RNA-seq profiling of U-2 OS osteosarcoma cells expressing scrambled control or Timeless shRNA
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE339246 | GEO | 2026/08/19
REPOSITORIES: GEO
Similar Datasets
Project description:Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1/Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Î mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of the FPC in regulation of telomere stability in cancer cells. Genome-wide distributions of Rad52 in wild type and in swi1 deletion in fission yeast The'SP1173_WT ChIP-seq' is an input sample (non-tagged data).
2016-02-25 | E-GEOD-74487 | biostudies-arrayexpress
Project description:Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1/Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Δ mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of the FPC in regulation of telomere stability in cancer cells.
2016-02-25 | GSE74487 | GEO
Project description:The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.
2020-10-08 | GSE144321 | GEO
Project description:RNA-seq profiling of U-2 OS osteosarcoma cells expressing scrambled control or Timeless shRNA
| PRJNA1498013 | ENA
Project description:Regions of DNA with the potential to form secondary structure pose a frequent and significant impediment to DNA replication that must be actively countered by cells in order to preserve genetic and epigenetic integrity. The fork protection complex (FPC), a conserved group of replisome-associated proteins, comprising Timeless, plays an important role in maintaining efficient replisome function. A previously unappreciated DNA binding domain in the C terminus of Timeless, which exhibits specificity towards G4 structures, acts in collaboration with the DDX11 helicase to ensure replication of G4 structures in vivo and maintenance of genetic and epigenetic stability. Using RNA-seq experiments, we show that (i) Timeless and DDX11 share common gene targets, (ii) the promoter of Timeless- and DDX11-dependent genes are enriched in G4 structures in the vicinity of their promoter and (iii) both Timeless and DDX11 share a common set of gene targets with the FANCJ helicase known to maintain epigenetic stability in the vicinity of G4 structures.
2020-07-01 | GSE139256 | GEO
Project description:Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel relatively large distances from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA.
2024-07-29 | GSE269383 | GEO
Project description:Replication forks temporarily or terminally pause at hundreds of hard-to-replicate regions around the genome. A conserved pair of budding yeast replisome components Tof1-Csm3 (fission yeast Swi1-Swi3 and human TIMELESS-TIPIN) acts as a ‘molecular brake’ and promotes fork slowdown at proteinaceous replication fork barriers (RFBs), while the accessory helicase Rrm3 assists the replisome in removing protein obstacles. Here we show that Tof1-Csm3 complex promotes fork pausing independently of Rrm3 helicase by recruiting topoisomerase I (Top1) to the replisome. Topoisomerase II (Top2) partially compensates for the pausing decrease in cells when Top1 is lost from the replisome. The C-terminus of Tof1 is specifically required for Top1 recruitment to the replisome and fork pausing but not for DNA replication checkpoint (DRC) activation. We propose that forks pause at proteinaceous RFBs through a ‘sTOP’ mechanism (‘slowing down with TOPoisomerases I-II’), which we show also contributes to protecting cells from topoisomerase-blocking agents.
2019-11-07 | GSE140034 | GEO
Project description:RNA-sequencing (RNA-seq) analysis was performed to identify TIMELESS-regulated gene signatures in mouse glioblastoma (GBM) CT2A cells. Timeless was originally identified as a core component of the circadian clock machinery, but its role in GBM stemness and malignancy has not been defined. To investigate the mechanisms underlying Timeless-driven GBM malignancy, we generated CT2A cells with stable Timeless knockdown (KD) using a lentivirus expressing Timeless- specific shRNA, alongside CT2A cells transduced with a non-targeting scramble shRNA (shScramble) as a control. Total RNA was extracted from three independent biological replicates of shScramble and Timeless-KD CT2A cells, and bulk RNA barcoding and sequencing (BRB-seq) libraries were prepared and sequenced on the Illumina NextSeq2000 platform. Among the genes detected, genes whose mean expression levels in Timeless-KD cells were reduced to half or less of those in shScramble cells were selected and subjected to enrichment analysis using ShinyGO, which revealed significant enrichment of stem cell- related gene signatures. These findings suggest that TIMELESS regulates cancer stem-like properties of GBM cells.
2026-09-01 | GSE336084 | GEO
Project description:A subset of cancer cells are intrinsically sensitive to inhibitors targeting PARG, the poly(ADP-ribose) glycohydrolase that degrades PAR chains. Sensitivity is accompanied by persistent DNA replication stress, and can be induced by inhibition of TIMELESS, a replisome accelerator. However, the nature of the oncogenic vulnerability responsible for intrinsic sensitivity remains undetermined. To understand PARG activity dependency, we analyzed Timeless model systems and intrinsically sensitive ovarian cancer cells. We show that nucleoside supplementation rescues all phenotypes associated with PARG inhibitor sensitivity, including replisome speed and fork-restart ability, S-phase completion and mitotic entry, proliferation dynamics and clonogenic potential, in both a TIMELESShaploinsufficient model and intrinsically sensitive cells. Importantly nucleoside supplementation restores PARG inhibitor resistance without reverting PAR chain accumulation, indicating that sensitivity correlates with PAR chain intolerance. We show that inhibition of thymidylate synthase, an enzyme required for dNTP homeostasis, induces PARG-dependency, and, using label-free, quantitative proteomics, we show that PKMYT1, a negative regulator of CDK1, is overexpressed in PARG inhibitor-sensitive cells. Together, these observations suggest that PARG inhibitor sensitivity reflects an inability to control replisome speed and/or maintain helicase-polymerase coupling in response to nucleotide imbalances, in turn applying selective pressure on mitotic entry controls to maintain genome integrity.
2024-06-28 | PXD049682 | Pride