Glucosylated Hydroxymethyluracil, DNA Base J, Prevents Transcriptional Readthrough in Leishmania

Abstract

SUMMARY 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 growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J in JBP2 knockout cells is accompanied by massive readthrough at RNA polymerase II termination sites. The readthrough 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 readthrough of transcriptional stops.

Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea

“…TET2 in mammals is reported to associate with the SET1/COMPASS complex, which travels with RNA polymerase II (44), and would therefore mark gene bodies of highly expressed genes (25); in contrast, 5fC/ 5caC have been reported, by using in vitro assays, to diminish the processivity of RNA polymerase II. A similar function has been attributed to base J (β-D-glucosyl-hydroxymethyluracil): a fraction of base J in Leishmania is found at transcription termination sites where two polycistronic transcription units that are transcribed in opposite directions converge, and the loss of base J is accompanied by a dramatic increase in read-through transcription at these sites (45).…”

Cross-species genetic exchange between visceral and cutaneous strains of Leishmania in the sand fly vector

“…Thus, even these few 2n progeny appear to have differentially inherited the genes controlling the respective tissue tropisms of their parents, suggesting that one or both of the parents are heterozygous for these genes(s). Alternatively, or in concert with specific inheritance patterns, epigenetic mechanisms involved in regulating transcription initiation or termination of the relevant genes (31,32), or changes in gene dosage due to aneuploidy, might contribute to the phenotypic differences observed. A possible effect of increasing gene dosage due to polyploidy was clearly evident, because the triploid hybrids displayed distinct skin or viscera tropisms depending on the parental origin of the extra chromosomes, although differences in allelic inheritance might still be influencing the behavior of these clones.…”

DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

“…These amplification and deletion of DNA loci appear to occur via homologous recombination between homologous regions of the genomic DNA. It has been proved that many repeated DNA sequences are widespread throughout the Leishmania genome and can therefore recombine with each other in order to amplify or delete some DNA regions (43). Considering the importance of HR events in resistant Leishmania parasites, a discussion on the involvement of DNA repair protein implicated in HR is warranted.…”

“…Approximately 1% of thymine is replaced by base J, which is present mostly in repetitive DNA, such as telomeric repeats. Base J regulates gene expression, as its loss leads to a readthrough of normal RNA polymerase II transcription termination sites in Leishmania (43). Using a specific ethidium bromide fluorescence assay, recombinant T. cruzi uracil DNA glycosylase (TcUNG) was shown to specifically excise uracil from DNA.…”