerasas de Tipo II/metabolismo , ADN Superhelicoidal/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Regulación de la Expresión Génica , Genes Inmediatos-Precoces , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/antagonistas & inhibidores , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Polimerasa II/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/enzimología , Tiobarbitúricos/farmacología , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Topoisomerase 2 (TOP2) DNA transactions proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer TOP2 drugs. How genotoxic TOP2 DNA-protein cross-links are resolved is unclear. We found that the SUMO (small ubiquitin-related modifier) ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a "split-SIM" SUMO2 engagement platform. These findings uncover a ZATT-TDP2-catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Aminoaciltransferasas , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Dominio Catalítico , ADN/genética , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN , Etopósido/farmacología , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Inmunoprecipitación , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Proteínas Nucleares/genética , Hidrolasas Diéster Fosfóricas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Inhibidores de Topoisomerasa II/farmacología , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Mutations in chromatin modifier genes are frequently associated with neurodevelopmental diseases. We herein demonstrate that the chromodomain helicase DNA-binding protein 7 (Chd7), frequently associated with CHARGE syndrome, is indispensable for normal cerebellar development. Genetic inactivation of Chd7 in cerebellar granule neuron progenitors leads to cerebellar hypoplasia in mice, due to the impairment of granule neuron differentiation, induction of apoptosis and abnormal localization of Purkinje cells, which closely recapitulates known clinical features in the cerebella of CHARGE patients. Combinatory molecular analyses reveal that Chd7 is required for the maintenance of open chromatin and thus activation of genes essential for granule neuron differentiation. We further demonstrate that both Chd7 and Top2b are necessary for the transcription of a set of long neuronal genes in cerebellar granule neurons. Altogether, our comprehensive analyses reveal a mechanism with chromatin remodellers governing brain development via controlling a core transcriptional programme for cell-specific differentiation.
Asunto(s)
Encéfalo/metabolismo , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Neuronas/metabolismo , Animales , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Humanos , Mamíferos/genética , Mamíferos/crecimiento & desarrollo , Mamíferos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuronas/citologíaRESUMEN
Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders.