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