Role of the TcDOT1a and TcDOT1b isoforms in H3K76 differential methylation and their impact in Trypanosoma cruzi life cycle

Abstract

Trypanosoma cruzi, the etiologic agent of Chagas Disease, affects a large number of the population in Latin America. It has a complex life cycle alternating between a mammalian host and the vector insect, Triatoma infestans. This cycle consists of three well-defined stages: amastigotes, epimastigotes and trypomastigotes. As the parasite faces different environments, it requires changes in gene expression in order to survive. Hence, gene expression regulation might be a key aspect to understand adaptation. Despite Trypanosomes gene expression is mainly regulated post transcriptionally, there are evidences that chromatin state influence it. In T. cruzi, there are two Dot1 methyltransferases homologues, called Dot1a and Dot1b, involved in the sequential mono, di and tri methylation of lysine 76 of histone H3 (H3K76me). Additionally, recent studies have shown that Dot1a deletion is not viable while Dot1b null mutant has aberrant morphology and decreased growth rate.In this project, we investigated the relevance of TcDot1a and TcDot1b during cell cycle and the metacyclogenesis process. Therefore, to analyze the isoforms subcelular location and their effects on cell cycle progression and differentiation, we have cloned the TcDot1 isoforms in a pRibotex vector with an N-terminal HA-tag and transfected epimastigotes of CL Brener strain. Nevertheless, the overexpression was toxic for the cell. Therefore, we decided to switch to an inducible vector.In order to elucidate the epigenetic implications of H3K76 differential methylation in the different stages of the life cycle of T. cruzi, we propose to map H3K76 mono, di and tri-methylation genome wide by MNase-ChIP-seq technique followed by paired-end sequencing.Overall, our data will be useful to further understand the role of the Dot1 isoforms and the epigenetic mechanism mediated by H3K76 in this unique parasite.