Nucleosome positioning and histone methylation in Trypanosoma cruzi

MASSIMINO STEPÑICKA M; VILCHEZ LARREA SC; BEATI P; BARRERA NM; ALONSO GD; OCAMPO J

Resistencia, Chaco

Congreso; XXX Reunión Anual de la Sociedad Argentina de Protozoología; 2018

Sociedad Argentina de Protozoología

Trypanosoma cruzi is a protozoan parasite with a complex life cycle that varies from infective and nonreplicative to replicative but non-infective forms. It is known that gene expression is barely regulated at the level of transcription, however some differences in gene transcription levels between life cycle stages have been observed and differential histone modication has been described in trypanosomatids. Based on these observations, we postulate that chromatin remodeling might influence the life cycle of T.cruzi. In the presentstudy we aim to map and compare the nucleosome positioning in epimastigotes and trypomastigotes using MNase-seq and complementarily, characterize a histone methyl transferase that has differential expression between life cycle stages. To carry on with the rst objective, we obtained mononucleosomes from CL Brener(DTU VI) and Sylvio X10 (DTUI) and mapped them genome-wide using paired-end sequencing. We found that the length distribution of the sequenced DNA had a pick at 147-148bp for CL Brener and at 148-159bp for Sylvio X10, indicating the good quality of the samples. Both by local and global analyses, we found thatstrand switch regions (SSR) are depleted of nucleosomes, consistent with previous observations that suggest these SSR as transcription initiation sites. Moreover, we divided up the genes into up- and down-regulated in epi- vs trypomastigotes, based on RNA-seq results. Up-regulated genes showed phased nucleosomes as opposed to the unphased disposition displayed at down regulated genes, suggesting the presence of a phasing complex upstream of the more active ones. Nucleosome assembly, positioning and its interaction with DNA and other protein complexes is influenced by histone posttranslational modications. DOT1 is a H3 methyltransferase that catalyzes mono, di and tri methylation on lys79. In mammals and yeast it's implicated in transcription regulation, cell cycle progression and DNA damage response. In T. brucei there are two homolog isoforms involved in the parasite cell cycle. Here, we initiate the characterization of the two isoforms present in T. cruzi, TcDOT1A and TcDOT1B. To test their catalytic activity, we cloned the HA-tagged isoforms and transform dot1D yeast to analyze the phenotype. We conrmed the expression by western blot.Our results show for the first time the nucleosome positioning in T. cruzi and represent a new approach to understand the regulation of gene expression in this organism.