Trypanosoma brucei, the causative pathogen of trypanosomiasis, threatens >60 million people and causes economic burdens in sub-Saharan Africa. Only a few drugs are available for treating its infection. All these drugs have severe side effects and some are difficult to administer. Therefore, identification and characterization of essential cellular processes with unique features in T. brucei will be invaluable for developing better anti-parasite agents in the future. DNA replication is essential for cell proliferation and genome integrity. Replication initiation and elongation must be tightly regulated in accordance with nucleosome disassembly and assembly. Importantly, we have discovered that simultaneous deletion of region-specific chromatin marks, histone variants H3v and H4v, and a Kinetoplastid-specific DNA modification, base J, are lethal with terminal phenotypes associated with replication defects, including accumulation of nuclear TbRPA1 foci, an indicative of abnormal exposure of ssDNA resulting from replication stress. Therefore, we hypothesize that dynamic interactions between replication and chromatin at specific loci are essential in maintaining genome and epigenome integrity. In this project, we will characterize the H3v? H4v? J? mutant in DNA replication at both chromosome internal regions (Aim 1) and telomere regions (Aim 2), focusing on changes in binding of TbORC1 with origins, origin firing choices, and fork migration. Our preliminary results suggest that H3v, H4v, and base J have roles in telomere maintenance. Thus, we will also examine whether deletion of these epigenetic marks disrupts telomere integrity (Aim 2). Transcription in T. brucei occurs polycistronically. Transcription start or termination sites (TSSs and TTSs) at boundaries of polycistronic transcription units are marked by specific nucleosome modifications and histone variants: H3K4me3, H4K10ac, H2Az, and H2Bv are at TSSs, H3v and H4v are at TTSs. Base J is located at both TSSs and TTSs. We have recently shown that simultaneous deletion of two chromatin marks, H3v and base J, disrupted transcription termination. Hence, T. brucei transcription relies greatly on chromatin structures. Furthermore, DNA replication is closely linked with transcription, because transcription and replication share their initiation sites. Depletion of TbMCM-BP, a replication protein, shares similar defects in replication and transcription termination as H3v? H4v? J? and H3v? J? mutant. Finally, in Aim 3, we will investigate whether the overall chromatin structure changes at both chromosome internal and telomere regions account for DNA replication and transcription defects in mutants lacking epigenetic marks or replication factors.
This aim will reveal mechanistic relationship between DNA replication and transcription that is mediated by the same epigenetic factors. Our studies will reveal unique features in regulation of DNA replication and transcription termination in T. brucei, which will help develop better means for eventual eradication of T. brucei in the future.

Public Health Relevance

Although Trypanosoma brucei causes fatal human African trypanosomiasis and threatens >60 million people in sub-Sahara Africa, only few drugs are available for the treatment of T. brucei infection, and most of these drugs have severe side effects and are hard to administer. Organized DNA replication is essential for cell proliferation and is tightly coordinated with other essential cellular processes such as transcription and nucleosome assembly/disassembly. As T. brucei has distinct factors and features in DNA replication, transcription, and nucleosome dynamics, we will investigate the interplay between these essential processes and decipher underlying molecular mechanisms, which will be applicable to development of new generation trypanocidal agents.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Joy, Deirdre A
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Cleveland State University
Schools of Arts and Sciences
United States
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