Trypanosoma cruzi chromatin milieu: an active or dormant character on reporter genes transcription?

• Grant number: 21/11419-9
• Support type: Scholarships in Brazil - Doctorate (Direct)
• Effective date (Start): October 01, 2021
• Effective date (End): May 31, 2024
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal researcher: Julia Pinheiro Chagas da Cunha
• Grantee: Herbert Guimarães de Sousa Silva
• Home 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

In trypanosomes, it is general accepted that gene expression is mainly regulated by post-transcriptional mechanisms due to the lack of classical RNA Pol II promoters regions and polycistronic transcription. However, previous low-scale nuclear run-on experiments indicate that satellites genes have lower expression, suggesting some layer of regulation (Elias, Marques-Porto et al. 2001), and a significant decrease in global transcription is observed when replicative forms are compared to non-replicative forms (ie. Metacyclics) (Ferreira, Dossin Fde et al. 2008). In addition, it was observed in Leishmania (an organism from trypanosomatid order) that genes associated to cell cycle can be regulated at transcription level (Chandra, Yadav et al. 2017). Thus, here we want to investigate if the genomic milieu (namely, chromatin content) interferes with the expression of a reporter gene positioned in distinct parts of T. cruzi genome. To evaluate that, we will insert luciferase-reporter genes (containing proper 5' and 3' UTR) by using the CRISPR/Cas9 system, at distinct parts of T.cruzi genome (namely, I. putative TTS and TSS, II. telomeric regions, III. DGF-1 family; IV. SL promoter regions; V. regions of RNA Pol I and Pol III transcripts; VI. Beginning, middle, and end of a selected polycistronic region). These experiments will reflect if chromatin packing (as well as histone PTMs and other chromatin-associated proteins) can influence repression/activation in a gene-dependent position. We envisage that differences will be obtained among clones whose luciferase where inserted at different loci, therefore, to increase the comprehensive of this regulation, we will evaluate the chromatin content of these regions by using CLASP (or enChIP) technology. (AU)