Authors
J Rosón1; MO Vitarelli2; H Costa-Silva1; DS Pires1; T Rodrigues1; B Cordeiro1; A Kraus3; S Calderano1; N Siegel3; MC Elias1; JP Cunha2; 1 INSTITUTO BUTANTAN, CELL CYCLE SPECIAL LAB, SÃO PAULO, Brazil; 2 Butantan Institute, Brazil; 3 Division of Experimental Parasitology, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universita¨ t in Munich, Munich, Germany, Brazil Discussion
Introduction: Histones variant deposition and histone posttranslational modifications (PTMs) can affect chromatin structure and gene expression. For Trypanosoma brucei, it is known that the histone variant H2B.V dimerizes with the H2A.Z and generates unstable nucleosomes in divergent switch strand sites. When nucleosomes harboring these variants associate with protein factors (e.g. bromodomains) and histone acetylation, a scenario of transcription activation is suggested. H3.V and H4.V are other examples of histone variants that affect nuclear architecture. In T. brucei, they are frequently positioned at transcription termination regions, often surrounded by J bases. Parasites lacking the H3.V/H4.V have shown profound changes in chromatin structure. In T. cruzi, H2B.V was found enriched in infective mammalian tissue culture trypomastigotes (TCT) compared to epimastigote forms by using quantitative proteomics. To date, H4.V was not identified in T. cruzi genome database. Objectives: First, to identify H4.V sequence candidates and generate parasites expressing H4.V and H2B.V tagged by CRISPR Cas9. Second, to compare histone variants (H4.V and H2B.V) associated with chromatin in distinct T. cruzi life forms, through gradient salt extraction. Third, to evaluate H2B.V genome location comparing epimastigotes with TCT forms, by ChIP-seq. Results: To retrieve a putative H4.V in T. cruzi (CL Brener Esmeraldo-like) we have performed BLAST and phylogenetic analysis by using T. brucei H4.V sequence. A strong candidate for H4.V was revealed (TcCLB.511681.20), homologous to T. brucei and Leishmania. To further evaluate histone variant genome location and chromatin interaction, lineages expressing a tagged version of H2B.V and H4.V were obtained by CRISPR-Cas9 system. By comparing the association of the canonical histone H3 and the variants H2B.V and H4.V to the epimastigote chromatin by salt extraction, we observed that variants associated weakly compared to histone H3. Also, H2B.V was weakly bonded to the TCTs’ chromatin. Moreover, the genomic location of H2B.V was performed by ChIP-seq assay. Our results showed H2B.V peak enrichment in dSSRs, tDNAs, and regions between conserved (mainly protein-coding genes) and disrupted (non-synthetic regions, mainly virulence factors) genome compartments. H2B.V peaks were more evident in epimastigotes than TCT forms in agreement with a weak chromatin association in the latter. Discussion/Conclusion: This ongoing research highlights the differential histone variants association with the chromatin and contributes unveil their function among the parasite life forms. The integration of methods explored in this work reinforces the role of the H2B.V and H4.V in the chromatin structure and suggests differences among the parasite life forms. 
