Identification of transcription start sites and possible promoter regions in Trypanosoma cruzi

• Grant number: 22/15610-8
• Support Opportunities: Scholarships in Brazil - Doctorate
• Effective date (Start): January 01, 2024
• Effective date (End): December 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Julia Pinheiro Chagas da Cunha
• Grantee: Letícia de Sousa Lopes
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 18/15553-9 - Going deeper into Trypanosoma cruzi chromatin regulation: identifying new players and quizzing its impact on a potential transcription control, AP.JP2

Abstract

Trypanosoma cruzi is the parasite that causes Chagas disease. Recently it was proposed that its genome is organized in two compartments: the central compartment - composed of highly conserved genes, and the disruptive compartment - composed of rapidly evolving genes, such as trans-sialidase, mucins and MASP virulence genes. Our group showed enrichment of histone H2B variant (H2B.V), a marker of transcription start regions (TSS), in the transition between these compartments. As a trypanosomatid, transcription of T. cruzi occurs in a polycistronic manner, i.e. RNA Polymerase II transcribes its protein-coding genes in long transcripts. Until now, no specific promoter regions have been described in T. cruzi, indicating that its gene regulation would happen mainly by post-transcriptional mechanisms. However, recently published studies have identified the presence of specific promoter sequences capable of driving the initiation of transcription by RNA Pol II in T. brucei. Thus, in this PhD project we propose to determine transcription start sites (TSSs) in T. cruzi, aiming to identify possible promoter sequences close to the disruptive compartment and possibly associated with the expression of virulence genes. Two large-scale methodologies will be employed to map the TSSs. First, we will use the chromatin immunoprecipitation technique followed by large-scale sequencing (ChIP-seq) to determine sites enriched with RNA Pol II, since enrichment of this enzyme correlates with transcriptional pausing downstream of promoter regions. We will compare the localization of RNA Pol II and H2B.V proteins, aiming to better identify delimiting TSSs in T. cruzi. In addition, we will map the distribution of small nascent primary transcripts to corroborate with previous assays. The identified TSSs will be evaluated in silico to delineate the smallest possible promoter sequence to initiate transcription in T. cruzi. For the completion of this PhD, we will functionally investigate the regions found in an in vivo assay using the luciferase reporter gene in order to identify minimal DNA sequences capable of driving transcription in T. cruzi. We believe that this PhD project will allow us to better elucidate the mechanisms of transcription in T. cruzi by determining TSSs and their possible promoter sequences.