Cytosine DNA Methylation and Transcriptional Repression in African Trypanosomes

描述(申请人提供)：布鲁氏锥虫是引起人类非洲锥虫病(HAT)的原生动物寄生虫，也称为非洲睡眠病。由于没有有效的疫苗，迫切需要对这种疾病采取新的控制策略。此外，用于治疗疾病晚期的药物是有毒的，而且往往无效。彻底了解这种寄生虫的生物化学及其与其涉及昆虫和哺乳动物的复杂生命周期的关系，可能会揭示治疗干预的新靶点。我们最近的生化研究表明，非洲锥虫含有一种名为5-甲基胞嘧啶的修饰DNA碱基。在包括人类在内的许多生物中，5-甲基胞嘧啶抑制基因转录。我们已经鉴定出一个基因TbDMT，该基因可能与布鲁氏毛滴虫5-甲基胞嘧啶的合成有关。因此，我们的主要假设是TbDMT使布氏毛滴虫DNA甲基化，从而影响布氏毛滴虫的转录过程。这一假设将在我们提议的项目中得到验证。首先，我们将比较野生型和TbDMT-/-寄生虫特定位点上5-甲基胞嘧啶的水平，我们预计在TbDMT-/-寄生虫中不会观察到5-甲基胞嘧啶。接下来，我们将评估TbDMT在基因表达中的作用。将比较野生型寄生虫和TbDMT-/-寄生虫的基因表达。这些寄生虫的变异表面糖蛋白(VSG)基因的表达将被详细评估，因为VSG的转换是没有疫苗的原因。还将测量SLACS反转录转座子的表达和基因组稳定性，因为许多生物中的反转录转座子是甲基化的，这阻止了反转录转座子。最后，将评估5-甲基胞嘧啶转化为5-羟甲基胞嘧啶的过程，因为这一过程最近在人类细胞中被发现，并由与布氏锥虫J碱基结合蛋白同源的蛋白质催化。为了确定这一过程是否发生在布氏毛滴虫中，将使用DNA免疫沉淀实验评估J碱基结合蛋白敲除寄生虫5-甲基胞嘧啶水平的增加和5-羟甲基胞嘧啶水平的降低。我们的研究可能会改善对HAT的控制。我们预测，5-甲基胞嘧啶和5-羟甲基胞苷是寄生虫调节表面抗原表达和逃避人类免疫系统所必需的。因此，缺乏5-甲基胞嘧啶或5-羟甲基胞嘧啶的基因工程寄生虫可能会过度表达VSG基因，并用作疫苗。 公共卫生相关性：迫切需要预防非洲人锥虫病，这是一种每年影响数万人的潜在致命疾病。我们对布鲁氏锥虫胞嘧啶DNA甲基化的研究可能揭示这种寄生虫调节表面抗原表达和逃避人类免疫系统的机制。缺乏DNA甲基化的工程寄生虫可能会过度表达表面抗原，并用作疫苗。

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.