Chromatin Biology of the African Trypanosome

项目摘要 动质体中的原生寄生虫是造成人类主要疾病的原因， 病（布氏锥虫，也称为非洲锥虫）、恰加斯病（锥虫病）。cruzi），以及 利什曼病（利什曼原虫属）。由于缺乏廉价和安全的药物， 目前的药物和有限的药物发现努力，迫切需要新的方法来对抗这些问题。 被忽视的热带病因为动质体是地球上最早分支的生物之一， 真核生物的生命树，他们表现出许多分子和细胞特征，是不同于后生动物， 真菌，并且可以用于药理学干预。通过结合结构，生物化学， 体内方法，我们寻求解决染色质生物学和基因调控的基本问题， 模式动质体T.布鲁塞。我们特别感兴趣的结构和机制的密切相关的 DOT 1A和DOT 1B酶是T.布鲁氏杆菌并催化 在球状核小体核心区域中的组蛋白H3赖氨酸76（H3 K76）的甲基化。虽然DOT 1A调节 细胞周期进程，抗原变异中的DOT 1B，寄生虫逃避宿主免疫的重要机制， 免疫系统由于锥虫DOT 1A/B与人和酵母DOT 1的显著机制差异， 尽管这些酶的基因表达水平很低，但它们如何使染色质甲基化以及如何调节染色质的分子机制仍然很差 明白因此，在目标1中，我们将研究DOT 1A-核小体底物识别的机制 及其对细胞周期控制的影响。在目标2中，我们将破译DOT 1A的调控机制， RNaseH 2，一种已知在RNA/DNA杂交体中切割RNA的酶， DNA复制和转录调控。我们的目标是确定RNaseH 2对DOT 1A活性的影响， 为它的调节功能提供了结构基础，并阐明了DOT 1A活性是如何被调节的机制。 与细胞周期相协调。RNaseH 2与DOT 1A/B的相互作用对锥虫是特异性的， 提示DOT 1酶的一种新的调节机制。总的来说，我们的研究将阐明长期以来- DOT 1A如何被招募到染色质以及它如何以时空方式被调节的问题。 我们的研究将产生染色质基本单位核小体的第一个原子结构， 原生动物的一个类群，在医学、生态学、进化论和科学上都具有重要意义。由于 对于RNaseH 2的新的调节功能，我们的研究结果将拓宽我们对DOT 1A机制的理解 和RNaseH 2酶。因为T.布氏杆菌DOT 1调节的过程对寄生虫至关重要， 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。 DOT 1结构和调节为昏睡病和其他疾病引起的新疗法提供信息 影响着5亿人的动质体

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.