Characterization of the TcDOT1a and TcDOT1b isoforms: implication of H3K76 differential methylation during Trypanosoma cruzi life cycle.

MALENA BALESTRASSE; MILENA MASSIMINO STEPÑICKA; GUILLERMO DANIEL ALONSO; JOSEFINA OCAMPO

Mar del Plata

Congreso; ANNUAL MEETING OF BIOSCIENCE SOCIETIES 2019, XXXI Annual Meeting of Sociedad Argentina de Protozoología (SAP); 2019

Sociedad Argentina de Protozoología en conjunto con otras sociedades

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. Recent studies have shown that DOT1 methyltransferases homologues, called DOT1a and DOT1b, are involved in the methylation of lysine 76 of histone H3 in T. cruzi. In T. brucei, DOT1a mediates H3K76 mono and di-methylation, whereas DOT1b catalyzes H3K76 tri-methylation. However, these two enzymes remain poorly characterized.In this project, we investigated the relevance of TcDOT1a and TcDOT1b during cell cycle and the metacyclogenesis process. Therefore, to evaluate the catalytic activity using heterologous complementation, we have successfully cloned and transformed a null DOT1 yeast strain with TcDOT1a. Additionally, 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.Overall, our data will be useful to further understand the role of the DOT1 isoforms and the differential methylation of H3K76 in T. cruzi. In the long term, our findings might unravel new targets for antiparasitic drugs.