Functional validation of candidate cis-regulatory elements in Trypanosoma cruzi using a reporter gene assay

• Grant number: 25/20165-1
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: December 29, 2025
• End date: June 28, 2026
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Julia Pinheiro Chagas da Cunha
• Grantee: Letícia de Sousa Lopes
• Supervisor: Martin Craig Taylor
• Host Institution: Instituto Butantan. São Paulo , SP, Brazil
• Institution abroad: London School of Hygiene and Tropical Medicine, England
• Associated to the scholarship: 22/15610-8 - Identification of transcription start sites and possible promoter regions in Trypanosoma cruzi, BP.DR

Abstract

The etiological agent of Chagas disease, Trypanosoma cruzi, has a genome organised into linear compartments: the central compartment, with housekeeping genes, and the disruptive compartment, with virulence genes. Its genes are transcribed into polycistronic units (PTUs) that have no functional correlations with each other and exhibit different patterns of gene expression. Over the last few years, our research group has aimed to understand the regulatory mechanisms at the chromatin level that are involved in T. cruzi gene expression. We have demonstrated that core regions have fewer DNA-DNA interactions, higher levels of open chromatin leading to more transcriptionally active chromatin, while disruptive regions have more chromatin loops, lower levels of open chromatin, which are closed and have decreased transcriptional activity. Our analyses revealed that divergent strand switch regions (dSSRs) are positioned near H2B.V-enriched domains and exhibit distinct epigenetic signatures and cis-regulatory elements, depending on whether they flank core or disruptive-enriched PTUs. In addition, Leticia's FAPESP PhD project experimentally mapped numerous TSSs both at dSSRs and outside these regions, using nascent RNAs obtained from sequencing small triphosphate RNAs (3p-sRNAs) in epimastigote forms. Building on this, in the present BEPE project we propose to assess the transcriptional activity of selected sequences enriched in 3p-sRNAs, in order to determine whether they are capable of activating transcription in vivo, functionally validating them through a reporter gene assay. For this, we will genetically engineer parasite cell lines to express a cassette containing a luciferase reporter gene inserted into a silent locus, with the selected cis-acting elements cloned upstream. With these analyses, we aim to advance the identification and characterization of regulatory elements in T. cruzi, combining strategies to clarify the mechanisms that control gene expression in this parasite.