Gene Regulation and Epigenetic Mechanisms in the Parasite Trypanosoma cruzi

Abstract: Trypanosomes are unicellular protozoan parasites responsible for several human diseases that affect millions of people and cause thousands of casualties every year. They also represent a primitive eukaryotic model system harboring unique processes and basic regulatory mechanisms such as RNA-editing, polycistronic transcription and trans-splicing, first described in these organisms. Unlike most eukaryotes where levels of gene expression are controlled at initiation of transcription, trypanosomes use post-transcriptional events as the main regulators. This thesis explores the epigenetic mechanisms involved in gene expression control in trypanosomes, providing the first evidences for a functional “histone-code” as well as the existence and location of DNA methylation in Trypanosoma cruzi.Chromatin immunoprecipitation (ChIP) was used for the profiling of acetylated and methylated histones in T. cruzi, showing that the modified histones were exclusively localized at bidirectional transcription start sites. In addition, promoters from highly transcribed genes were found depleted of nucleosomes, while DNA regions expected to be silent were not enriched in the investigated modified histones. Furthermore, we showed that the histone patterns were developmentally regulated.The first in depth characterization of the DNA methylation patterns in T. cruzi was presented in this work. We detected m5C in regions of transcriptional initiation and termination, retrotransposons, pseudogenes and the kinetoplast minicircle. We also showed that the amount of methylation changes during development, with an increase in non-replicative forms.We also characterized the DNA-interacting properties of a T. cruzi polypyrimidine-tract binding protein (TcPTB), and its potential role as a transcription factor. TcPTB was found to interact with single-stranded DNA present in promoters bound by its mammalian homologue as well as to the region of transcriptional initiation in Leishmania major. We also demonstrated that T. cruzi polypyrimidine stretches were able to confer ssDNA conformations.Overall, these results provide new insights into the biology of ancient pathogenic parasites, which might be exploited for drug development.