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Genomic uracil in early embryonic development of Zebrafish

ABSTRACT: Uracil incorporation into genomic DNA (U-DNA) can arise under conditions of nucleotide imbalance and may have regulatory consequences. Here, we report an unexpectedly high uracil content in early Danio rerio embryos, which decreases markedly by the time of zygotic genome activation (ZGA). Genome-wide profiling of U-DNA at 2.5 hpf revealed enrichment at specific satellite repeats, including MOSAT, BRSAT1, and SAT-12. These observations suggest that U-DNA and its processing could play roles either in supporting rapid cleavage divisions or in facilitating chromatin remodeling required for ZGA. To test this, we microinjected fertilized eggs with either active or catalytically inactive uracil-DNA glycosylase (UNG), thereby promoting or inhibiting U-DNA removal. Both interventions resulted in embryonic lethality, consistent with disruption of the normal uracil-processing balance and consequent perturbation of ZGA timing. Our findings indicate a potential regulatory function of U-DNA in early vertebrate development.

ORGANISM(S): Danio rerio

PROVIDER: GSE307307 | GEO | 2026/06/10

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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Genomic uracil in early embryonic development of Zebrafish

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