Base J is a hypermodified DNA base localized primarily to telomeric regions of 
the genome of Trypanosoma brucei. We have previously characterized two 
thymidine-hydroxylases (TH), JBP1 and JBP2, which regulate J-biosynthesis. JBP2 
is a chromatin re-modeling protein that induces de novo J-synthesis, allowing 
JBP1, a J-DNA binding protein, to stimulate additional J-synthesis. Here, we 
show that both JBP2 and JBP1 are capable of stimulating de novo J-synthesis. We 
localized the JBP1- and JBP2-stimulated J by anti-J immunoprecipitation and 
high-throughput sequencing. This genome-wide analysis revealed an enrichment of 
base J at regions flanking polymerase II polycistronic transcription units (Pol 
II PTUs) throughout the T. brucei genome. Chromosome-internal J deposition is 
primarily mediated by JBP1, whereas JBP2-stimulated J deposition at the 
telomeric regions. However, the maintenance of J at JBP1-specific regions is 
dependent on JBP2 SWI/SNF and TH activity. That similar regions of Leishmania 
major also contain base J highlights the functional importance of the modified 
base at Pol II PTUs within members of the kinetoplastid family. The regulation 
of J synthesis/localization by two THs and potential biological function of J in 
regulating kinetoplastid gene expression is discussed.