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 Summary Trypanosomatids 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.


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BDF2 has already been shown to interact with histone H4 acetylated residues (K10 and K14),

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showing increased levels after exposure to UV in epimastigote forms and likely being involved 270 in the response to DNA damage [49].

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The H2B.V and BDF2 interaction was validated by reverse pulldown assays using 272 recombinant BDF2-6xHis-HA and WCE of H2B.V-myc-tagged parasites (epimastigotes and 273 TCTs) (Fig 3 B). In contrast to what was observed in H2B.V pulldowns, here the BDF2 interacted 274 with H2B.V in both life forms. In addition, the BDF2 interaction with H2B.V seemed to be 275 specific and not a spurious histone interaction, as histone H3 was not eluted from the BDF2 276 pulldown.

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It is worth mentioning that besides H2B.V and H2B, we only detected H4 peptides in our 278 pulldown assays. Here, the proteomics protocol was not optimized for histone identification, 279 which is often overdigested by trypsin due to high amounts of lysine and arginine residues. In T.

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Furthermore, in the 96-h supernatant after the induction of metacyclogenesis, almost 3 times more 337 trypomastigote metacyclics were found in H2B.V-HtzKO when compared to wild-type parasites 338 ( Fig 4C). This supernatant was also enriched in nonmetacyclic parasites (classified as "others", 339 which included epimastigote and intermediate forms), suggesting that changes in H2B.V levels 340 promoted loss of adhesion to substrates.

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We also evaluated whether the ability to infect mammalian cells would be affected in

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H2B.V-HtzKOs TCTs. Surprisingly, we found that cl2 and cl4 infected approximately twice as 343 many cells as wild type ( Fig 4D). Taken together, these data suggested that decreasing H2B.V 344 levels might interfere with parasite differentiation and infection of mammalian cells.

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Discussion 347 H2B has few variants described in eukaryotes, but trypanosomatids have a H2B.V that 348 has been shown to dimerize with H2A.Z [50] and to be predominantly located at dSSRs [12].

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Here, we investigated the genomic location, interaction partners, and phenotypic changes in single 350 knockout H2B.V parasites in both replicative (epimastigote) and nonreplicative (TCT) life forms.

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To our knowledge, our work is not just the first histone ChIP-seq analysis performed in T. cruzi 352 but also the first to compare, from a genome-wide perspective, the differences in replicative and 353 nonreplicative forms in trypanosomatids, shedding light on epigenetic changes in these 354 organisms, as discussed above.

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We found that H2B.V was preferentially located at T. cruzi dSSR edges extending ~5 kb 356 and included the first few CDSs at a polycistron. Importantly, H2B.V enrichment was also found 357 at a few non-SSRs, which were preferentially close to tDNAs and, strikingly, between the 358 disrupted and conserved genome compartments, with enrichment mainly in mucin and trans-359 sialidase genes. Moreover, the great majority of dSSRs flanking polycistrons containing protein-coding genes showed an enrichment of H2B.V. In contrast, dSSRs flanking monocistrons and 361 short polycistrons (less than 4 CDSs) did not contain a significant enrichment of H2B.V. Future 362 studies should clarify why these dSSRs are not enriched in H2B.V and whether its absence has 363 any transcriptional consequences for the flanked polycistron. Generation of H2B.V-HtzKO parasites. All primers sequences are described in Table S1. Cloning and recombinant protein expression. All primer sequences are described in Table   526 S1. The H2B gene (TcCLB.511635.10), BDF2 (TcCLB.506553.20) and H2A.Z

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HA_BDF2_Forward and BDF2_Reverse, and H2A.Z_Forward and H2A.Z_Reverse. BDF2 was 530 amplified by fusion with HA from the vector pDEST17, which was kindly provided by Dr.

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Esteban Serra. The PCR products were purified and cloned first at pJET1/2blunt (Invitrogen), and 532 after SANGER sequencing confirmation, the insert was subcloned either at pET28(a)+ (Novagen)

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DH5α and/or at BL21 DE3 for recombinant expression as described previously [35]. Briefly, E. Protein extracts. A total of 5 × 10 8 parasites were used to obtain basic protein extracts [32].

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Extracts were quantified using the PierceTM BCA Protein Assay Kit protocol (Thermo Scientific) 552 and stored at -20°C.

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Pulldown assays. The pulldown protocol was based on the principle of immobilization of iodoacetamide followed by digestion with trypsin (1:100, w:w) (Promega) as described in [80].