RESUMO
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.
Assuntos
DNA Topoisomerases Tipo II/genética , DNA Super-Helicoidal/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas Proto-Oncogênicas c-fos/genética , RNA Polimerase II/genética , Transcrição Gênica , Linhagem Celular Transformada , DNA Topoisomerases Tipo II/metabolismo , DNA Super-Helicoidal/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Regulação da Expressão Gênica , Genes Precoces , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Polimerase II/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/enzimologia , Tiobarbitúricos/farmacologia , Inibidores da Topoisomerase II/farmacologiaRESUMO
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.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Aminoaciltransferases , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , DNA/genética , DNA/metabolismo , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA , Etoposídeo/farmacologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Proteínas Nucleares/genética , Diester Fosfórico Hidrolases , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Inibidores da Topoisomerase II/farmacologia , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
Encéfalo/metabolismo , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , Mamíferos/genética , Mamíferos/crescimento & desenvolvimento , Mamíferos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/citologiaRESUMO
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.