Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors

Pessina, Fabio; Giavazzi, Fabio; Yin, Yandong; Gioia, Ubaldo; Vitelli, Valerio; Galbiati, Alessandro; Barozzi, Sara; Garré, Massimiliano; Oldani, Amanda; Flaus, Andrew; Cerbino, Roberto; Parazzoli, Dario; Rothenberg, Eli; Fagagna, Fabrizio d’Adda di

doi:10.1038/s41556-019-0392-4

Cited by 275 publications

(285 citation statements)

References 86 publications

(81 reference statements)

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“…53BP1 requires its oligomerization domain for recruitment to sites of DNA damage and shows hallmarks of dynamic self-assembly by phase separation [25][26][27] . A reduced concentration of H4K20me2, as present in replicated nascent chromatin, therefore likely increases the threshold for efficient 53BP1 accumulation.…”

Section: Resultsmentioning

confidence: 99%

Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and -deficient cells

Mitxelena

Pellegrino

Spegg

et al. 2020

Preprint

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DNA double-strand breaks can be repaired by two competing mechanisms, nonhomologous end-joining (NHEJ) and homologous recombination (HR). Whether one or the other repair pathway is favored depends on the availability of an undamaged template DNA that allows for homology-directed repair. The tumor suppressor proteins 53BP1 and BRCA1 are considered antagonistic players in this repair pathway choice, as 53BP1 restrains DNA end resection, whereas BRCA1, together with its partner protein BARD1, displaces 53BP1 from damaged replicated chromatin and promotes HR. How cells switch from a 53BP1-dominated to a BRCA1-dominated response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells, in comparison to 53BP1 accumulation at damaged unreplicated chromatin. These findings substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1-BARD1-dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1's binding mark H4K20me2 functionally cooperates with BRCA1-BARD1mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, e.g. to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

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Section: Resultsmentioning

confidence: 99%

Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and -deficient cells

Mitxelena

Pellegrino

Spegg

et al. 2020

Preprint

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“…In this case it might prevent hybridization of putative exposed single stranded DNA with RNA, thus facilitating alternative end joining pathways. Interestingly, the initial formation of 53BP1 foci has been described to require the recruitment of RNA polymerase II and transcription of long non-coding DNA in the vicinity of a DSB [49][50][51] and processing them into small non-coding DNA-damage response RNA (DDRNA, reviewed in [52]). Although the evidence of these processes after high-LET irradiation have not yet been presented, such transcriptional activity is fully compatible with the open EC-l DNA organization within DNA damage domains.…”

Section: Discussionmentioning

confidence: 99%

Correlative Light and Electron Microscopy (CLEM) Analysis of Nuclear Reorganization Induced by Clustered DNA Damage Upon Charged Particle Irradiation

Tonnemacher

Eltsov

Jakob

2020

IJMS

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Chromatin architecture plays major roles in gene regulation as well as in the repair of DNA damaged by endogenous or exogenous factors, such as after radiation. Opening up the chromatin might provide the necessary accessibility for the recruitment and binding of repair factors, thus facilitating timely and correct repair. The observed formation of ionizing radiation-induced foci (IRIF) of factors, such as 53BP1, upon induction of DNA double-strand breaks have been recently linked to local chromatin decompaction. Using correlative light and electron microscopy (CLEM) in combination with DNA-specific contrasting for transmission electron microscopy or tomography, we are able to show that at the ultrastructural level, these DNA damage domains reveal a chromatin compaction and organization not distinguishable from regular euchromatin upon irradiation with carbon or iron ions. Low Density Areas (LDAs) at sites of particle-induced DNA damage, as observed after unspecific uranyl acetate (UA)-staining, are thus unlikely to represent pure chromatin decompaction. RNA-specific terbium-citrate (Tb) staining suggests rather a reduced RNA density contributing to the LDA phenotype. Our observations are discussed in the view of liquid-like phase separation as one of the mechanisms of regulating DNA repair.

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“…In contrast with the well documented inhibition of transcription reported above, recent studies have revealed that de novo transcription occurs at free DNA ends in a process that generates non‐polyadenylated damage‐induced long non‐coding RNAs (dilncRNAs). [ 19–23 ] DilncRNAs were identified using strand specific RT‐qPCR (ssRT‐qPCR) and were postulated to be the precursors of damage‐induced small RNAs (diRNAs). [ 19,20,22,24 ] Production of dilncRNAs is sensitive to RNAPII inhibitors, but not to inhibitors specific to other RNA polymerases.…”

Section: De Novo Rna Synthesis At Dsbs: An Unexpected Discoverymentioning

confidence: 99%

“…[ 25 ] Moreover, the MRN complex is involved in the assembly of a transcription pre‐initiation complex at DSBs. [ 23 ] Despite these recent observations, the specifics of the initiation mechanism are not well understood. Interestingly, a detailed RNA analysis using deep sequencing revealed that dilncRNAs emerge only a few nucleotides away from the break site.…”

Section: De Novo Rna Synthesis At Dsbs: An Unexpected Discoverymentioning

confidence: 99%

RNA at DNA Double‐Strand Breaks: The Challenge of Dealing with DNA:RNA Hybrids

2020

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RNA polymerase II is recruited to DNA double-strand breaks (DSBs), transcribes the sequences that flank the break and produces a novel RNA type that has been termed damage-induced long non-coding RNA (dilncRNA). DilncRNAs can be processed into short, miRNA-like molecules or degraded by different ribonucleases. They can also form double-stranded RNAs or DNA:RNA hybrids. The DNA:RNA hybrids formed at DSBs contribute to the recruitment of repair factors during the early steps of homologous recombination (HR) and, in this way, contribute to the accuracy of the DNA repair. However, if not resolved, the DNA:RNA hybrids are highly mutagenic and prevent the recruitment of later HR factors. Here recent discoveries about the synthesis, processing, and degradation of dilncRNAs are revised. The focus is on RNA clearance, a necessary step for the successful repair of DSBs and the aim is to reconcile contradictory findings on the effects of dilncRNAs and DNA:RNA hybrids in HR.

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Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors

Cited by 275 publications

References 86 publications

Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and -deficient cells

Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and -deficient cells

Correlative Light and Electron Microscopy (CLEM) Analysis of Nuclear Reorganization Induced by Clustered DNA Damage Upon Charged Particle Irradiation

RNA at DNA Double‐Strand Breaks: The Challenge of Dealing with DNA:RNA Hybrids

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