Abstract
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. 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 Leishmania major and Trypanosoma brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination at the end of polycistronic gene clusters 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 termination and increased expression of downstream genes. Thus, J regulation of RNAP II transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates termination and gene expression at specific sites within polycistronic gene clusters.
Highlights
Members of the Kinetoplastida order include the human parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, which cause African sleeping sickness, Chagas’ disease and leishmaniasis, respectively
We found that DMOG reduced chromosome internal J, though J loss was more pronounced at some convergent strand switch regions (cSSRs) than others (Figure 1B)
We have clearly demonstrated an important function for base J in the regulation of RNA polymerase (RNAP) II termination in L. major, as was observed in L. tarentolae
Summary
Members of the Kinetoplastida order include the human parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, which cause African sleeping sickness, Chagas’ disease and leishmaniasis, respectively. Base J reduction in T. cruzi, following the deletion of either JBP1 or JBP2, resulted in the formation of more active chromatin at transcription initiation sites, increased RNAP II recruitment and PTU transcription rate, and global changes in gene expression [26,36]. It was argued that J loss resulted in readthrough transcription at cSSR termination sites, which led to the production of RNAs antisense to the genes on the opposite strand. We show that reduction of base J using DMOG in L. major resulted in genome-wide transcriptional readthrough at cSSRs and HT sites, without cell death. We localized base J at sites prior to the end of a PTU where the loss of J led to upregulated expression of the downstream genes within the same PTU. While termination occurs at the end of each PTU in T. brucei in a J-independent manner, J-dependent termination within a PTU allows developmentally regulated expression of downstream genes
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