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Thymidylate synthase RIP-seq from DNA-repair-deficient HCT116 cells treated with RTX or 5FdUR [RIP-Seq]

ABSTRACT: Thymidylate synthase (TS) is a key enzyme in thymidylate biosynthesis and an established target of chemotherapeutics such as 5-fluoro-2’-deoxyuridine (5FdUR) and raltitrexed (RTX). Inhibition of TS disrupts the dUTP:dTTP balance, leading to uracil misincorporation, futile base excision repair cycles, DNA strand breaks, and ultimately cell death. Beyond its catalytic role, TS also binds RNA, autoregulating its own translation and interacting with transcripts such as p53 and c-myc, thereby linking TS activity to broader post-transcriptional regulatory networks. To better understand how TS inhibitory drugs influence this network, we performed TS-coupled RNA-coimmunoprecipitation (TS-RIP) and sequencing from mismatch repair-deficient, UNG-inhibited HCT116 colon cancer cells treated with 5FdUR or RTX. TS-RIP revealed RNA-binding targets of TS, in addition to the known p53 and TS mRNAs. Our findings underscore the multifaceted impact of TS inhibition, linking enzymatic disruption to RNA-level regulation and revealing drug-specific differences in cellular responses.

ORGANISM(S): Homo sapiens

PROVIDER: GSE307531 | GEO | 2026/07/08

REPOSITORIES: GEO

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Whole transcriptome sequencing of HCT116 cells with altered DNA repair capacities treated with RTX or 5FdUR

Project description:Thymidylate synthase (TS) is a key enzyme in thymidylate biosynthesis and an established target of chemotherapeutics such as 5-fluoro-2’-deoxyuridine (5FdUR) and raltitrexed (RTX). Inhibition of TS disrupts the dUTP:dTTP balance, leading to uracil misincorporation, futile base excision repair cycles, DNA strand breaks, and ultimately cell death. However, active U-DNA repair is apparently not required for the cytotoxic effect of these drugs, indicating that alternative pathways contribute to the observed S-phase arrest. To dissect these mechanisms, we investigated the transcriptomic effects of TS inhibition in HCT116 colon cancer cell lines with altered DNA repair capacities treated with 5-fluoro-2′-deoxyuridine (5FdUR) or raltitrexed (RTX). Both drugs induced similar DNA damage responses and S-phase arrest, yet displayed distinct transcriptional signatures. Our findings underscore the multifaceted impact of TS inhibition and reveal drug-specific differences in cellular responses.

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Thymidylate synthase RIP-seq from DNA-repair-deficient HCT116 cells treated with RTX or 5FdUR [RIP-Seq]

Project description:Thymidylate synthase RIP-seq from DNA-repair-deficient HCT116 cells treated with RTX or 5FdUR [RIP-Seq]

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Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

Project description:We report a new immunoprecipitation-coupled sequencing (DIP-Seq) application termed U-DNA-Seq, where a tailored and catalytically inactive uracil-DNA glycosylase (UNG) was used as uracil-DNA sensor to immunoprecipitate uracil containing genomic DNA fragments. Genomic uracil was profiled in drug-treated (5-fluoro-2'-deoxyuridine (5FdUR) or raltitrexed (RTX)) or non-treated (NT) HCT116 cells expressing the UNG inhibitor (UGI). The same experiments were also performed in the mismatch repair proficient version of the HCT116 cells (HCT116MMR), where chromosome 3 is reinserted to restore functional MMR (PMID: 8044777). Moreover, wild-type HCT116 or K562 cells were also measured. We found that regions of uracil enrichment in this assay were rather broad as compared to the sharp peaks typical in ChIP-seq. Therefore, we applied an approach alternative to the conventional peak calling. Namely, we calculated genome scaled coverage tracks and log2 ratio tracks of the enriched versus the input samples using deepTools package (bamCoverage and bigwigCompare tools, respectively) to provide a more appropriate description of uracil-enriched genomic regions. Interval (bed) files were also derived from these log2 ratio tracks to be able to screen large datasets for colocalizing features with them. For wider context of the study, see the related publication.

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