Histone proteins form nucleosomes that regulate packaging of DNA, thereby affecting DNA transcription, replication, and repair. In addition to the four canonical histones (H2A, H2B, H3 and H4), variant histones exist that can be substituted into the nucleosome, affecting its properties. Canonical and variant histones are also subject to a wide array of post-translational modifications (PTMs) that can alter the nucleosome or recruit chromatin remodeling machinery. The combination of PTMs and exchange of histone variants is believed to be important for gene regulation in the protozoan parasite Toxoplasma gondii, offering new targets for drug development [, , ].
T. gondii is a member of phylum Apicomplexa and infects between 10 and 90% of the population depending on the country , presumably influenced by dietary habits and environmental conditions . T. gondii converts from rapidly growing tachyzoites, which cause acute infection, to slow or non-growing bradyzoites, which cause chronic infection and typically reside in the brain . Although T. gondii infection is asymptomatic in healthy people, untreated clinical toxoplasmosis can be lethal in immunocompromised individuals such as transplant or HIV patients, and can produce severe disease during congenital infection, especially in early stages of development [7,8]. Chronic toxoplasmosis has also been linked to brain tumor predisposition, attention-deficit/hyperactivity disorder, obsessive-compulsive disorder and schizophrenia [, , , , ].
The T. gondii life cycle is comprised of sexual and asexual stages. While sexual replication takes place only in felids, T. gondii is able to replicate asexually as tachyzoites in any nucleated cell in any warm-blooded vertebrate. Following infection, tachyzoites convert into bradyzoites, which are housed in tissue cysts that are impervious to immunity and current drug treatments. Recently, a myb-like transcription factor (BFD1) was described to be necessary and sufficient to induce bradyzoite differentiation . How BFD1 is recruited to stage-specific promoters remains to be determined, but likely involves interplay with chromatin remodelers and epigenetic processes that include histone PTMs and/or variant histone exchange . In support of this idea, treatment of tachyzoites with inhibitors of histone deacetylase 3 (HDAC3) initiates bradyzoite differentiation . The roles of histone variants in T. gondii remain poorly understood.
We have previously shown that T. gondii expresses histone variants that mark functional regions of the genome . H2A.Z is a ubiquitous variant that has been implicated in both transcriptional activation and gene silencing in eukaryotes; it also contributes to the regulation of DNA damage repair [, , ], which is an important process in T. gondii tachyzoites . Unlike H2A.Z, most eukaryotes possess only specialized isoforms of H2B. By contrast, apicomplexan parasites [, , , ] and trypanosomatids  contain H2B-like variants called H2B.Z and H2Bv, respectively, but their functions remain poorly understood.
Histones H2A.Z and H2B.Z form a dimer that localizes with the transcriptional activation mark H3K4me3 in promoter/transcriptional start site (TSS) regions surrounding the nucleosome-free region upstream of the transcription start site . In addition, H2B.Z and H2A.Z localize to the gene bodies of silent genes, including repressed stage-specific genes, suggesting a role in the regulation of stage transitions. In T. gondii, both H2A.Z (TGGT1_300200) and H2B.Z (TGGT1_209910) are essential during the lytic cycle with CRISPR fitness scores of −5.08 and −4.05, respectively . By mass spectrometry, both histone variants were found to be hyperacetylated at the N-terminal domain, whereas few or no acetylation marks were identified on canonical H2A, H2B and H2A.X . T. gondii H2B.Z was shown to be acetylated at 5 lysine residues in its N-terminal tail. The H2A.Z N-terminal tail has 10 acetylatable lysines, in which lysine 18 can also be methylated . H2A.Z N-terminal tail acetylation has been widely shown to be a hallmark for active chromatin whereas N-terminal methylation is associated with gene silencing [, , , ], suggesting it is essential for changes in gene expression during cell differentiation [, , , , ]. While the role of N-terminal lysine acetylation in H2B.Z has not yet been studied, N-terminal acetylated canonical H2B is associated with some, but not all, active genes in vertebrates . Interestingly, H2A.Z in most eukaryotes only bears 4–5 acetylatable lysine residues in the N-terminus, while in protists like T. gondii, Plasmodium or Tetrahymena termophila, this number is considerably higher (between 5 and 16). Moreover, T. gondii could harbor double variant nucleosomes that collectively contain 15 acetylatable lysines that could be involved in regulating chromatin and DNA processes.
In the present work we studied the role of the five T. gondii H2B.Z N-terminal lysines using a mutagenesis strategy; we also pursued a gene knockout strategy for H2B.Z. In an RH strain background, we generated T. gondii that possess mutated versions of H2B.Z: c-Myc-R, in which the five N-terminal acetylatable lysines were replaced by arginines, and c-Myc-A, in which they were replaced by alanines. These lines were analyzed for their ability to grow in vitro, differentiate to bradyzoites in vitro and produce virulence in mice. In addition, expression of key genes was studied, as well as nucleosome composition and DNA damage sensitivity. Our results suggest that the aforementioned processes are regulated through the neutralization of the positive charge patch on the N-terminus of H2B.Z. We have also found some proteins that specifically interact with acetylated N-terminal H2B.Z peptide and not an unacetylated version, shedding new light on its role during the lytic cycle. The implications of these data on the putative role of H2B.Z are discussed.
Sequence alignment of Apicomplexa H2B.Z N-tail
H2B.Z is a variant histone of the H2B family that appears early in the phylum Apicomplexa. Previously, it was observed that T. gondii and Plasmodium falciparum H2B.Z N-terminal tails contain five acetylated lysines (K4, K9, K13, K14, and K18) [28,39,40]. The conservation of the five acetylatable lysines is clear from the alignment of H2B.Z N-terminal tail sequences from different Apicomplexa (Fig. 1A). This profuse number of modifications in the N-terminal tail, concomitant with the nucleosome
In T. gondii, the proper regulation of gene expression is crucial for progression through the cell cycle, lytic cycle, and stage differentiation [15,, , , , , ]. Chromatin remodeling is a major contributing factor in the regulation of gene expression and is mediated in part by histone variant exchange and histone PTMs. In addition to regulating chromatin, these processes affect chromosomal organization, DNA repair, and DNA replication . Apicomplexan parasites possess
Parasite culture and manipulation
RHΔhxgprt strain was used in all cases and grown in standard tachyzoite conditions in vitro: hTERT (ATCC® CRL-4001, USA) monolayers were infected with tachyzoites and incubated with Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 1% fetal bovine serum (FBS, Internegocios S.A., Argentina) and penicillin (10,000units/ml)-streptomycin (10mg/ml) solution (Gibco, Argentina) at 37°C and 5% CO2.
Cloning over-expression strategy
To generate the c-Myc-H2B.Z WT construct, the open reading frame (ORF) was
This work was supported by the Ministerio Nacional de Ciencia y Tecnología (MINCyT): PICT 2015 1288 (S.O.A.), PICT 2018 2434 (L.V.), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET): PIP 11220150100145CO and 11220210100572CO (S.O.A., L.V.) and by National Institutes of Health: NIH-NIAID 1R01AI129807 (S.O.A.) and AI152583 (to W.J.S.).
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CRediT authorship contribution statement
L.V. participated in all the experiments, analyses and design of the study and wrote the first draft; D.M. performed Crispr/Cas9 and some of the characterization experiments along with the pull-down experiments with L.V. supervision; C.C. performed the DNA damage experiments and some of the transfections; A.G. was involved in many of the experiments with technical support, and was in charge of RNA and cDNA preparation; R.N. performed RT-PCR experiments and ELISAs with LV supervision; M.C.B.
Declaration of competing interest
The authors declare that there are no conflict of interest.
L. Vanagas (Researcher), D. Muñoz (Fellow), C. Cristaldi (Fellow), D. Ganuza (Technician), V. Turowski (Researcher), and S.O. Angel (Researcher) are members of CONICET. S.O. Angel (Full) and L. Vanagas (Adjunct) are Professors at Universidad Nacional General de San Martin (UNSAM). The Vermont Biomedical Research Network (VBRN) Proteomics Facility is supported through NIH grant P20GM103449 from the INBRE Program of the National Institute of General Medical Science (BD).
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