Biology subjects

da Cunha, J. P. C.

Publications and source records attributed to da Cunha, J. P. C..

5 recordsLinked to original sources

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↗

bioRxiv2024

Hidden origami in Trypanosoma cruzi nuclei highlights its nonrandom 3D genomic organization

The protozoan Trypanosoma cruzi, the causative agent of Chagas disease, exhibits polycistronic transcription and unidimensional genome compartmentalization of core (conserved) and disruptive (virulence factors from multigenic families) genes. Approximately 50% of its genome is repetitive, mainly virulence factor genes. Genomic sequences, including repeats, motifs of architectural proteins, and noncoding RNA loci are crucial for genome folding. Here, we evaluated the genomic features associated with higher-order chromatin organization in T. cruzi through extensive computational processing of high-throughput chromosome conformation capture (Hi-C) data, accounting for repetitive regions and improvements in genome annotation. Our study revealed that repetitive DNA (multimapped reads) influences 3D chromatin folding, particularly in determining the boundaries of topologically associated domains (TAD)-like structures. Virulence factor genes, unlike core genes, form shorter and more compact TAD-like structures enriched in loops, suggesting a gene expression regulatory mechanism. We found nonprotein-coding RNA loci (e.g., tRNAs) and transcription termination sites preferentially located at the boundaries of the TAD-like structures, while pseudogenes and multigenic family genes located in unstructured genomic regions. Our data indicate 3D clustering of tRNA loci, likely optimizing transcription by RNA polymerase III, and a complex interaction between spliced-leader RNA and 18S rRNA loci. Our findings provide insights into 3D genome organization in T. cruzi, contributing to the understanding of supranucleosome-level chromatin organization and suggesting possible links between 3D architecture and gene expression. We draw an analogy to the art of origami (e.g., papers folded into various shapes) resembling the DNA packed in chromatin fibers assuming distinct folds within the nucleus. ImportanceDespite the knowledge about the linear genome sequence and the identification of numerous virulence factors in the protozoan parasite Trypanosoma cruzi, there has been a limited understanding of how these genomic features are spatially organized within the nucleus and how this organization impacts gene regulation and pathogenicity. By providing a detailed analysis of the three-dimensional chromatin architecture in T. cruzi, our study contributed to filling this gap. We deciphered part of the origami structure hidden in the T. cruzi nucleus, showing the unidimensional genomic features are nonrandomly organized in the nuclear 3D landscape. We revealed the possible role of non-protein-coding RNA loci (e.g., tRNAs, SL-RNA, and 18S RNA) in shaping the genomic architecture. These findings provide insights into an additional epigenetic layer that may influence gene expression. Graphical abstractThe spatial organization of chromatin within the nuclei of T. cruzi and its resemblance to origami art. A. Identification of the 3D nuclear architectures within T. cruzi nuclei: topologically associating domains (TADs) and their boundaries; chromatin loops; and 3D networks. Inter- and intrachromosomal interactions reflect DNA-DNA contacts on the same (cis) and between different (trans) chromosomes. B. Resemblance between origami art and chromatin folding. Steps "a" to "l" show the process of folding a flat piece of paper from its unidimensional view up to its 3D boat form. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/601582v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1ca38b4org.highwire.dtl.DTLVardef@150bc99org.highwire.dtl.DTLVardef@18de8a9org.highwire.dtl.DTLVardef@1a5efa4_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Branched chain amino acids modulate the proteomic profile of Pro-induced differentiation for the infective stages of Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas disease, undergoes a complex life cycle involving triatomine insects as vectors and mammals. The differentiation of epimastigote forms into metacyclic trypomastigotes within the insect vector is crucial for the parasites life cycle progression. Factors influencing this process, including temperature, pH, and nutritional stress, along with specific metabolite availability, play a pivotal role. Amino acids like Pro, His, and Gln support cell differentiation, while branched-chain amino acids (BCAAs) inhibit it. Interestingly, combining the pro-metacyclogenic amino acid Pro with one of the anti-metacyclogenic BCAAs results in viable metacyclics with significantly reduced infectivity. To explore the characteristics of metacyclic parasites differentiated in the presence of BCAAs, proteomics analyses were conducted. Metacyclics obtained in triatomine artificial urine (TAU) supplemented with Pro alone and in combination with Leu, Ile, or Val were compared. The analyses revealed differential regulation of 40 proteins in TAU-Pro-Leu, 131 in TAU-Pro-Ile, and 179 in TAU-Pro-Val, as compared to metacyclics from TAU-Pro. Among these, 22%, 11%, and 13% of the proteins were associated with metabolic processes, respectively. Notably, enzymes related to glycolysis and the tricarboxylic acid (TCA) cycle were reduced in metacyclics with Pro-BCAAs, while enzymes involved in amino acid and purine metabolic pathways were increased. Furthermore, metacyclics with Pro-Ile and Pro-Val exhibited elevated enzymes linked to lipid and redox metabolism. These findings suggest that the presence of BCAAs can reshape the metabolism of metacyclics, contributing to the observed reduction in infectivity in these parasites.

biochemistry↗

bioRxiv2024

Comprehensive Analysis of Nascent Transcriptome Reveals Diverse Transcriptional Profiles Across the Trypanosoma cruzi Genome Underlining the Regulatory Role of Genome Organization, Chromatin Status, and Cis-Acting Elements

Trypanosomatids are eukaryotic parasites exhibiting polycistronic transcription and trans-splicing. Post-transcriptional mechanisms are acknowledged as pivotal in gene expression regulation of their protein-coding genes. To comprehensively investigate the impact of transcription on gene expression in Trypanosoma cruzi and the association with the epigenetic landscape, we conducted a genome-wide nascent transcriptomic analysis. Our findings reveal significant asymmetrical transcriptional abundance across the genome, notably between polycistronic transcription units (PTUs) enriched in conserved genes (core PTUs) and those containing virulence genes (disruptive PTUs). We found that trypanosomes exploit linear genome organization to regulate transcription abundance by embedding virulence genes into highly transcribed core-enriched PTUs, by positioning PTUs near non-coding regions of small non-coding RNAs (e.g., tRNAs, snoRNAs), and by placing core CDSs in PTUs of various sizes. Additionally, we found correlations between open chromatin status and nascent transcript levels, both globally and particularly at transcription starting regions (divergent strand switch regions - dSSRs), indicating a crucial role for chromatin architecture in transcriptional regulation. While both core and disruptive dSSRs exhibit similar levels of some epigenetic marks (H2B.V deposition and 5mC), disruptive dSSRs display significantly higher 5hmC content and nucleosome occupancy compared to core dSSRs. Furthermore, we identified distinct conserved motifs within dSSRs of core and disruptive PTUs. These findings challenge the notion of constitutive and uniform transcription in T. cruzi, underscoring the paramount importance of linear genome organization, cis-acting motifs, and chromatin landscape in transcriptional regulation.

molecular biology↗

bioRxiv2021

{-}{-} Histone H2B.V demarcates strategic regions in the Trypanosoma cruzi genome, associates with a bromodomain factor and affects parasite differentiation and host cell invasion

Histone variants play a crucial role in chromatin structure organization and gene expression. Trypanosomatids have an unusual H2B variant (H2B.V) that is known to dimerize with the variant H2A.Z generating unstable nucleosomes. Previously, we found that H2B.V protein is enriched in nonreplicative life forms of Trypanosoma cruzi, suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among parasite life forms. Here, we performed the first genome-wide profiling of histone localization in T. cruzi using replicative and nonreplicative life forms, and we found that H2B.V was preferentially located at the edges of divergent switch regions, which encompass putative transcriptional start regions; at some tDNA loci; and between the conserved and disrupted genome compartments, mainly at trans-sialidase, mucin and MASP genes. Remarkably, the chromatin of nonreplicative forms was depleted of H2B.V-enriched peaks in comparison to replicative forms. Interactome assays indicated that H2B.V associated specifically with H2A.Z, bromodomain factor 2, nucleolar proteins and a histone chaperone, among others. Parasites expressing reduced H2B.V levels were associated with higher rates of parasite differentiation and mammalian cell infectivity. Taken together, H2B.V demarcates critical genomic regions and associates with regulatory chromatin proteins, suggesting a scenario wherein local chromatin structures associated with parasite differentiation and invasion are regulated during the parasite life cycle. Author SummaryTrypanosomatids have to adapt to different environmental conditions, changing their morphology, gene expression and metabolism. These organisms have many unique features in terms of gene expression regulation. The genomic organization includes polycistronic regions with the absence of well-defined transcription start sites. In T. brucei, histone variants mark the start and ending sites of transcription; however, little is known about whether these proteins change their genome location, expression levels and interactors along life forms and what the impact is of these changes on parasite differentiation and infection. In T. cruzi, the causative agent of Chagas disease, we previously found that the histone variant of H2B is enriched in nonreplicative and infective forms, suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among these life forms. Here, we aimed to go one step further and performed the first histone ChIP-seq analysis in T. cruzi, in which we found that H2B.V was enriched at divergent strand switch regions, some tDNA loci and other critical genomic regions associated with T. cruzi genome compartments. We found that H2B.V interacts with a bromodomain factor, suggesting an intricate network involving chromatin acetylation around H2B.V enriched sites. Moreover, parasites expressing reduced H2B.V levels were associated with higher rates of differentiation and mammalian cell infectivity.

microbiology↗