Cytosine DNA Methylation and Transcriptional Repression in African Trypanosomes


Principal Investigator: KEVIN MILITELLO
Abstract: DESCRIPTION (provided by applicant): Trypanosoma brucei is the protozoan parasite responsible for Human African Trypanosomiasis (HAT), otherwise known as African Sleeping Sickness. There is an urgent need for new control strategies for this disease, as there is no efficacious vaccine. Furthermore, drugs used to treat the late stages of the disease are toxic, and often ineffective. A thorough understanding of the parasite's biochemistry and its relationship to its complex life cycle involving insects and mammals will potentially reveal novel targets for therapeutic intervention. Our recent biochemical studies indicate that African trypanosomes contain a modified DNA base called 5-methylcytosine. In many organisms including humans, 5-methylcytosine represses gene transcription. We have identified a gene, TbDMT, which may be responsible for 5-methylcytosine synthesis in T. brucei. Therefore, our main hypothesis is that TbDMT methylates T. brucei DNA and that this influences the process of transcription in T. brucei. This hypothesis will be tested in our proposed project. First, the leves of 5- methylcytosine at specific loci in wild-type and TbDMT-/- parasites will be compared, and we expect to observe no 5-methylcytosine in the TbDMT-/- parasites. Next, the role of TbDMT in gene expression will be evaluated. Gene expression in wild-type parasites and TbDMT-/- parasites will be compared. Expression of variant surface glycoprotein (VSG) genes in these parasites will be evaluated in detail, as VSG switching is the reason there is no vaccine. Expression and the genomic stability of the SLACS retrotransposon will also be measured, as retrotransposons in numerous organisms are methylated and this blocks retrotransposition. Last, the conversion of 5-methylcytosine to 5-hydroxymethylcytosine will be evaluated, as this process has been recently discovered in human cells and is catalyzed by proteins homologous to the T. brucei J-base binding proteins. To determine if this process occurs in T. brucei, J-base binding protein knockout parasites will be evaluated for increased levels of 5-methylcytosine and decreased levels of 5-hydroxymethylcytosine using DNA immunoprecipitation experiments. Our studies will potentially improve control of HAT. We predict that 5-methylcytosine and 5-hydroxymethylcyotsine are required for the parasite's ability to regulate surface antigen expression and evade the human immune system. Therefore, genetically engineered parasites lacking 5-methylcytosine or 5-hydroxymethylcytosine may over-express VSG genes and serve as a vaccine. PUBLIC HEALTH RELEVANCE: There is an urgent need to prevent Human African Trypanosomiasis, a potentially fatal disease that impacts tens of thousands of people each year. Our studies of cytosine DNA methylation in Trypanosoma brucei may reveal the mechanism by which the parasites regulate expression of surface antigens and evade the human immune system. Engineered parasites lacking DNA methylation may over-express surface antigens and serve as a vaccine.
Funding Period: 2007-04-01 - 2015-01-31
more information: NIH RePORT