Division of Gene Regulation, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands.
J Cell Sci. 2010 Dec 1;123(Pt 23):4019-23. doi: 10.1242/jcs.073882.
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.
Dot1 是一种高度保守的甲基转移酶，可在核小体核心表面修饰组蛋白 H3。与酵母、果蝇和人类不同，这些生物中只有一种 Dot1 酶负责组蛋白 H3 赖氨酸 79（H3K79）的所有甲基化，而非洲锥虫表达两种 DOT1 蛋白，以细胞周期调控的方式甲基化组蛋白 H3K76（对应于其他生物中的 H3K79）。虽然 DOT1A 对于正常的细胞周期进展是必不可少的，但 DOT1B 参与了这种原生动物寄生虫的分化和抗原变异的控制。分析锥虫中的 DOT1A 和 DOT1B 或体外，以了解 H3K76 甲基化在细胞周期中是如何被控制的，由于缺乏遗传工具和生化测定而变得复杂。为了解决这些问题，我们在酵母中开发了一种异源表达系统。虽然布氏锥虫 DOT1A 主要二甲基化 H3K79，但 DOT1B 甚至在没有 DOT1A 二甲基化的情况下也三甲基化 H3K79。此外，通过消除 H2B 的泛素化，选择性地降低了 DOT1A 的活性。组蛋白 H4 的尾部对于 DOT1A 或 DOT1B 的活性不是必需的。这些在酵母中的发现为布氏锥虫 H3K76 甲基化的可能调控机制提供了新的见解。
