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

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 response^1,2.

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).

   

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 ALS^5,6. 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: 5th February 2021

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

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