Release of paused RNA polymerase II at specific loci favors DNA double-strand-break formation and promotes cancer translocations

Abstract

It is not clear how spontaneous DNA double-strand breaks (DSBs) form and are processed in normal cells, and whether they predispose to cancer-associated translocations. We show that DSBs in normal mammary cells form upon release of paused RNA polymerase II (Pol II) at promoters, 5′ splice sites and active enhancers, and are processed by end-joining in the absence of a canonical DNA-damage response. Logistic and causal-association models showed that Pol II pausing at long genes is the main predictor and determinant of DSBs. Damaged introns with paused Pol II-pS5, TOP2B and XRCC4 are enriched in translocation breakpoints, and map at topologically associating domain boundary-flanking regions showing high interaction frequencies with distal loci. Thus, in unperturbed growth conditions, release of paused Pol II at specific loci and chromatin territories favors DSB formation, leading to chromosomal translocations.

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Fig. 1: Genome-wide mapping of digested AsiSI sites and endogenous DSBs in diploid mammary epithelial cells.
Fig. 2: Pol II-pS5 accumulates at fragile promoters.
Fig. 3: Effect of etoposide (Eto) treatment on TOP2B ChIP-seq signals, and ChIP-seq signals of Pol II-pS5 and DNA-repair factors around the TSSs of fragile and control promoters.
Fig. 4: Pol II-pS5, topoisomerases and DNA-repair factors at fragile and control promoters from different transcription classes, and effect of etoposide treatment on gene expression.
Fig. 5: In situ localization of TOP2B-XRCC4 interaction sites, and length of genes with fragile or control promoters.
Fig. 6: Mechanisms of endogenous DSB formation/processing, and effect of DRB administration and washout on Pol II-pS5 and XRCC4 ChIP-seq signals at fragile and control promoters.
Fig. 7: Characterization of fragile and control enhancers.
Fig. 8: Characterization of the damaged introns associated with translocation breakpoints.

Data availability

Raw and processed data are available under accession number GSE93040. Previously published data used in this work are: GRO-seq: E-MTAB-742; γH2AX ChIP-seq data of 4-OHT-treated cells (t = 2 h, replicate no. 1 in Supplementary Fig. 2a) are available under accession number GSE71447.

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Acknowledgements

We thank R. Mirzazadeh for initial training on the BLISS method; I. Pallavicini and T. Kallas for technical assistance with cell culture; L. Rotta and T. Capra of the Sequencing Facility at the IEO Genomic Unit; E. Colombo for helpful discussions; and P. Dalton and S. Averaimo for critical review of the manuscript. F.P. was supported by a fellowship from Fondazione Umberto Veronesi (grant no. FUV 2018). N.C. acknowledges support from the Karolinska Institutet, the Swedish Research Council (grant no. 521-2014-2866), the Swedish Cancer Research Foundation (grant no. CAN 2015/585) and the Ragnar Söderberg Foundation. M.F. acknowledges support from Italian Ministry of Health grant no. RF-2011-02347946. This study was supported by European Research Council advanced grant no. 341131 (to P.G.P.).

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R.P. and B.A.M.B. performed the BLISS assays under the supervision of N.C. R.P., G.I.D. and G.D.C. performed the ChIP-seq and RNA-seq assays. F.P. performed statistical analyses and machine learning-based approaches. G.I.D., F.P., L.L., G.M. and D.C. analyzed the sequencing data. A.M.C. and S.B. performed the Hi-C analyses under the supervision of M.N. D.G. contributed to the statistical analyses. L.G. aligned the sequencing data. L.F. performed the immunofluorescence. M.F. performed the imaging analyses. P.G.P. and G.I.D. wrote the manuscript. G.I.D. and P.G.P. contributed to study design and oversaw the study.

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Correspondence to Gaetano Ivan Dellino or Pier Giuseppe Pelicci.

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Dellino, G.I., Palluzzi, F., Chiariello, A.M. et al. Release of paused RNA polymerase II at specific loci favors DNA double-strand-break formation and promotes cancer translocations. Nat Genet 51, 1011–1023 (2019). https://doi.org/10.1038/s41588-019-0421-z

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