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FUS-dependent liquid-liquid phase separation is an early event in double-strand break repair

Brunno R. Levone, Silvia C. Lenzken, Marco Antonaci, Andreas Maiser, Alexander Rapp, Francesca Conte, Stefan Reber, Antonella E. Ronchi, Oliver Mühlemann, Heinrich Leonhardt, M. Cristina Cardoso, Marc-David Ruepp, Silvia M.L. Barabino

Preprint posted on 24 August 2020 https://www.biorxiv.org/content/10.1101/798884v2

Article now published in Journal of Cell Biology at http://dx.doi.org/10.1083/jcb.202008030

FUSzy ways of DNA damage response.

Selected by Sree Rama Chaitanya, Giuseppina D'Alessandro

Categories: biochemistry, cell biology, molecular biology, neuroscience

Context

Liquid-Liquid phase separation (LLPS) coalesces macromolecules from the local cellular milieu to facilitate many biological processes. LLPS has emerged as an important biophysical mechanism to regulate a myriad of the biological process from organizing non-membranous cellular compartments to macromolecules that facilitate cellular and genome function. Notably, in the nucleus, LLPS promotes the spontaneous organization of proteins and nucleic acids with shared functions to facilitate genomic processes like chromatin organization, transcription, RNA processing, DNA replication, and DNA damage response1,2.

In response to DNA damage, cells often commission the proteins available in the local vicinity of the damage for the repair process. This could be the reason for many RNA processing factors to play a crucial role in DNA damage response pathways. Dysregulation of one or many RNA processing factors is known to cause DNA damage and impede genome integrity3. In light of this evidences, the current work investigated the role of LLPS in mediating a DNA/RNA binding protein Fused in sarcoma (FUS) driven DNA damage response.

Key findings

  1. The investigators made FUS depleted HeLa and SH-SY5Y cells using CRISPR (and shRNA) based technologies to evaluate the role of FUS in the DNA damage response. They found that both cell lines manifested higher levels of DNA damage, as assayed by measuring immunofluorescence staining of DNA damage marker γH2AX. This damage is rescued by ectopic expression of FUS. They also report perturbed DNA damage signaling (assayed by DNA damage marker proteins like γH2AX, 53BP1, pATM, pATR) and low survival rates in FUS depleted cells pharmacologically treated with DNA damaging agents (etoposide and camptothecin).

    (1) & (2) FUS recruitment to DNA damage sites precedes SFPQ and its absence strongly reduces SFPQ accumulation. (3) FUS is required for γH2AX nano-foci clustering. Taken and modified directly from Levone B et. al., 2020 under a CC-BY 4.0 international license.
  2. Quantification of γH2AX foci or intensities of γH2AX foci is often used as a read-out to measure DNA damage in human cells. However, in earlier work4, the team demonstrated that γH2AX nano-foci could reveal the 3D organization of γH2AX marked nucleosomes at the DNA damaged chromatin. Building on this, they now show that FUS depletion impedes γH2AX nano-foci clustering that can be rescued by ectopic expression of FUS. Thus, they suggest that FUS is necessary for efficient nano-clustering of γH2AX (and other DNA repair factors) at the DNA damaged chromatin.
  3. To understand the role of FUS in the early DNA damage response, they analyzed the recruitment kinetics of DNA repair factors at laser-induced microirradiated DNA damage sites. They found that FUS was recruited to DNA damage sites that are prominently marked by γH2AX. FUS recruitment to sites of DNA damage was swift (~5s after damage induction) and preceded SFPQ (~20S after damage induction), an interacting protein of FUS. Furthermore, FUS depletion compromised SFPQ recruitment at DNA damage sites. FUS depletion also modified the recruitment dynamics of other DNA repair factors (KU80, NBS1, 53BP1, BRCA1). Thus, they suggest that FUS acts at early time points of DNA damage response.
  4. Based on earlier studies, the authors hypothesized whether the ability of FUS and SFPQ to phase separate is necessary for their recruitment to damaged DNA. The investigators demonstrate that LLPS acts as an important driver for FUS and SFPQ mediated DNA damage response by evaluating the recruitment kinetics of FUS and SFPQ to DNA damage sites in the presence of LLPS inhibiting chemicals (1,6-hexanediol and ammonium acetate). They also found that γH2AX and 53BP1 foci were reduced in the presence of LLPS inhibiting chemicals. Furthermore, LLPS-compromised FUS variants did not localize to sites of DNA damage and also hindered SFPQ and KU80 recruitment to the DNA damage sites. Thus, they report that LLPS is crucial to recruit DNA repair factors, and FUS-mediated DNA damage response depends on its LLPS nature.

Conclusion and perspective

Previous reports demonstrate the role of FUS, a DNA/RNA binding protein in DNA damage response5. The current study reports that LLPS is an important property of FUS to act at the onset of damaged DNA, and this is an early event for the repair of damaged DNA. Toxic loss of function mutations in multiple RNA processing genes including FUS causes familial amyotrophic lateral sclerosis (ALS). Moreover, cytoplasmic inclusions of FUS protein aggregates were observed in patients with sporadic ALS5,6. If and how phase separation of toxic FUS variants explain the etiology of FUS-ALS is yet to be elucidated.

Acknowledgments: Thanks to Silvia Barabino and all the authors of this work for their support.

References

  1. https://doi.org/10.1242/jcs.235093
  2. https://doi.org/10.1016/j.tibs.2020.06.007
  3. https://doi.org/10.1016/j.molcel.2009.06.021
  4. https://doi.org/10.1038/ncomms15760
  5. https://doi.org/10.1073/pnas.1611673113
  6. https://doi.org/10.1016/j.brainres.2014.10.009

Tags: dna damage response, fus, llps

Posted on: 5 February 2021

doi: https://doi.org/10.1242/prelights.25121

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Author's response

Silvia Barabino (SB) shared

1. The authors demonstrate that the recruitment of SFPQ to DNA damage sites was inhibited in FUS depleted cells. However, why do the authors think FUS depletion resulted in anomalies in the recruitment of other DNA repair factors (KU80, NBS1, 53BP1, or any other HR proteins)? Does the ability of FUS to phase separate required for this? (related to Fig 3).

SB: Others and we observed that FUS silencing affects HR and NHEJ-mediated repair of GFP reporter constructs directly implicating FUS in the repair process. In the accepted version of the manuscript, we show that FUS stabilizes KU at DNA damage sites and is necessary for the proper formation of H2AX clusters. Our data also indicated that FUS-dependent LLPS is necessary for this context. Further studies will clarify the molecular mechanism.

2. Previous reports suggest that FUS regulates transcription-associated DNA damage5. Would the authors try using transcription inhibitors in their experimental setting to test the role of transcription in mediating γH2AX nano-foci or LLPS of FUS?. his could be especially relevant considering the role of transcription or RNA in mediating LLPS.

SB: Increasing evidence implicates transcription both in DNA damage generation and in DNA repair. In their paper Hill et al5 observed increased R-loop formation upon FUS silencing. Consistent with their work, we also found an accumulation of R-loops in FUS-KO cells but we also observed that RNAseH1 overexpression does not completely eliminate DDR activation suggesting that R-loops are not the only cause of increased DDR in these cells. Indeed, we are currently addressing the role of transcription by performing similar experiments.

3a. The authors’ data about γH2AX nano-foci is interesting. Would the authors investigate the role of LLPS in orchestrating the dynamics of γH2AX nano-foci?

SB: We would certainly like to further explore the role of  LLPS in γH2AX nano-foci clustering.

3b. Also, is there a connection between reduced DDR signaling and γH2AX nano-clustering?

SB: Literature evidence indicates that γH2AX foci formation is important for ATM activation

4. How do the authors reconcile their data considering that dysregulation of FUS (and TDP-43) cause ALS?

SB: Aberrant phase separation triggers the formation of aggregates that are the pathological hallmark of many neurodegenerative disorders, including ALS. Both FUS and TDP-43 are nucleocytoplasmic shuttling proteins. The cytoplasmic aggregates that are found in the neurons of patients may not only be toxic per se (for example by activating the unfolded protein response) but could deplete the nuclear protein pools affecting the functions that FUS (and TDP-43) have in the nucleus. Indeed, accumulating evidence has described chronic DDR activation in neurodegenerative disorders (AD, and also ALS), which may contribute to neuronal death.

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