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.

Figure 1.

H3K76me1 is undetectable in S phase cells. (A) Indirect IFA of different cell-cycle stages in PCF expressing PCNA-TY (green) and H3K76me1 (red). DNA was stained with DAPI. Scale bar, 2 µm. (B) Quantification of cell cycle-dependent expression of PCNA-TY, H3K76me1 and H3K76me2. Three independent experiments were analysed.

Figure 2.

RNAi-mediated depletion of DOT1A inhibits replication. (A) Western blot analysis of different H3K76 methylation states in DOT1A-depleted cells. Two major proteins of the paraflagellar rod (PFR) were used as loading control. (B) Flow cytometry analysis of PI-stained non-induced BSF cells (upper left panel) and cells 19 h post-induction of DOT1A-RNAi (lower left panel). Non-induced cells with a DNA content of 2n and DOT1A-depleted cells with a DNA content of 1n (both indicated by a red bar) were sorted and incorporation of BrdU (green) was analysed (right panels). Cells were incubated with BrdU for 7 h before fixation. DNA was stained with DAPI. Scale bars, 2 µm.

Figure 3.

DOT1A over-expression generates aneuploid cells and disturbs the H3K76 methylation pattern. (A) Cumulative growth curves of wild-type, non-induced and induced DOT1A over-expressing cell lines. (B) Flow cytometry analysis of PI-stained DOT1A over-expressing cells. Relative amount of DNA content is indicated. The population of enucleated cells (‘zoids’) is marked by an arrow. (C) Quantitative analysis of the number of nuclei (N) and kinetoplasts (K) in wild-type and DOT1A over-expressing cells. Mean values of three independent experiments are presented with standard deviation (n > 100). (D) Western blot analysis of DOT1A expression and different H3K76 methylation states in DOT1A over-expressing cells. Histone H3 was used as loading control. (E) Percentage of H3K76me2 positive cells in different cell cycle stages. Mean values of three independent experiments are presented with standard deviation (n > 70 for each cell cycle phase). 2N2K cells were only analysed in wild-type cells and 4 h after induction because of low amounts of 2N2K cells at later time points.

Figure 4.

DOT1A over-expression causes continuous replication of nuclear DNA. Incorporation analysis of EdU and BrdU in replicated DNA during S phase of (A) wild-type and (B) DOT1A over-expressing cells (14 h post-induction). Top panels show a schematic representation of the experimental design. Labelled cells are shown as coloured dots. Bottom panels show IFA of labelled parasites. Detection of EdU (red) is based on a ‘click reaction’, BrdU (green) was detected by an antibody. DNA was stained with DAPI (blue). Scale bars, 2 µm.

Figure 5.

DOT1A over-expression causes re-initiation of replication. FISH of (A) wild-type and (B) DOT1A over-expressing cells. A probe specific for the tubulin gene array (red) was used for hybridization. DNA was stained with DAPI. Aneuploid nuclei show several distinguishable signals of the tubulin locus. Scale bars, 2 µm.

Figure 6.

Distribution of H3K76 mono- and di-methylation. Representative regions of chromosome 10 showing the distribution of H3K76me1 (black, upper panels), H3K76me2 (red, lower panels) and histone H3 (green, lower panels) are displayed. The distribution of H3K76 methylation is normalized with histone H3 distribution. Orange boxes represent ORFs and orange arrows indicate direction of transcription. Displayed are examples of (A) a convergent transcription termination site (TTS, marked by an arrow), (B) a divergent transcription start site (TSS, marked by an arrow) and (C) an internal part of a polycistronic unit.