The histone methyltransferase DOT1B is dispensable for stage differentiation in Leishmania mexicana but not for efficient infection of macrophages

Abstract text

1 Introduction

One of the greatest challenges for eukaryotic cells is to organise and maintain a dynamic genome architecture. This allows precise temporal and spatial access to the chromatin fibres to regulate nuclear processes such as transcription, DNA repair and replication. Post-translational histone modifications such as methylation of lysine or arginine residues are one device in the nuclear tool box to facilitate required changes of chromatin structure. DOT1 (disruptor of telomeric silencing 1) is a conserved histone methyltransferase that mediates methylation of lysine 79 of histone H3 (H3K79me) in many eukaryotes. In trypanosomatids, two homologues of DOT1, DOT1A and DOT1B, have been identified and are best characterised in Trypanosoma brucei. While TbDOT1A mediates mono- and di-methylation of H3K76, TbDOT1B can additionally catalyse tri-methylation of this residue (H3K76me3). TbDOT1A is necessary for replication initiation and thus essential for the survival of the cells. TbDOT1B is not essential for parasite growth in cell culture but is required for efficient in situ switching of variant surface glycoproteins. Interestingly, TbDOT1B-depleted bloodstream forms are not able to differentiate to procyclic forms. Substantially less is known about the functions of DOT1 enzymes in T. cruzi and to date nothing is known about their role in Leishmania parasites.

2 Objectives

Here, we aimed to elucidate whether the functions of DOT1 histone methyltransferases are conserved in Leishmania mexicana.

3 Results

As in T. brucei, the H3K76 methylation pattern is also cell cycle-regulated in L. mexicana with H3K76me1/2 only detectable in G2 phases and mitosis of the cell cycle. As in T. brucei, we observed that LmxDOT1B is not essential for growth of procyclic promastigotes in vitro. CRISPR Cas9-mediated LmxDOT1B deletion resulted in a loss of H3K76me3, indicating that LmxDOT1B is solely responsible for this histone modification. Strikingly, and in contrast to T. brucei, LmxDOT1B is not essential for differentiation of axenic procyclic promastigotes to amastigotes in vitro, but important to establish an infection of bone marrow-derived macrophages.

4 Conclusion

These data suggest that although the enzymatic activity of LmxDOT1 enzymes is conserved, L. mexicana parasites have developed novel functions specific to their life cycle.