Cytosine DNA Methylation and Transcriptional Repression in African Trypanosomes

非洲锥虫中的胞嘧啶 DNA 甲基化和转录抑制

• 批准号: 8289380
• 负责人: KEVIN MILITELLO
• 金额: $ 38.78万
• 依托单位: COLLEGE AT GENESEO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8289380
• 关键词: African; African Trypanosomiasis; Animals; Award; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Cells; Complex; Custom; Cytosine; DNA; DNA Methylation; DNA Methyltransferase; DNA Microarray Chip; DNA Modification Methylases; Differentiation and Growth; Disease; Drug usage; Engineering; Enzymes; Gene Expression; Gene Targeting; Generations; Genes; Genetic Transcription; Genetic Variation; Genome; Genome Stability; Genomics; Homologous Gene; Homologous Protein; Human; Human Cell Line; Immune system; Immunoblotting; Immunoprecipitation; Insecta; Knock-out; Life Cycle Stages; Liquid Chromatography; Mammals; Mass Spectrum Analysis; Measures; Membrane Glycoproteins; Methylation; Modeling; Northern Blotting; Nuclear; Organism; Parasites; Pathway interactions; Positioning Attribute; Process; Property; Proteins; Pseudogenes; RNA; Retrotransposition; Retrotransposon; Role; Staging; Surface Antigens; Testing; Therapeutic Intervention; Tissues; Transcription Process; Trypanosoma; Trypanosoma brucei brucei; United States National Institutes of Health; Vaccines; Variant; base; design; gene repression; homologous recombination; improved; nagana; novel; prevent; research study; tandem mass spectrometry

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.

描述（由申请方提供）：布氏锥虫是引起人类非洲锥虫病（HAT）的原生动物寄生虫，也称为非洲昏睡病。由于没有有效的疫苗，因此迫切需要针对这种疾病的新的控制策略。此外，用于治疗疾病晚期的药物是有毒的，并且通常无效。深入了解寄生虫的生物化学及其与昆虫和哺乳动物复杂生命周期的关系，将有可能揭示治疗干预的新靶点。我们最近的生化研究表明，非洲锥虫含有一种被称为5-甲基胞嘧啶的修饰DNA碱基。在包括人类在内的许多生物体中，5-甲基胞嘧啶抑制基因转录。我们已经确定了一个基因，TbDMT，这可能是负责5-甲基胞嘧啶合成T。布鲁塞。因此，我们的主要假设是TbDMT甲基化T。布氏杆菌DNA，这影响了T.布鲁塞。这一假设将在我们提出的项目中得到检验。首先，将比较野生型和TbDMT-/-寄生虫中特定基因座处5-甲基胞嘧啶的水平，并且我们预期在TbDMT-/-寄生虫中没有观察到5-甲基胞嘧啶。接下来，将评估TbDMT在基因表达中的作用。将比较野生型寄生虫和TbDMT-/-寄生虫中的基因表达。将详细评价这些寄生虫中变异表面糖蛋白（VSG）基因的表达，因为VSG转换是没有疫苗的原因。还将测量SLACS逆转录转座子的表达和基因组稳定性，因为许多生物体中的逆转录转座子被甲基化，这阻断了逆转录转座。最后，将评价5-甲基胞嘧啶向5-羟甲基胞嘧啶的转化，因为该过程最近已在人类细胞中发现，并且由与T.布鲁氏菌J-碱基结合蛋白。为了确定这一过程是否发生在T.对于布氏杆菌，J-碱基结合蛋白敲除寄生虫将使用DNA免疫沉淀实验评价5-甲基胞嘧啶水平的增加和5-羟甲基胞嘧啶水平的降低。我们的研究将有可能改善HAT的控制。我们预测，5-甲基胞嘧啶和5-羟甲基胞嘧啶所需的寄生虫的能力，调节表面抗原的表达和逃避人类免疫系统。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过表达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。我们对布氏锥虫胞嘧啶DNA甲基化的研究可能揭示该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作为疫苗。