Abstract
Fused in sarcoma (FUS) encodes an RNA-binding protein with diverse roles in transcriptional activation and RNA splicing. While oncogenic fusions of FUS and transcription factor DNA-binding domains are associated with soft tissue sarcomas, dominant mutations in FUS can cause amyotrophic lateral sclerosis. FUS has also been implicated in genome maintenance. However, the underlying mechanisms of its actions in genome stability are unknown. Here, we applied gene editing, functional reconstitution, and integrated proteomics and transcriptomics to illuminate roles for FUS in DNA replication and repair. Consistent with a supportive role in DNA double-strand break repair, FUS-deficient cells exhibited subtle alterations in the recruitment and retention of double-strand break–associated factors, including 53BP1 and BRCA1. FUS−/− cells also exhibited reduced proliferative potential that correlated with reduced speed of replication fork progression, diminished loading of prereplication complexes, enhanced micronucleus formation, and attenuated expression and splicing of S-phase–associated genes. Finally, FUS-deficient cells exhibited genome-wide alterations in DNA replication timing that were reversed upon re-expression of FUS complementary DNA. We also showed that FUS-dependent replication domains were enriched in transcriptionally active chromatin and that FUS was required for the timely replication of transcriptionally active DNA. These findings suggest that alterations in DNA replication kinetics and programming contribute to genome instability and functional defects in FUS-deficient cells.
Highlights
ORF cause dominant forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [5,6,7]
FET proteins are capable of promoting invasion and pairing of a homologous ssDNA sequence with a dsDNA molecule in vitro [22,23,24], which suggests a possible role for FET proteins in the D-loop formation step of homology-directed repair (HDR) of DNA double-strand breaks (DSBs)
Using Fused in sarcoma (FUS)−/− cells with and without reconstitution, we found that, while FUS may play a supporting role in DSB repair, it is more prominently required for timely DNA replication, which plausibly contributes to genome instability and DNA damage response (DDR)-related phenotypes ascribed to FUS-deficient cells
Summary
To discern roles of FUS in genome protection, we disrupted FUS gene loci in U-2 OS osteosarcoma cells using CRISPR–CAS9 followed by genetic reconstitution with a retroviral vector encoding the untagged FUS ORF (see Experimental procedures section). FUS−/− U-2 OS reconstituted with a FUS construct lacking the N-terminal LCD exhibited reduced colony growth rates relative to FUS−/−:FUS cells (Fig. S4, C and D) This finding implies that biochemical activities associated with the LCD, including transcriptional activation [15, 33] and phase separation/oligomerization [19, 34], contribute to its replication-associated functions. While DNA repair was not overrepresented in Gene Ontology (GO) terms, we identified a handful of genes with annotated roles in DNA repair and replication that exhibited FUS-dependent splicing changes (Fig. S6, G and H) Both origin recognition complex 3 (ORC3) and suppressor of cancer cell invasion (SCAI) saw increased inclusion of poison cassette exons predicted to terminate their respective ORFs and/or promote mRNA degradation via nonsensemediated mRNA decay (Fig. S6, I–N). TRIP12 has been implicated in the ubiquitylation of the p53 regulator ARF and RNF168 [39, 40], and its FUSdependent alternative splicing may alter its activity toward ARF, RNF168, or other targets
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
