ABSTRACT
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.
Subject(s)
Chromatin , DNA Topoisomerases, Type II , Genome/genetics , Models, Genetic , Animals , Cells, Cultured , Chromatin/chemistry , Chromatin/genetics , Chromatin/metabolism , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Genomics , Humans , MCF-7 Cells , Machine Learning , Mice , Protein Binding , ThymocytesABSTRACT
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.
Subject(s)
DNA Damage , Gene Regulatory Networks/physiology , Aminoquinolines/pharmacology , Animals , CRISPR-Cas Systems/genetics , Cell Line , Cytochrome-B(5) Reductase/genetics , Cytochrome-B(5) Reductase/metabolism , DNA Damage/drug effects , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Repair , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Humans , Mice , Picolinic Acids/pharmacology , RNA, Guide, Kinetoplastida/metabolism , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/geneticsABSTRACT
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.
Subject(s)
Androgens/toxicity , DNA Breaks, Double-Stranded , DNA-Binding Proteins/metabolism , Epithelial Cells/metabolism , Genomic Instability/genetics , Phosphoric Diester Hydrolases/metabolism , Prostate/metabolism , Animals , Cell Line , Cell Proliferation/drug effects , Cell Proliferation/genetics , Chromosome Breakage , DNA End-Joining Repair/drug effects , DNA End-Joining Repair/genetics , DNA-Binding Proteins/genetics , Epithelial Cells/drug effects , G1 Phase Cell Cycle Checkpoints/drug effects , G1 Phase Cell Cycle Checkpoints/genetics , Genomic Instability/drug effects , Histones/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphoric Diester Hydrolases/genetics , Prostate/drug effects , Prostatic Neoplasms/genetics , RNA, Small Interfering , Receptors, Androgen/metabolismABSTRACT
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.