Chromatin Biology of the African Trypanosome

项目总结 原生动物中的动质体类寄生虫是人类主要疾病的罪魁祸首，如致命的睡眠 疾病(布氏锥虫，又称非洲锥虫)、恰加斯病(克氏锥虫)和 利什曼病(利什曼原虫)。由于缺乏廉价和安全的药物，抗药性不断上升 目前的药物，以及有限的药物发现努力，迫切需要新的方法来对抗这些 被忽视的热带疾病。因为动叶绿体是最早的分枝生物之一 真核生物的生命之树，它们表现出许多不同于后生动物和 真菌，这可以被开发用于药物干预。通过结合结构、生化和In 活体方法，我们试图解决染色质生物学和基因调控中的基本问题 模式动质T.brucei。我们特别感兴趣的是密切相关的 DOT1A和DOT1B酶是布鲁氏毛滴虫基本功能的关键调节因子，它们催化 组蛋白H3赖氨酸76(H3K76)在球状核小体核心区的甲基化。而DOT1A则负责监管 细胞周期进展，抗原变异中的DOT1B，寄生虫逃避宿主的基本机制 免疫系统。由于锥虫体DOT1A/B与人和酵母DOT1有显著的机制差异 酶，它们如何甲基化染色质以及它们如何被调控的分子机制仍然很差。 明白了。因此，在目标1中，我们将研究DOT1A-核小体底物识别的机制 以及它对细胞周期控制的影响。在目标2中，我们将破译DOT1A的调控机制，由 RNaseH2，一种已知在RNA/DNA杂交物中裂解RNA的酶，它与这两种 DNA复制和转录调控。我们的目标是定义RNaseH2对DOT1A活动的影响， 为其调控功能提供了结构基础，并阐明了DOT1A活性的机制 与细胞周期相协调。RNaseH2与DOT1A/B的相互作用是锥体所特有的， 提示了DOT1酶的一种新的调节机制。总体而言，我们的研究将阐明- DOT1a如何被招募到染色质中，以及它是如何以时空方式调节的，这是一个悬而未决的问题。 我们的研究将产生染色质的基本单位--核小体的第一个原子结构 原生动物的一组，在医学上、生态上、进化上和科学上都是重要的真核生物。到期 对于RNaseH2的新的调控功能，我们的结果将拓宽我们对DOT1A的机制的理解 和RNaseH2酶。因为布氏锥虫DOT1调节的过程对寄生虫是必不可少的，所以这 通过利用原生动物的独特属性，研究可能最终对全球健康产生重大影响 DOT1结构和调控为昏睡病和其他由以下原因引起的疾病提供新的治疗方法 影响5亿人的动质粒。

PROJECT SUMMARY Protozoan parasites of the group kinetoplastids are responsible for major human maladies such as fatal sleeping sickness (Trypanosoma brucei, also termed the African trypanosome), Chagas disease (T. cruzi), and leishmaniasis (Leishmania species). Due to the lack of inexpensive and safe drugs, rising resistance against current drugs, and limited drug discovery efforts, novel approaches are urgently needed to combat these neglected tropical diseases. Because kinetoplastids constitute one of the earliest-branching organisms in the eukaryotic tree of life, they exhibit numerous molecular and cellular features that are distinct from metazoa and fungi, and that can be exploited for pharmacological intervention. By combining structural, biochemical, and in vivo approaches, we seek to address fundamental questions in chromatin biology and gene regulation in the model kinetoplastid T. brucei. We are particularly interested in the structure and mechanism of the closely related DOT1A and DOT1B enzymes that are key regulators of essential functions in T. brucei and that catalyze the methylation of histone H3 lysine 76 (H3K76) in the globular nucleosome core region. While DOT1A regulates cell-cycle progression, DOT1B in antigenic variation, an essential mechanism for the parasite to evade the host’s immune system. Due to significant mechanistic differences of trypanosome DOT1A/B to human and yeast DOT1 enzymes, the molecular mechanisms of how they methylate chromatin and how they are regulated remain poorly understood. In Aim 1, we will therefore investigate the mechanism of DOT1A-nucleosome substrate recognition and its impact on cell cycle control. In Aim 2, we will decipher the regulatory mechanism of DOT1A governed by RNaseH2, an enzyme that is known to cleave RNA in RNA/DNA hybrids and that has been implicated in both DNA replication and transcriptional regulation. Our goal is to define the impact of RNaseH2 on DOT1A activity, provide a structural basis for its regulatory function, and elucidate the mechanism of how DOT1A activity is coordinated with the cell cycle. The interaction of RNaseH2 with DOT1A/B is specific to trypanosomes, suggesting a novel regulatory mechanism of DOT1 enzymes. Collectively, our studies will illuminate the long- standing question of how DOT1A is recruited to chromatin and how it is regulated in a spatiotemporal manner. Our studies will yield the first atomic structures of the fundamental unit of chromatin, the nucleosome, of the vast group of protozoa, which are medically, ecologically, evolutionarily, and scientifically important eukaryotes. Due to the novel regulatory function of RNaseH2, our results will broaden our mechanistic understanding of DOT1A and RNaseH2 enzymes. Because T. brucei DOT1-regulated processes are essential for the parasite, this research may ultimately have a large impact on global health by exploiting the unique attributes of protozoan DOT1 structure and regulation to inform novel therapies for sleeping sickness and other diseases caused by kinetoplastids that affect half a billion of people.