Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2022, College of Sciences and Health Professions

Trypanosoma brucei (T. brucei) causes sleeping sickness in humans and regularly switches its major surface antigen, variant surface glycoprotein (VSG), to evade the host immune response. TbRAP1, a telomere protein, is essential for VSG silencing, suppresses the level of telomeric repeat containing RNA (TERRA) and the telomeric R-loop, and suppresses DNA recombination-mediated VSG switching events. However, the mechanism of TbRAP1-mediated VSG silencing is still poorly understood. Like other RAP1 orthologs, TbRAP1 has conserved functional domains. We have identified that the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal (NLS) that is required for its interaction with importin-alpha and its nuclear localization. However, how is TbRAP1 localized to the telomere was unclear. Known RAP1 homologs either bind telomere DNA directly through their Myb and Myblike domains or are recruited to the telomere through their interaction with duplex telomere DNA binding factors. We have identified both a double-stranded and a single-stranded DNA binding activities in TbRAP1. Both these activities depend on a short, highly positively charged peptide that resides in the MybLike domain and overlaps with the TbRAP1 NLS. Surprisingly, we have also identified an RNA Recognition Motif (RRM)-mediated RNA binding activity in TbRAP1, a novel finding in RAP1 homologs. TbRAP1 RRM recognizes consensus sequences of VSG 3'UTRs in vitro and binds the active VSG RNA in vivo. Concentration-dependent competition between TbRAP1's RNA and dsDNA binding activities suggests a novel mechanism where the active VSG RNA can antagonize the local TbRAP1 dsDNA binding-mediated VSG silencing to sustain VSG monoallelic expression. Recently we also have identified TbTRF-Interacting Factor 3 (TbTIF3) in a yeast 2- (open full item for complete abstract)

Committee: Bibo Li Ph.D. (Advisor); Aaron Severson Ph.D. (Committee Member); Donny D. Licatalosi (Committee Member); Zihua Gong M.D., Ph.D. (Committee Member); Valentin Börner Ph.D. (Committee Member); Merlin Nithya Gnanapragasam Ph.D. (Committee Member) Subjects: Biology; Genetics; Molecular Biology; Parasitology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2016, College of Sciences and Health Professions

Trypanosoma brucei is a parasite that causes fatal African sleeping sickness. Antigenic variation is an obligatory mechanism for long-term survival of T. brucei inside its mammalian host. T. brucei expresses VSG as its major surface antigen and regularly switches its VSG coat to evade the host immune response. Although T. brucei genome has more than 2,500 VSG genes and pseudogenes, it only expresses one VSG from one of 15 subtelomeric VSG expression sites (ESs) at any time. VSG switching can be transcriptional (in situ switching) or be mediated by DNA homologous recombination (such as gene conversion and reciprocal DNA crossover/telomere exchange). However, regulation of VSG switching is poorly understood. We previously found that T. brucei RAP1, a telomere protein, is essential for silencing subtelomeric VSG genes. Here we found that transient depletion of TbRAP1 increases the VSG switching frequency, and most switchers in TbRAP1-depleted cells arose thorough VSG-associated gene conversion events. Also, we detected increased amount of DSBs in the active and silent ESs upon TbRAP1 depletion. However, the underlying mechanisms of how TbRAP1 suppresses DSBs at telomeres/subtelomere remained unknown. T. brucei telomeres are transcribed, generating long, non-coding RNA called TERRA (Telomeric repeat-containing RNA). Now, we found that depletion of TbRAP1 not only leads to derepression of telomeric silent VSGs, but also results in increased TERRA levels. In addition, we observed a sixteen-fold increase in telomeric RNA:DNA hybrids (R-loops) in TbRAP1-depleted cells. R-loop has been shown to induce DSBs in yeast, mouse, and human cells and are sensitive to RNaseH, a ribonuclease that cleaves the RNA strand of the RNA:DNA hybrid. Ectopic expression of TbRNaseH1 in TbRAP1 RNAi cells resulted in reduction of the telomeric R-loop levels and reduced the DSB amount back to the WT level and suppressed the elevated VSG switching frequency phenotype. Therefore, we propose that (open full item for complete abstract)

Committee: Bibo Li Ph.D. (Advisor); Valentine Boerner Ph.D. (Committee Member); Aaron Severson Ph.D. (Committee Member); Michelle Longworth Ph.D. (Committee Member); Sailen Barik Ph.D. (Committee Member); Derek Taylor Ph.D. (Committee Member) Subjects: Biology; Genetics; Molecular Biology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2014, College of Sciences and Health Professions

Subtelomeres consist of sequences adjacent to telomeres and contain genes involved in important cellular functions, as subtelomere instability is associated with several human diseases. Balancing between subtelomere stability and plasticity is particularly important for Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major surface antigen, variant surface glycoprotein (VSG), to evade the host immune response. VSGs are expressed exclusively from subtelomeres in a strictly monoallelic fashion. Telomere proteins are important for protecting chromosome ends from illegitimate DNA processes. However, whether they contribute to subtelomere integrity and stability has not been well studied. We have identified a novel T. brucei telomere protein, T. brucei TRF-Interacting Factor 2 (TbTIF2), as a functional homolog of mammalian TIN2. A transient depletion of TbTIF2 led to an elevated VSG switching frequency and an increased amount of DNA double strand breaks (DSBs) in both active and silent subtelomeric VSG expression sites (BESs). Therefore, TbTIF2 plays an important role in VSG switching regulation and is important for subtelomere integrity and stability. TbTIF2 depletion increased the association of TbRAD51 with the telomeric and subtelomeric chromatin, and TbRAD51 deletion further increased subtelomeric DSBs in TbTIF2-depleted cells, suggesting that TbRAD51-mediated DSB repair is the underlying mechanism of subsequent VSG switching. Surprisingly, significantly more TbRAD51 associated with the active BES than with the silent BESs upon TbTIF2 depletion, and TbRAD51 deletion results in much more DSBs in the active BES than in the silent BESs in TbTIF2-depleted cells, suggesting that TbRAD51 preferentially repairs DSBs in the active BES. Interestingly, depletion of TbTIF2 affects the protein's level of its interacting TTAGGG Repeat binding Factor, TbTRF. In addition, a transient depletion of TbTRF led to a s (open full item for complete abstract)

Committee: Li Bibo Ph.D (Advisor); Boerner Valentin Ph.D (Committee Member); Kondratov Roman Ph.D (Committee Member); Almasan Alexandru Ph.D (Committee Member); Severson Aaron Ph.D (Committee Member); Taylor Derek Ph.D (Committee Member) Subjects: Molecular Biology; Parasitology