African trypanosomes (Trypanosoma brucei) are protozoan parasites that cause a fatal disease known as African sleeping sickness in humans and related diseases in animals. These diseases have devastating health and economic consequences. Pathogenesis of T. brucei is critically dependent on the phenomena of antigenic variation. During infection of the mammalian host the majority of the trypanosome population is destroyed by the immune response to the Variant Surface Glycoprotein (VSG) coat. In the process of antigenic variation, individual trypanosomes are able to switch the VSG gene that is expressed, from a repertoire of several hundred different VSG genes, and evade immune destruction. To be expressed, a VSG gene has to be located within a telomeric expression site (ES). However, there are approximately 20 ESs and only one is active at a time. The mechanism of selective expression/repression and switching of silent copies of VSG is not entirely understood. Previous analysis indicates that regulation of a given VSG ES correlates with the absence or presence of a uniquely modified DMA base, represented by the glycosylation of thymine residues and called base J. The association of J with transcriptional repression of telomeric VSG genes suggests its role in regulation of antigenic variation. In this proposal we aim to examine the molecular mechanisms underlying J activity by defining the protein/enzymatic activities associated with J and the consequences of J incorporation within the chromosomes of bloodstream form trypanosomes. Towards this end we have isolated a protein, JBP2, that regulates J-biosynthesis, determining both the developmental stage-specific and telomeric site-specific synthesis of the modified base. Preliminary analysis indicates that JBP2 is an essential gene suggesting that base J is an essential modification of the bloodstream chromosome. The broad, long-term objectives of this proposal are to use the biochemical and genetic analyses of JBP2 function as tools to advance ourunderstanding of the mechanism and biology of J-function in T. brucei. These analyses will allow us to directly address the role of telomeric DMAglucosylation on the regulation of antigenic variation. The potentially significant role of J to the survival of the parasite, and the absence of base J in their mammalian hosts, indicates attractive targets for the design of inhibitors with broad specificity and low toxicity. Enzymes and co-factors involved in J-biosynthesis would represent such targets. These studies may prove useful in identifying novel approaches to prevention, treatment and diagnosis of the debilitating and deadly diseases caused by these parasites.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Pathogenic Eukaryotes Study Section (PTHE)
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Rogers, Martin J
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University of Georgia
Schools of Arts and Sciences
United States
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Reynolds, David; Cliffe, Laura; Förstner, Konrad U et al. (2014) Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei. Nucleic Acids Res 42:9717-29
Cliffe, Laura J; Hirsch, Gwen; Wang, Jin et al. (2012) JBP1 and JBP2 proteins are Fe2+/2-oxoglutarate-dependent dioxygenases regulating hydroxylation of thymidine residues in trypanosome DNA. J Biol Chem 287:19886-95
Ekanayake, Dilrukshi; Sabatini, Robert (2011) Epigenetic regulation of polymerase II transcription initiation in Trypanosoma cruzi: modulation of nucleosome abundance, histone modification, and polymerase occupancy by O-linked thymine DNA glucosylation. Eukaryot Cell 10:1465-72
Ekanayake, Dilrukshi K; Minning, Todd; Weatherly, Brent et al. (2011) Epigenetic regulation of transcription and virulence in Trypanosoma cruzi by O-linked thymine glucosylation of DNA. Mol Cell Biol 31:1690-700
Cliffe, Laura J; Siegel, T Nicolai; Marshall, Marion et al. (2010) Two thymidine hydroxylases differentially regulate the formation of glucosylated DNA at regions flanking polymerase II polycistronic transcription units throughout the genome of Trypanosoma brucei. Nucleic Acids Res 38:3923-35
Cliffe, Laura J; Kieft, Rudo; Southern, Timothy et al. (2009) JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes. Nucleic Acids Res 37:1452-62
Borst, Piet; Sabatini, Robert (2008) Base J: discovery, biosynthesis, and possible functions. Annu Rev Microbiol 62:235-51
Kieft, Rudo; Brand, Verena; Ekanayake, Dilrukshi K et al. (2007) JBP2, a SWI2/SNF2-like protein, regulates de novo telomeric DNA glycosylation in bloodstream form Trypanosoma brucei. Mol Biochem Parasitol 156:24-31