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Fused in sarcoma regulates DNA replication timing and progression

ABSTRACT: Fused in sarcoma (FUS) encodes a low complexity RNA-binding protein with diverse roles in transcriptional activation and RNA processing. While oncogenic fusions of FUS and transcription factor DNA-binding domains are associated with soft tissue sarcomas, dominant mutations in FUS cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FUS has also been implicated in DNA double-strand break repair (DSBR) and genome maintenance. However, the underlying mechanisms are unknown. Here we employed quantitative proteomics, transcriptomics, and DNA copy number analysis (Sort-Seq), in conjunction with FUS-/- cells to ascertain roles of FUS in genome protection. FUS-/- cells exhibited alterations in the recruitment and retention of DSBR factors BRCA1 and 53BP1 but were not overtly sensitive to genotoxins. By contrast, FUS-deficient cells had reduced proliferative potential that correlated with reduced replication fork speed, diminished loading of pre-replication complexes, and attenuated expression of S-phase associated genes. FUS interacted with lagging strand DNA synthesis factors and other replisome components, but did not translocate with active replication forks. Using a Sort-Seq workflow, we show that FUS contributes to genome-wide control of DNA replication timing and is essential for the early replication of transcriptionally active DNA. These findings illuminate new roles for FUS in DNA replication initiation and timing that may contribute to genome instability and functional defects in cells harboring disease-associated FUS fusions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE147784 | GEO | 2020/05/13

REPOSITORIES: GEO

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Fused in sarcoma regulates DNA replication timing and progression

Project description: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 cause amyotrophic lateral sclerosis (ALS). FUS has also been implicated in genome maintenance. However, the underlying mechanisms are unknown. Here, we applied gene editing, functional reconstitution and integrated proteomic and transcriptomics to illuminate roles for FUS in DNA replication and repair. Consistent with a supportive role in DNA double-strand break (DSB) repair FUS deficient cells exhibited subtle alterations in the recruitment and retention of DSB-associated factors, including 53BP1 and BRCA1. FUS-/- cells also exhibited reduced proliferative potential that correlated with reduced replication fork speed, diminished loading of pre-replication 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 reeexpression of FUS cDNA. FUS-dependent replication domains were enriched in transcriptionally active chromatin and FUS was required for the timely replication of transcriptionally active DNA. These findings suggest that alterations DNA replication kinetics and programming contribute to genome instability and functional defects in FUS deficient cells.

2021-06-24 | MSV000087698 | MassIVE

A global profile of replication polymerase usage

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A global profile of replication polymerase usage

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Project description:Counting DNA reads using whole genome sequencing is providing new insight into DNA double-strand break repair (DSBR) in the model organism Escherichia coli. We describe the application of RecA chromatin immunoprecipitation coupled to genomic DNA sequencing (RecA-ChIP-seq) and marker frequency analysis (MFA) to analyse the genomic consequences of DSBR.

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Multistep mechanism of G-quadruplex resolution during DNA replication

Project description:G-quadruplex (or G4) structures are non-canonical DNA structures that form in guanine-rich sequences and threaten genome stability when not properly resolved. G4 unwinding occurs during S phase via an unknown mechanism. Using Xenopus egg extracts, we define a three-step G4 unwinding mechanism that acts during DNA replication. First, the replicative helicase (CMG) stalls at a leading strand G4 structure. Second, the DHX36 helicase mediates the bypass of the CMG past the intact G4 structure, which allows approach of the leading strand to the G4. Third, G4 structure unwinding by the FANCJ helicase enables the DNA polymerase to synthesize past the G4 motif. A G4 on the lagging strand template does not stall CMG, but still requires active DNA replication for unwinding. DHX36 and FANCJ have partially redundant roles, conferring robustness to this pathway. Our data reveal a novel genome maintenance pathway that promotes faithful G4 replication thereby avoiding genome instability.

2022-02-17 | PXD027623 | Pride

The Effects of Rif1 Loss on Gene Expression During Early Zebrafish Development [SLAM-seq]

Project description:Deregulated DNA replication is a major contributor to human developmental disorders and cancer, yet our understanding of how replication is coordinated with changes in transcription and chromatin structure is limited. Our lab has employed the zebrafish model to investigate the mechanisms driving changes in the replication timing program during development. Previous studies have identified changes in replication timing patterns from the onset of zygotic transcription through gastrulation in zebrafish embryos. The protein Rif1 is crucial for replication timing in a wide range of eukaryotes, yet its role in establishing the replication timing program and chromatin structure during early vertebrate development is not well understood. Using Rif1 mutant zebrafish and performing RNA sequencing and whole-genome replication timing analysis, we found that Rif1 mutants were viable but had a defect in female sex determination. Interestingly, Rif1 loss primarily affected DNA replication timing after gastrulation, while its impact on transcription was more pronounced during zygotic genome activation. Our results indicate that Rif1 has distinct roles in regulating DNA replication and transcription at different stages of development.

2023-08-15 | GSE226222 | GEO

The Effects of Rif1 Loss on Gene Expression During Early Zebrafish Development

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Decoding the Replication Timing Program in Zebrafish: The Role of Rif1

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The role of mammalian DNA methyltransferases in the regulation of temporal order of DNA replication [BrdU-IP]

Project description:Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the context of development and disease. DNA methylation by DNA methyltransferases (DNMTs) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell biological and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various DNMT-mutant mouse ES cell lines that include a line with a drug-inducible DNMT3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) correlates with redistribution of H3K27me3 induced upon DNA hypomethylation: later replicating PH coincides with H3K27me3 enriched regions. In contrast, this relationship with H3K27me3 was not evident at chromosomal arm regions that undergo either early-to-late (EtoL) or late-to-early (LtoE) replication timing switching upon loss of DNMTs. Interestingly, transcriptional up- and down-regulation frequently coincide with earlier and later shifts in replication timing of chromosomal arm regions, respectively. Our study revealed previously unrecognized complex and diverse roles of DNMTs in shaping the mammalian DNA replication landscape.

2021-02-12 | GSE119405 | GEO

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