Chromatin Biology of the African Trypanosome

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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.