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. 2022 Feb 18;18(2):e1009694.
doi: 10.1371/journal.ppat.1009694. eCollection 2022 Feb.

H2B.V demarcates divergent strand-switch regions, some tDNA loci, and genome compartments in Trypanosoma cruzi and affects parasite differentiation and host cell invasion

Juliana Nunes Rosón  1   2   3 , Marcela de Oliveira Vitarelli  1   2 , Héllida Marina Costa-Silva  1   2 , Kamille Schmitt Pereira  4   5 , David da Silva Pires  1   2 , Leticia de Sousa Lopes  1   2 , Barbara Cordeiro  1   2 , Amelie J Kraus  6   7 , Karin Navarro Tozzi Cruz  1   2 , Simone Guedes Calderano  1   2 , Stenio Perdigão Fragoso  4   5 , T Nicolai Siegel  6   7 , Maria Carolina Elias  1   2 , Julia Pinheiro Chagas da Cunha  1   2
Affiliations

H2B.V demarcates divergent strand-switch regions, some tDNA loci, and genome compartments in Trypanosoma cruzi and affects parasite differentiation and host cell invasion

Juliana Nunes Rosón et al. PLoS Pathog. .

Abstract

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 tissue-derived trypomastigote (TCT) life forms, a nonreplicative stage 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 epimastigotes and TCT life forms, and we found that H2B.V was preferentially located at the edges of divergent transcriptional strand 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 TCT forms was depleted of H2B.V-enriched peaks in comparison to epimastigote 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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. H2B.V is enriched at dSSRs of epimastigote forms.
A. IGV snapshot of whole chr 6S of the CL Brener Esmeraldo-like haplotype using the wig files from the ChIP H2B.V/input and ChIP H3/input ratios obtained by COVERnant v.0.3.2. Note that H2B.V, but not H3, is enriched at dSSRs. Blue bar arrows indicate the transcription direction in each polycistron. Genes in the same polycistron are stained with the same color. dSSRs, purple bars; cSSRs, yellow bars. Red bars represent H2B.V peaks (fold equal to 4 over the input, Poisson p-value over input required = 1.00e-04) obtained by HOMER in the epimastigote form. Rectangles with interrupted lines highlight dSSRs. Summary plot obtained by deepTools for H2B.V and H3 (ChIP/input) enrichment at polycistrons (B), dSSRs and cSSRs (C). Ten kilobases upstream and downstream of the abovementioned regions were also analyzed. All genomic features were automatically scaled as indicated (Start-End). Polycistron start and end is related, respectively, to the first ATG (from the first CDS) and the last stop codon (from the last CDS). dSSR and cSSR start refer to the first coordinate downstream, respectively, to the start and end of the left polycistron. dSSR and cSSR end refer to the first coordinate upstream, respectively, to the start and end of the right polycistron. D. Percentual distribution of H2B.V peaks (Fold 4) on all 41 chromosomes of the CL Brener Esmeraldo-like haplotype.
Fig 2
Fig 2. H2B.V enrichment is also found at the border of conserved and disrupted genome compartments and around tDNA loci.
A. IGV snapshot of a 120-kb region from Chr 4S highlighting H2B.V enrichment around tDNA loci (black arrow) and between conserved and disrupted genome compartments (rectangle with interrupted lines). Wig files from the ChIP H2B.V/input and ChIP H3/input were obtained by COVERnant. Blue bar arrows indicate the transcription direction in each polycistron. Genes in the same polycistron are stained with the same color. Black and red bars represent respectively, tDNA loci and H2B.V -peaks (fold equal to 4 over the input, Poisson p-value over input required = 1.00e-04) obtained by HOMER in epimastigote form. The conserved and disrupted compartments are represented by green and orange bars, respectively. B. Heatmaps of multigenic family members showing ratios of ChIP H2B.V/input and ChIP H3/input in two epimastigote replicates. Note an enrichment (red) of H2B.V at MASP, mucin and TS genes. C. Summary plot obtained by deepTools of the normalized (ChIP/input) H2B.V reads at tDNA loci and the adjacent 2 kb upstream and downstream regions. The tDNA locus was automatically scaled as indicated (first (S) and last (E) nucleotides of each locus). Normalized data were classified into 3 hierarchical clusters (dark blue– 13 genes, light blue– 11 genes and yellow -31 genes) based on the H2B.V enrichment pattern.
Fig 3
Fig 3. H2B.V in vitro interactors.
A. Venn diagram showing the number of common and specific interactors of H2B.V and H2B using epimastigote and TCT extracts. The total number of eluted proteins in each pulldown is shown between parentheses. Some relevant proteins are highlighted. B. BDF2 pulldown assays. Recombinant BDF2 tagged with HA and 6xHis was incubated with NTA-Ni resins with or without H2B.V-myc-tagged parasite extracts (epimastigotes or TCTs). Eluates were fractionated by 15% SDS-PAGE, and protein interactors were revealed by WB with anti-HA (against rBDF2), anti-myc (against H2B. V-myc tagged), and anti-H3 (as a chromatin control). C. H2A.Z interacts with H2B.V in vitro. H2A.Z-MBP and H2B.V-6xHis recombinants were incubated together or separately with amylose resin, known to specifically interact with MBP. Eluates were fractionated by 15% SDS-PAGE and stained with Coomassie. H2A.Z-MBP is approximately 60 kDa, and H2B.V-6xHis is 16.5 kDa. The * indicates a contaminant of lysed extraction.
Fig 4
Fig 4. Phenotypic evaluation of H2B.V-HtzKO parasites.
A. H2B.V relative expression levels were evaluated by qPCR in wild-type and H2B.V-HtzKO (cl 2 and 4) parasites. H2B.V transcript levels were assessed relative to GAPDH levels (ΔCtH2BV–ΔCtGAPDH). One-way ANOVA: * p-value < 0.05. B. Mean growth curves (in log10) for wild-type and H2B.V-HtzKO (cl 2 and 4) parasites. C. Metacyclogenesis assay. The number of metacyclic trypomastigotes, epimastigotes and intermediate forms (herein, classified as other) were counted in 96-h supernatant upon nutritional stress as described in the Materials and Methods. Values were normalized to their respective numbers in wild-type cells. Experiments were performed in biological triplicates. Unpaired T-test: *p < 0.05 and ***p < 0.01 with Bonferroni correction. D. H2B.V-HtzKO parasites are more infective. LLCMK2 cells were infected with TCTs (1:40; cells:parasite). Slides were fixed, and nuclei were labeled with DAPI. The number of infected cells was counted relative to the total cells after 1 h of infection by fluorescence microscopy. Unpaired t-test with Welch’s correction *** p < 0.0001.
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