Unveiling new perspectives on Trypanosoma cruzi chromatin through locus-specific immunoprecipitation

Abstract

The regulation of gene expression is crucial for the adaptation and survival of Trypanosoma cruzi, the etiological agent of Chagas disease. Specifically, post-transcriptional regulation has been described as fundamental in this process, as gene transcription occurs polycistronically (PTU), meaning that multiple genes are transcribed on a single messenger RNA (mRNA) strand. However, mechanisms associated with chromatin status and genome organization suggest the existence of a yet underexplored transcriptional level of regulation. Despite significant changes in the nucleus and euchromatic and heterochromatic regions between life forms, we have not yet identified the proteins (or their set) present at a specific gene locus. Thus, our objective is to standardize in vitro and in vivo chromatin immunoprecipitation (ChIP) techniques for specific regions of the T. cruzi genome using dead Cas9 protein (dCas9) fused to a Flag-tag guided, initially, to the promoter regions of the spliced leader (SL) gene as a proof of concept for the technique. After standardizing the technique, we will select genes with multiple copies from the core compartment versus genes from the disruptive compartment for analysis. Genes like MASP, present in the disruptive compartment, are less transcribed and are located in compacted chromatin regions. The standardization of the proposed technique will allow us to identify proteins associated with heterochromatin responsible for gene expression silencing at these loci. Thus, we aim to contribute to the understanding of the factors involved in maintaining gene silencing and/or activation in T. cruzi, as well as understanding how genome organization contributes to this process (AU)