Heterologous expression reveals distinct enzymatic activities of two DOT1 histone methyltransferases of Trypanosoma brucei

Dot1 is a highly conserved methyltransferase that modifies histone H3 on the nucleosome core surface. In contrast to yeast, flies, and humans where a single Dot1 enzyme is responsible for all methylation of H3 lysine 79 (H3K79), African trypanosomes express two DOT1 proteins that methylate histone H3K76 (corresponding to H3K79 in other organisms) in a cell-cycle-regulated manner. Whereas DOT1A is essential for normal cell cycle progression, DOT1B is involved in differentiation and control of antigenic variation of this protozoan parasite. Analysis of DOT1A and DOT1B in trypanosomes or in vitro, to understand how H3K76 methylation is controlled during the cell cycle, is complicated by the lack of genetic tools and biochemical assays. To eliminate these problems, we developed a heterologous expression system in yeast. Whereas Trypanosoma brucei DOT1A predominantly dimethylated H3K79, DOT1B trimethylated H3K79 even in the absence of dimethylation by DOT1A. Furthermore, DOT1A activity was selectively reduced by eliminating ubiquitylation of H2B. The tail of histone H4 was not required for activity of DOT1A or DOT1B. These findings in yeast provide new insights into possible mechanisms of regulation of H3K76 methylation in Trypanosoma brucei.