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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE126822 | GEO | 2020/09/20

REPOSITORIES: GEO

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Project description:HDAC inhibitors (HDACi) belong to a new group of chemotherapeutics that are increasingly used to treat B-cell-derived malignancies. Such malignancies regularly carry mutational signatures that conform to off-target induction of uracil by the AID/APOBEC family of cytidine deaminases, or downstream processing of uracil. . HDACi suppress thymidylate synthase increasing the cellular dUTP/dTTP ratio and leading to increased pressure on uracil repair machinery due to misincorporated uracil lesions. To investigate potential effect upon other enzymes involved in genomic uracil induction and processing, Jurkat (T-cell lymphoma) and SUDHL5 (B-cell lymphoma) cells were treated with pan-HDACi SAHA prior to SILAC based MS/MS investigation. HDACi treatment mediated significant differential expression of xx and xx proteins in Jurkat and SUDHL5, respectively, and had a substantial impact upon enzymes involved in in pyrimidine metabolism. Surprisingly, uracil N-glycosylase, UNG, was strongly downregulated by HDACi treatment. Further analysis in HEK and HeLa cells revealed that HDACis induce specific loss of the nuclear isoform UNG2 independent of transcription and cell-cycle alterations. More than 80% of UNG2 is degraded proteasomally after 24 hours treatment with SAHA, MS275, Valproate or Na-butyrate, indicating a universal ability of HDACis to mediate loss of UNG2. Targeted MS/MS analysis in HEK cells against a panel of proteins involved in DNA repair, translesion synthesis and nucleotide metabolism, revealed that UNG2 was the most pronounced differentially expressed among these after HDACi treatment. 48 hour treatment lead to a 30-40% increase in uracil lesions in the nuclear genome of HeLa and HEK cells and MS275 treatment in murine CH12F3 cell line mediated robust UNG2-loss accompanied by reduced class switch recombination. Furthermore, our analysis identified the PCNA-associated factor PAF15 among the downregulated proteins. PAF15 is overexpressed in many cancers and suppress TLS by inducing double monoubiquitinylation of PCNA and recruitment of the replicative polymerases. In summary, our findings demonstrate that HDAC inhibition affects the levels of proteins involved in DNA base excision repair, translesion synthesis and pyrimidine metabolism. These findings are important for a wide range of clinical applications of HDACi, such as in rheumatology, HIV-, and cancer treatment.

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

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