Histone variant H2B.Z acetylation is necessary for maintenance of Toxoplasma gondii biological fitness (2023)

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 [[1], [2], [3]].

T. gondii is a member of phylum Apicomplexa and infects between 10 and 90% of the population depending on the country [4], presumably influenced by dietary habits and environmental conditions [5]. 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 [6]. 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 [[9], [10], [11], [12], [13]].

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 [14]. 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 [15]. In support of this idea, treatment of tachyzoites with inhibitors of histone deacetylase 3 (HDAC3) initiates bradyzoite differentiation [16]. 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 [17]. 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 [[18], [19], [20]], which is an important process in T. gondii tachyzoites [21]. Unlike H2A.Z, most eukaryotes possess only specialized isoforms of H2B. By contrast, apicomplexan parasites [[22], [23], [24], [25]] and trypanosomatids [26] 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 [17]. 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 [27]. 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 [28]. 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 [28]. 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 [[29], [30], [31], [32]], suggesting it is essential for changes in gene expression during cell differentiation [[33], [34], [35], [36], [37]]. 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 [38]. 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.