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An enzyme-mediated bioorthogonal labeling method for genome-wide mapping of 5-hydroxymethyluracil† † Electronic supplementary information (ESI) available: Tables S1–S3; Fig. S1–S13. See DOI: 10.1039/d1sc03812e

ABSTRACT: DNA 5-hydroxymethyluracil (5hmU) is a thymine modification existing in the genomes of various organisms. The post-replicative formation of 5hmU occurs via hydroxylation of thymine by ten-eleven translocation (TET) dioxygenases in mammals and J-binding proteins (JBPs) in protozoans, respectively. In addition, 5hmU can also be generated through oxidation of thymine by reactive oxygen species or deamination of 5hmC by cytidine deaminase. While the biological roles of 5hmU have not yet been fully explored, determining its genomic location will highly assist in elucidating its functions. Herein, we report a novel enzyme-mediated bioorthogonal labeling method for selective enrichment of 5hmU in genomes. 5hmU DNA kinase (5hmUDK) was utilized to selectively install an azide (N3) group or alkynyl group into the hydroxyl moiety of 5hmU followed by incorporation of the biotin linker through click chemistry, which enabled the capture of 5hmU-containing DNA fragments via streptavidin pull-down. The enriched fragments were applied to deep sequencing to determine the genomic distribution of 5hmU. With this established enzyme-mediated bioorthogonal labeling strategy, we achieved the genome-wide mapping of 5hmU in Trypanosoma brucei. The method described here will allow for a better understanding of the functional roles and dynamics of 5hmU in genomes. We developed an enzyme-mediated bioorthogonal labeling strategy for the enrichment and genome-wide mapping of 5hmU. With this strategy, we provided the first map of 5hmU in the whole Trypanosoma brucei genome.

SUBMITTER: Ma C

PROVIDER: S-EPMC8565368 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Genome-wide mapping of 5-hydroxymethyluracil in Trypanosoma brucei

Project description:5-Hydroxymethyluracil (5hmU) is a thymine modification existing in the genomes of a number of living organisms. The post-replicative formation of 5hmU occurs via hydroxylation of thymine, which can be mediated by the ten-eleven translocation (TET) dioxygenases in mammalian and J-binding proteins (JBPs) in protozoan genomes, respectively. In addition, 5hmU also can be generated through oxidation of thymine by reactive oxygen species or from deamination of 5hmC by activation-induced cytidine deaminase (AID) or APOBEC family enzymes. While the biological roles of 5hmU have not been fully explored, identifying its genomic location will assist in elucidating its functions. Herein, we report a method of enzyme-mediated bioorthogonal labeling to selectively enrich genomic regions containing 5hmU. 5hmU DNA kinase (5hmUDK) was utilized to selectively install an azide group or alkynyl group into the hydroxyl group of 5hmU followed by incorporation of the biotin linker through click chemistry and capture of 5hmU-containing DNA fragments via streptavidin pull-down. The enriched fragments were applied to deep sequencing to map the location of 5hmU. With this established enzyme-mediated bioorthogonal labeling strategy, we achieved the genome-wide mapping of 5hmU in Trypanosoma brucei (T. brucei) genomes. The method described here will allow for a better understanding of the functional roles and dynamics of 5hmU in genomes

2021-07-11 | GSE179809 | GEO

Genome-wide mapping of 5-hydroxymethyluracil in eukaryote parasite Leishmania

Project description:5-hydroxymethyluracil (5hmU) is a thymine base modification found in genomes of a diverse range of organisms. To explore the functional importance of 5hmU, we developed a method for the genome-wide mapping of 5hmU-modified loci based on a chemical tagging strategy for the hydroxymethyl group. We applied the method to generate genome-wide maps of 5hmU in parasitic protozoan Leishmania, where 5hmU forms enzymatically via hydroxylation of thymine. In the genus, another thymine modification 5-(β-glucopyranosyl) hydroxymethyluracil (base J) plays key roles during transcription. To elucidate relationships between 5hmU and base J, we also mapped base J loci by introducing a chemical tagging strategy for the glucopyranoside residue. Results: Observed 5hmU peaks were highly consistent among technical replicates, confirming the robustness of the method. 5hmU were particularly enriched in strand switch regions, telomeric regions and intergenic regions. Over 90% of 5hmU-enriched loci overlapped with base J-enriched loci, which occurred mostly within strand switch regions. We also identified loci comprising 5hmU but not base J. These 5hmU-specific loci were enriched with motifs consisting of a stretch of thymine bases and associated with higher RNA levels. Conclusions: By chemically detecting 5hmU we provide the first genome-wide map of 5hmU, which will help addressing the emerging interest in the role of 5hmU. The presence of 5hmU-specific loci may suggest that 5hmU has unique roles.

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Genome-wide mapping of 5-hydroxymethyluracil in Trypanosoma brucei

Project description:Genome-wide mapping of 5-hydroxymethyluracil in Trypanosoma brucei

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