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Glucosylated hydroxymethyluracil (DNA base J) prevents transcriptional read-through in Leishmania

ABSTRACT: 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.

ORGANISM(S): Leishmania tarentolae Leishmania major

PROVIDER: GSE32976 | GEO | 2011/10/15

SECONDARY ACCESSION(S): PRJNA146423

REPOSITORIES: GEO

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Glucosylated hydroxymethyluracil (DNA base J) prevents transcriptional read-through in Leishmania

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

Glucosylated hydroxymethyluracil (DNA base J) prevents transcriptional read-through in Leishmania

Project description:Glucosylated hydroxymethyluracil (DNA base J) prevents transcriptional read-through in Leishmania

| PRJNA146423 | ENA

Regulation of Transcription Termination by Glucosylated Hydroxymethyluracil, Base J, in L. major and T. brucei

Project description:Base J, β-D-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA Polymerase (RNAP) II initiation and termination. We have previously demonstrated a role of J in regulating RNAP II initiation in Trypanosoma cruzi. Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in L. major and T. brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription elongation and increased expression of downstream genes. Thus, J regulation of transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates RNAP II elongation and gene expression at specific genomic loci. We studied the effect of loss of base J on transcription using WT and WT cells grown in DMOG-containing medium. We used 2 RNA-seq libraries containing processed RNA products and 4 small RNA libraries representing the entire transcriptome.

2015-05-18 | E-GEOD-57621 | biostudies-arrayexpress

Regulation of Transcription Termination by Glucosylated Hydroxymethyluracil, Base J, in L. major and T. brucei

2015-05-18 | GSE57621 | 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.

2016-12-15 | GSE83384 | GEO

Regulation of Transcription Termination by Glucosylated Hydroxymethyluracil, Base J, in L. major and T. brucei

Project description:Regulation of Transcription Termination by Glucosylated Hydroxymethyluracil, Base J, in L. major and T. brucei

| PRJNA247217 | ENA

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

Project description:Genome-wide mapping of 5-hydroxymethyluracil in eukaryote parasite Leishmania

| PRJNA325744 | ENA

Sequencing 5-hydroxymethyluracil via chemical modification and mismatch formation

Project description:This is an oligonucleotide-based proof-of-concept study for a method to detect 5-hydroxymethyluracil (5hmU) in a single base-resolution on NGS platform. The method will be useful to identify the effect of 5hmU to genome functions.

2018-06-08 | E-MTAB-6456 | biostudies-arrayexpress

Sequencing 5-hydroxymethyluracil via chemical modification and mismatch formation

Project description:Sequencing 5-hydroxymethyluracil via chemical modification and mismatch formation

| PRJEB24754 | ENA

Genome-wide mapping of 5-hydroxymethyluracil in Trypanosoma brucei

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

| PRJNA745154 | ENA