A novel approach to study epigenetic marks in Trypanosoma cruzi

Abstract

T. cruzi is the etiologic agent of Chagas disease and has a complex life cycle. Epimastigotes are the replicativeform in the vector, trypomastigotes are the infective stage, and amastigotes are a replicative intracellular stage.Proper cell cycle progression requires multiple factors.Epigenetics could be relevant for gene regulation. Particularly, lysine 76 of histone 3 (H3K76), could be mono,di or tri methylated by the methyltransferases TcDot1a and TcDot1b. It is not known how H3K76 methylationinfluences cell cycle progression, but deletion of TcDOT1b arrests the cell cycle in G2/M. Additionally,overexpression of Aurora kinase 1 (TcAUK1), affects the same stage.One of the main problems to study the epigenetic role of H3K76 methylation, or TcAUK1 in cell cycle progressionis that transgenic parasites for these proteins had been hard to obtain or maintain. Therefore, we used Flowcytometry that allowed us to identify the differential methylation of H3K76 and to make quantitative analysisusing low amounts of antibodies. Remarkably, we have been able to fine-tune the technique to this end. Wehave used three strain of T. cruzi, Dm28c; K98 and Tulahuen that differ by their virulence. We could detect byFlow cytometer that H3K76 could be mono, di and trimethylated, being the last one the most abundant mark inevery strain tested. This result is consistent with previous outcomes that showed that H3K76me3 is present inevery step of the cell cycle. Additionally, we could detect TcAUK1 in Dm28c, K98 and Tulahuen. This isimportant because the subcellular location and expression levels of this protein are finely regulated and it isevasive to Western blot detection.Furthermore, to check the potential connection between TcAUK1 and TcDOT1 activity, we are testing differentialH3K76 methylation in parasites that overexpress TcAUK1.Our findings surpass our study, since this approach will be useful for the whole community working intrypanosome epigenetics.