RESUMO
DNA topoisomerase II-ß (TOP2B) is fundamental to remove topological problems linked to DNA metabolism and 3D chromatin architecture, but its cut-and-reseal catalytic mechanism can accidentally cause DNA double-strand breaks (DSBs) that can seriously compromise genome integrity. Understanding the factors that determine the genome-wide distribution of TOP2B is therefore not only essential for a complete knowledge of genome dynamics and organization, but also for the implications of TOP2-induced DSBs in the origin of oncogenic translocations and other types of chromosomal rearrangements. Here, we conduct a machine-learning approach for the prediction of TOP2B binding using publicly available sequencing data. We achieve highly accurate predictions, with accessible chromatin and architectural factors being the most informative features. Strikingly, TOP2B is sufficiently explained by only three features: DNase I hypersensitivity, CTCF and cohesin binding, for which genome-wide data are widely available. Based on this, we develop a predictive model for TOP2B genome-wide binding that can be used across cell lines and species, and generate virtual probability tracks that accurately mirror experimental ChIP-seq data. Our results deepen our knowledge on how the accessibility and 3D organization of chromatin determine TOP2B function, and constitute a proof of principle regarding the in silico prediction of sequence-independent chromatin-binding factors.
Assuntos
Cromatina , DNA Topoisomerases Tipo II , Genoma/genética , Modelos Genéticos , Animais , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Genômica , Humanos , Células MCF-7 , Aprendizado de Máquina , Camundongos , Ligação Proteica , TimócitosRESUMO
The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.
Assuntos
Dano ao DNA , Redes Reguladoras de Genes/fisiologia , Aminoquinolinas/farmacologia , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Camundongos , Ácidos Picolínicos/farmacologia , RNA Guia de Cinetoplastídeos/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Androgens stimulate the proliferation of epithelial cells in the prostate by activating topoisomerase 2 (TOP2) and regulating the transcription of target genes. TOP2 resolves the entanglement of genomic DNA by transiently generating double-strand breaks (DSBs), where TOP2 homodimers covalently bind to 5' DSB ends, called TOP2-DNA cleavage complexes (TOP2ccs). When TOP2 fails to rejoin TOP2ccs generating stalled TOP2ccs, tyrosyl DNA phosphodiesterase-2 (TDP2) removes 5' TOP2 adducts from stalled TOP2ccs prior to the ligation of the DSBs by nonhomologous end joining (NHEJ), the dominant DSB repair pathway in G0 /G1 phases. We previously showed that estrogens frequently generate stalled TOP2ccs in G0 /G1 phases. Here, we show that physiological concentrations of androgens induce several DSBs in individual human prostate cancer cells during G1 phase, and loss of TDP2 causes a five times higher number of androgen-induced chromosome breaks in mitotic chromosome spreads. Intraperitoneally injected androgens induce several DSBs in individual epithelial cells of the prostate in TDP2-deficient mice, even at 20 hr postinjection. In conclusion, physiological concentrations of androgens have very strong genotoxicity, most likely by generating stalled TOP2ccs.
Assuntos
Androgênios/toxicidade , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/metabolismo , Instabilidade Genômica/genética , Diester Fosfórico Hidrolases/metabolismo , Próstata/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Quebra Cromossômica , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/genética , Células Epiteliais/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Instabilidade Genômica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Diester Fosfórico Hidrolases/genética , Próstata/efeitos dos fármacos , Neoplasias da Próstata/genética , RNA Interferente Pequeno , Receptores Androgênicos/metabolismoRESUMO
The ATM kinase is a master regulator of the DNA damage response to double-strand breaks (DSBs) and a well-established tumour suppressor whose loss is the cause of the neurodegenerative and cancer-prone syndrome Ataxia-Telangiectasia (A-T). A-T patients and Atm-/- mouse models are particularly predisposed to develop lymphoid cancers derived from deficient repair of RAG-induced DSBs during V(D)J recombination. Here, we unexpectedly find that specifically disturbing the repair of DSBs produced by DNA topoisomerase II (TOP2) by genetically removing the highly specialised repair enzyme TDP2 increases the incidence of thymic tumours in Atm-/- mice. Furthermore, we find that TOP2 strongly colocalizes with RAG, both genome-wide and at V(D)J recombination sites, resulting in an increased endogenous chromosomal fragility of these regions. Thus, our findings demonstrate a strong causal relationship between endogenous TOP2-induced DSBs and cancer development, confirming these lesions as major drivers of ATM-deficient lymphoid malignancies, and potentially other conditions and cancer types.
