Gene expression in Trypanosoma cruzi parasites expressing a TcBDF2 dominant negative mutant

VILLANOVA GABRIELA VANINA; LARESE MÓNICA; PAVONI DANIELA; BAYA ARIEL; TAPIA ELISABETH; SERRA ESTEBAN

Foz de Iguazú

Congreso; XXVI Reunião Anual da Sociedade Brasileira de Protozoologia XXXVII Reunião Anual sobre Pesquisa básica em Doença de Chagas; 2010

Sociedad Brasilera de Protozoología

Histones postranslational modifications, their role in gene expression regulation and others nuclear events in trypanosomatids are currently an active field of research. Recently it has been proposed a model for RNA Polymerase II transcription initiation in Trypanosoma brucei which involves histone posttranslational modifications that would serve as binding site for a bromodomain factor (BDF). In turn, this BDF would recruit chromatin remodelling proteins involved in incorporating variants of histones into nucleosomes, resulting in less stable nucleosomes, permissive for polymerase binding and transcription initiation. We have reported that Trypanosma cruzi Bromodomain Factor2 TcBDF2 is expressed in discrete regions inside the nucleus, between dense and less dense chromatin regions. It binds H4, with preference for K10 and K14 acetylated residues. In order to analyze its function, TcBDF2 fused to c-myc tag and deleted at its C-term (TcBDF2C) were expressed in epimastigotes from a tetracycline regulated promoter. TcBDF2C mutant acts as a dominant negative, inhibiting parasite growth and enhancing their sensitivity to UV irradiation. Global transcriptome from TcBDF2C, TcBDF2-c-myc and control parasites was evaluated 48 h after tetracycline addition by using a 10 K oligonucleotide microarray. Limma software (R) was used to determine mRNA relative abundances (LFC>1.5 and p value =0.01) among the three strains. Just 25 genes were up- and down-regulated between TcBDF2myc and control parasites. However, 100 genes were differentially expressed in TcBDF2C respect to control parasites and this quantity increased to more than 1000 when mRNA relative abundances of TcBDF2C and TcBDF2myc parasites were compared. RT-PCR assays are in progress to corroborate these data. Neither up-regulated nor down-regulated genes showed a pattern of localization in chromosomes respect to strand switch region. Our results support the model proposed and the hypothesis that acetylated histones and BDF2 are taking part in gene expression process throw chromatin remodeling. Supported by ANPCyT and CONICET