Describing new players at Trypanosoma cruzi chromatin by locus-specific chromatin immunoprecipitation

• Grant number: 23/08391-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: July 01, 2023
• End date: October 31, 2023
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Julia Pinheiro Chagas da Cunha
• Grantee: Ana Paula de Jesus Menezes
• 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

At JP1, our group described many chromatin-associated proteins as well as more than 40 histone PTMs differentially expressed in replicate versus non-replicative life forms in Trypanosoma cruzi. Classical eu- and heterochromatin regions are presented at their nucleus and changes dramatically during differentiation from epimastigotes to metacyclics (EàM). Traditionally, these chromatin regions (and some histone PTMs) are associated to transcription regulation. These dataset, however, do not allow the association of a given protein (or a set of) to a specific genomic locus. That would, in turn, increase the comprehensive of chromatin biology possibly describing unpredicted functions of both protein and the genomic context. Recently, cutting-edge methodologies have been developed to recover proteins associated to a given locus. Thus, here we will evaluate the chromatin content of specific regions of T.cruzi genome by using dCas9-Flag (both in vitro and in vivo strategies) guided to target specific genomic regions (namely, TTS, TSS, telomeric regions, replication origins, SL promoter regions; 24S and small-subunit rRNA (RNA Pol I transcripts); tRNA and soRNA regions (RNA Pol III transcripts)). The comparison among the associated-chromatin content of each locus, together with the evaluation of (possible) changes during differentiation EàM (and cell cycle) may contribute for a better understanding of factors involved on the establishment and maintenance of gene silencing/activation as well as describe key protein components associated to differentiation/cell cycle. Finally, the role of 1-2 targets will be explored through CRISPR/Cas9 system for generation of knockouts (KO) parasites and tagged proteins.