Gachet-Castro, C. | bioRxiv Science

bioRxiv2025

Nuclear proteome analysis reveals spatial and temporal compartmentalization of metabolic enzymes during Trypanosoma cruzi cell cycle

Trypanosoma cruzi, the etiological agent of Chagas Disease (CD), represents a significant public health concern and serves as a valuable model for investigating the cell cycle in early-diverging eukaryotes. Its unique cellular features--including the absence of chromosome condensation and the coordination of nuclear division with specialized organelles--provide insights into non-canonical regulatory mechanisms, and underscore the pivotal role of the nucleus in maintaining genomic integrity and regulating fundamental processes such as DNA replication and gene expression. To investigate nuclear dynamics throughout the cell cycle, we synchronized T. cruzi epimastigotes at G1/S, S, and G2/M using hydroxyurea (HU), and isolated nuclear proteins were quantitative evaluated by LC-MS/MS. We identified 2,937 nuclear-enriched proteins, revealing distinct, phase-specific expression patterns. The G1/S phase was marked by increased levels of metabolic enzymes including those related to energy and nucleotide/nucleoside metabolisms. The S phase showed elevated abundance of canonical and variant histones, consistent with chromatin remodeling and DNA replication. The G2/M phase was enriched in proteins involved in protein synthesis and microtubule dynamics, essential for mitosis. Notably, [~]6% of the nuclear proteome consisted of metabolic enzymes, a finding further supported by other public proteomics datasets and in vitro nuclear activity assays. These results suggest that metabolic enzymes and/or their metabolites may modulate nuclear processes in T. cruzi, potentially influencing the epigenome and gene expression regulation, as proposed in other eukaryotic models. Altogether, our findings reveal dynamic remodeling of the nuclear proteome during the T. cruzi cell cycle and point to a previously underappreciated role for metabolic enzymes likely regulating nuclear functions in a cell cycle-dependent manner.

microbiology↗

bioRxiv2025

New Histone H4 variant and H2B variant Exhibits Distinct Genomic Locations, Chromatin Affinities, and Dynamics Throughout Life Cycle 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 telomeric regions, demarcates convergent strand-switch regions (cSSRs), and determines new transcription termination sites at codirectional PTUs interrupted by tDNA loci. Throughout the cell cycle, H4.V transcript levels remain stable, while protein abundance increases in G2/M, as shown by immunofluorescence and image flow cytometry. In contrast, the histone H2B variant (H2B.V) transcripts peak at S-phase, and protein 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 demonstrated differing chromatin binding affinities across life stages for both variants, with H4.V displaying a more permanent chromatin association than H2B.V, indicating that H4.V may be associated with a more compact chromatin state. 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, underscoring its potential function as an epigenetic marker in transcriptional regulation and adaptation to diverse host environments. Author summaryChromatin is composed of a group of proteins called histones that are involved in compacting DNA and thereby affecting many DNA-associated mechanisms, such as replication and transcription. Histone variants share distinct primary sequences from the canonical ones and display different roles and genomic locations. While variants of histone H3 and H2A are common in all eukaryotes, variants of H2B and H4 are unusual, except in trypanosomatids, a group of parasites that cause human and veterinary diseases. Among them, Trypanosoma cruzi, the causative agent of Chagas disease, exhibits variants for three core histones, but a variant of histone H4 (H4.V) had not been described. Here, we identify H4.V and demonstrate its preferential deposition at telomeres, convergent strand-switch regions (cSSRs), and codirectional polycistronic transcription units (PTUs) interrupted by tDNA loci, likely marking transcription termination sites. Both variants are enriched in the nuclei of metacyclic trypomastigotes; however, H4.V is barely detectable in amastigotes and bloodstream trypomastigotes, while H2B.V is consistently present in all forms. Additionally, each variant shows distinct chromatin-binding affinities across life stages, with H4.V potentially linked to a more compact chromatin state. Our findings reveal a novel histone variant with unique dynamics and epigenetic roles distinct from H2B.V in T. cruzi.

microbiology↗