New Histone H4 variant and H2B variant Exhibits distinct genomic distributions, chromatin affinities, and dynamics throughout life and cell cycle of Trypanosoma cruzi

Histone variants play crucial roles in chromatin organization and transcriptional regulation in eukaryotes. Unusually, trypanosomatids display histone variants for all histones, although a functional homolog of histone H4 variant (H4.V) had not yet been described in Trypanosoma cruzi. In this study, we identified a H4.V in T. cruzi that is encoded by a single-copy gene located apart from the typical tandem arrays of canonical histone H4. Functional characterization using ChIP-seq assays revealed that H4.V is located at telomere-subtelomere interface, demarcates convergent strand-switch regions (cSSRs), and determines potential new transcription termination sites at codirectional PTUs interrupted by tDNA loci. Throughout the cell cycle, H4.V abundance increases in G2/M, as shown by immunofluorescence and imaging flow cytometry. In contrast, the histone H2B variant (H2B.V) abundance accumulates progressively. H4.V is more abundant in the nuclei of metacyclic trypomastigotes but barely detectable in amastigotes and bloodstream trypomastigotes. In contrast, H2B.V shows a punctate nuclear pattern and is present in all life stages, with the highest levels also observed in metacyclics. During metacyclogenesis, both variants show a progressive increase in expression, particularly H4.V, suggesting a role in parasite differentiation. Ultimately, salt extraction experiments revealed life stage-dependent differences in histone distribution profiles, particularly between insect-stage and mammalian-stage parasites, with H4.V displaying greater retention in the salt-resistant chromatin fraction, whereas H2B.V was more readily released under low-salt conditions compared to canonical histone H3. Our data reveal H4.V as a novel histone variant in T. cruzi, characterized by unique genomic localization, expression profiles, and chromatin-binding dynamics in contrast to H2B.V. These findings underscore H4.V as a marker associated with transcriptional termination, and possibly contributing to metacyclogenesis or vector-stage adaptation.