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

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 (E-M). 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 E-M (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.