Genome-wide mapping of 5-hydroxymethyluracil in eukaryote parasite Leishmania
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PROVIDER: PRJNA325744 | ENA |
REPOSITORIES: ENA
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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.
2016-12-15 | GSE83384 | GEO
Project description:Some T's in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (β-D-glucosyl-hydroxymethyluracil). In Leishmania about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA Polymerase II termination sites. Both this internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J is accompanied by massive read-through at RNA Polymerase II termination sites. The degree of read-through varies between transcription units, but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive read-through of transcriptional stops.
2011-10-15 | GSE32976 | GEO
Project description:Some T's in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (?-D-glucosyl-hydroxymethyluracil). In Leishmania about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA Polymerase II termination sites. Both this internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J is accompanied by massive read-through at RNA Polymerase II termination sites. The degree of read-through varies between transcription units, but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive read-through of transcriptional stops. We determined the exact location of base J in the genome of Leishmania by high-throughput sequencing of J containing DNA fragments. Samples were enriched for J-containing fragments by two independent methods: ChIP using an anti-J DNA antibody or by binding to a the J-binding protein JBP1. We studied the effect of loss of J on transcription using WT, JBP2 knockout (30-37% of WT level J), and JBP2 knockout cells grown in BrdU containing medium (13-16% of WT level of J). We used 6 RNA-seq libraries (three samples & two replica each) containing processed RNA products (transspliced and poly-adenylated) and 3 small RNA libraries representing the entire transcriptome.
2011-10-15 | E-GEOD-32976 | biostudies-arrayexpress
Project description:To investigate the effect of infection of osteoclast by Leishmania infantum, we infected diffentiating bone marrow derived osteoclast with the parasite at day 2 of differentiation We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cultures of cells infected for 24 hours compared to 4 control cultures.
2022-10-26 | GSE216141 | GEO
Project description:Leishmania major strain:LV39 | breed:Eukaryote Raw sequence reads
| PRJNA504783 | ENA
Project description:Glucosylated hydroxymethyluracil (DNA base J) prevents transcriptional read-through in Leishmania
| PRJNA146423 | ENA
Project description:Leishmania major strain:hybrid FV1xLV39 | breed:Eukaryote Raw sequence reads
| PRJNA504788 | ENA
Project description:Leishmania major strain:hybrid FV1xLV39 | breed:Eukaryote Raw sequence reads
| PRJNA504791 | ENA
Project description:Genome-wide mapping of 5-hydroxymethyluracil in Trypanosoma brucei
| PRJNA745154 | ENA
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