Histone Methylation & Replication Initiation

Scanning EM image of T. brucei

Co-IP of DOT1 methyltransferases with RNaseH2

DOT1-mediated methylation of histone H3 regulates several nuclear processes in eukaryotes, including transcription regulation, DNA damage response, and telomeric silencing. In contrast to yeast and mammals, trypanosomes possess two DOT1 paralogs, DOT1A and DOT1B, with different enzymatic activities. The mono- and dimethylation of histone H3 on lysine 76 (H3K76) is mediated by DOT1A and is essential for replication initiation. Trimethylation of H3K76 is catalyzed by DOT1B. Deletion of DOT1B causes defects in defelopmental differentiation from the mammalian-infective stage to the insect-infective stage. In addition, DOT1B plays an important role in VSG transcription regulation.

Cell cycle progression requires careful regulation to ensure accurate propagation of genetic material to the daughter cells. Although many cell cycle regulators are evolutionarily conserved in trypanosomes, novel regulatory mechanisms seem to have evolved. We discovered that histone H3 methylation by DOT1A is a master regulater of replication initiation in T. brucei. Over-expression of DOT1A generates a population of cells with aneuploid nuclei as well as enucleated cells. Detailed analysis shows that DOT1A over-expression causes continuous re-initiation of replication of the nuclear DNA in S phase. In contrast, depletion of DOT1A by RNAi abolishes replication but does not prevent karyokinesis. H3K76 methylation is cell cycle-dependent with a peak of H3K76me1/2 in G2/M phase of the cell cycle. Recently, we discovered that the ribonuclease H2 complex (RNaseH2) interacts with DOT1 enzymes and might serve as a negative regulator of DOT1 enzymatic activity. In this project, we aim to understand how cell cycle-dependent activity of DOT1 enzymes is regulated to understand the mechanism of replication initiation in trypanosomes.