Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

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 (?-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.

ORGANISM(S): Leishmania major

SUBMITTER: Gowthaman Ramasamy

PROVIDER: E-GEOD-32976 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Glucosylated hydroxymethyluracil, DNA base J, prevents transcriptional readthrough in Leishmania.

van Luenen Henri G A M HG Farris Carol C Jan Sabrina S Genest Paul-Andre PA Tripathi Pankaj P Velds Arno A Kerkhoven Ron M RM Nieuwland Marja M Haydock Andrew A Ramasamy Gowthaman G Vainio Saara S Heidebrecht Tatjana T Perrakis Anastassis A Pagie Ludo L van Steensel Bas B Myler Peter J PJ Borst Piet P

Cell 20120801 5

Some Ts 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. 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 grow ...[more]

PMID: 22939620

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Project description:Protein-coding genes in kinetoplastid protists are transcribed from polycistronic arrays, yielding RNA precursors which are processed to form mature transcripts bearing a 5M-bM-^@M-^Y spliced leader (SL) and 3M-bM-^@M-^Y poly(A) tract. Regions of transcription initiation and termination lack known eukaryotic promoter and terminator elements, and current data suggest that transcription is instead regulated predominantly through epigenetic mechanisms. Several epigenetic marks, including histone modifications, histone variants, and an atypical DNA modification known as base J have been localized to regions of transcription initiation or termination in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Despite this conservation, the phenotypes of base J mutants vary significantly across trypanosomatids, suggesting that the specific epigenetic networks governing transcription initiation and termination have diverged significantly during evolution. In this light, we sought to characterize and compare the roles of the histone variants H2A.Z, H2B.V, and H3.V in L. major. As in T. brucei, the histone variants H2A.Z and H2B.V were shown to be essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast and again similar to T. brucei, H3.V is not essential in Leishmania as H3.V-null lines grew normally, resembled WT, and remained infectious. Using SL-primed RNA-seq, we found that H3.V-null parasites have steady-state transcript levels comparable to WT parasites and display no defects in the efficiency of transcription termination at convergent strand switch regions (SSRs). Our results show a conservation of histone variant phenotypes between L. major and T. brucei, in contrast to the phenotypes associated with the epigenetic DNA base J modification. Total RNA from Four LmjF samples were analyzed using RNA-Seq. One of them is wildtype parasites, one is single knockout for H3V gene and two independent double knockouts for H3V gene.

Dataset Information

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

Publications

Glucosylated hydroxymethyluracil, DNA base J, prevents transcriptional readthrough in Leishmania.

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