DOT1A-dependent H3K76 methylation is required for replication regulation in Trypanosoma brucei

Abstract

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 the protozoan parasite Trypanosoma brucei, novel regulatory mechanisms seem to have evolved. Here, we analyse the function of the histone methyltransferase DOT1A during cell-cycle progression. 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 replication of the nuclear DNA. In contrast, depletion of DOT1A by RNAi abolishes replication but does not prevent karyokinesis. As histone H3K76 methylation has never been associated with replication control in eukaryotes before, we have discovered a novel function of DOT1 enzymes, which might not be unique to trypanosomes.

Structure-guided mutational analysis reveals the functional requirements for product specificity of DOT1 enzymes

“…Different methylation levels exhibit completely different functions in trypanosomes. DOT1A-mediated H3K76me1/me2 seems to regulate replication initiation 30 , which has also been shown for higher eukaryotes recently 31,32 . In contrast, H3K76me3 appears to be involved in transcriptional regulation 33 and developmental differentiation 29 .…”

“…As controls, we mutated the central glycine residue of the DxGxGxG signature motif to arginine (G138R and G121R for DOT1A and DOT1B, respectively), generating catalytically inactive enzymes 29,37 . A histone methyltransferase assay was performed and H3K76 methylation levels (me1, me2 and me3) were detected by western blot analysis using specific antibodies 29,30 . Consistent with our previous observations 30 , DOT1A WT, but not its catalytically inactive G138R mutant, mono-and dimethylates H3K76 (Fig.…”

“…A histone methyltransferase assay was performed and H3K76 methylation levels (me1, me2 and me3) were detected by western blot analysis using specific antibodies 29,30 . Consistent with our previous observations 30 , DOT1A WT, but not its catalytically inactive G138R mutant, mono-and dimethylates H3K76 (Fig. 1c).…”

“…Next, we wanted to understand the molecular basis for the different product specificities of DOT1A and DOT1B observed in vivo and in vitro 29,30 ( Fig. 1c).…”

“…This observation is perfectly compatible with the different functions of DOT1A and DOT1B in the parasite. DOT1A-mediated H3K76me1/2 appears slowly after incorporation of new H3 into the chromatin fibre restricting these marks to G2 phase and mitosis, whereas DOT1B seems to convert all H3K76 quickly to a trimethylated state at the end of mitosis 30 .…”