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Updated: Apr 28, 2026
Author Spotlight: Combining Proximity Ligand Assay with Gamma-H2AX Staining to Characterize Protein Interactions in DNA Damage Response
Published on: August 2, 2024
FUS is phosphorylated by DNA-PK and accumulates in the cytoplasm after DNA damage
Qiudong Deng1, Christopher J Holler1, Georgia Taylor1
1Department of Pharmacology.
Abstract:
Abnormal cytoplasmic accumulation of Fused in Sarcoma (FUS) in neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). FUS is a member of the FET protein family that includes Ewing's sarcoma (EWS) and TATA-binding protein-associated factor 2N (TAF15). FET proteins are predominantly localized to the nucleus, where they bind RNA and DNA to modulate transcription, mRNA splicing, and DNA repair. In ALS cases with FUS inclusions (ALS-FUS), mutations in the FUS gene cause disease, whereas FTLD cases with FUS inclusions (FTLD-FUS) do not harbor FUS mutations. Notably, in FTLD-FUS, all FET proteins accumulate with their nuclear import receptor Transportin 1 (TRN1), in contrast ALS-FUS inclusions are exclusively positive for FUS. In the present study, we show that induction of DNA damage replicates several pathologic hallmarks of FTLD-FUS in immortalized human cells and primary human neurons and astrocytes. Treatment with the antibiotic calicheamicin γ1, which causes DNA double-strand breaks, leads to the cytoplasmic accumulation of FUS, TAF15, EWS, and TRN1. Moreover, cytoplasmic translocation of FUS is mediated by phosphorylation of its N terminus by the DNA-dependent protein kinase. Finally, we observed elevated levels of phospho-H2AX in FTLD-FUS brains, indicating that DNA damage occurs in patients. Together, our data reveal a novel regulatory mechanism for FUS localization in cells and suggest that DNA damage may contribute to the accumulation of FET proteins observed in human FTLD-FUS cases, but not in ALS-FUS.
Insights
DNA damage triggers cytoplasmic accumulation of Fused in Sarcoma (FUS) and other FET proteins, mimicking FTLD-FUS pathology. This suggests DNA damage contributes to FET protein buildup in FTLD-FUS, but not ALS-FUS.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Abnormal cytoplasmic accumulation of Fused in Sarcoma (FUS) protein is a hallmark of certain neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).
- FET proteins, including FUS, Ewing's sarcoma (EWS), and TATA-binding protein-associated factor 2N (TAF15), are typically nuclear but can mislocalize in disease.
- FTLD with FUS inclusions (FTLD-FUS) differs from ALS with FUS inclusions (ALS-FUS) in that FTLD-FUS involves the accumulation of all FET proteins with Transportin 1 (TRN1), while ALS-FUS is FUS-specific.
Purpose of the Study:
- To investigate the role of DNA damage in the pathogenesis of FTLD-FUS.
- To determine if DNA damage can induce the cytoplasmic accumulation of FET proteins observed in FTLD-FUS.
- To elucidate the molecular mechanisms underlying FUS mislocalization in response to cellular stress.
Main Methods:
- Induction of DNA double-strand breaks using the antibiotic calicheamicin γ1 in immortalized human cells and primary neurons/astrocytes.
- Analysis of the subcellular localization of FUS, EWS, TAF15, and TRN1 following DNA damage.
- Investigation of FUS phosphorylation by DNA-dependent protein kinase (DNA-PK) in response to DNA damage.
- Assessment of phospho-H2AX levels in post-mortem FTLD-FUS brain tissue.
Main Results:
- Treatment with calicheamicin γ1 induced cytoplasmic accumulation of FUS, TAF15, EWS, and TRN1 in human cells and neurons.
- Phosphorylation of FUS's N-terminus by DNA-PK was identified as the mechanism mediating its cytoplasmic translocation.
- Elevated levels of phospho-H2AX, a marker of DNA damage, were detected in FTLD-FUS brains.
Conclusions:
- DNA damage can replicate key pathological features of FTLD-FUS by causing cytoplasmic mislocalization of FET proteins.
- DNA-dependent protein kinase-mediated phosphorylation of FUS is a critical step in its stress-induced cytoplasmic translocation.
- These findings suggest that DNA damage may be a significant contributing factor to FET protein aggregation in FTLD-FUS, distinguishing it from ALS-FUS pathogenesis.
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