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

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.