**5. H2A.Z-H2B.Z: the double variant nucleosome**

H2A family also has a canonical H2A and several variants: H2A.Z, H2A.X, both exchangeable by H2A.Z-H2B or H2A.X-H2B, allowing the modulation of gene transcription, DNA replication, and/or DNA damage repair [58, 59]. In vertebrates, the H2A family has two more variants: H2Abd and macro-H2A. When talking about H2A-H2B and the incorporation of variants into such nucleosomes, there are vast differences if we take a glance at Apicomplexan parasites compared to most other eukaryotes. One of the most surprising discoveries in these parasites was the presence of a novel H2B variant (formerly named H2Bv, but recently reclassified as H2B.Z [60]), a histone, which is known to be deficient in variants, similar to H4 [58, 61]. Variants of this histone family, though, are not only found in these parasites, but also in *Trypanosomatids* (even though they are not evolutionary related), and some rare testis-specific variants in human and other mammalian species (reviewed in [55]).

lower abundance of histone modifications associated with heterochromatin [53]. However, it has been shown that the epigenome in *P. falciparum* is highly dynamic, and dependent on the stage, and, for example, H3K4me3 and H3K9ac are cycle regulated at *P. falciparum* genes [49]. This could also probably be true for *T. gondii*, where the tachyzoite to bradyzoite conversion is regulated at an epigenetic level. In this sense, it has been speculated that the H3R17me2 mark may have significance during the tachyzoite to bradyzoite differentiation process, as it was found only restricted to a subset of promoters, and taking into account the importance of arginine methylation during early development of mouse embryo [54]. In this study, using ChIP-on-chip technique, they found that H3K9ac, H4ac, and H3K4me3 modifications colocalize at focused loci in the *T. gondii* genome and correlate with significant gene expression, while the H3K4me1 and the H3K4me2 modifications were found at equal amounts in active

CenH3, the centromere-specific H3, has been observed in animals, fungi, and plants [24] and also in Apicomplexa, including *T. gondii*, *Plasmodium spp.*, and *N. caninum* [55]. This fact was recently confirmed by Fraschka et al. [42] who found the centromeres depleted of PfH3.3 and PfH3, but occupied by PfCenH3. In *T. gondii*, this histone variant was characterized with the aim to understand the way in which chromosomes are delivered to the daughter cells after mitosis, a process that is still intriguing [21]. In this work, the authors labeled all the histone H3 variants, and used TgCenH3 as a marker of centromeres, to perform ChIP and microarray assays [21]. They found a particular combination of histone PTMs surrounding centromeres; this region had a huge concentration of H3K9 di- and trimethylation, marks usually associated to heterochromatin and found in subtelomeric regions in *P. falciparum* but not in *T. gondii.* In this parasite, these modifications concentrate in two peaks directly flanking the center of the centromere in each chromosome, while H3K4me3 or H3K9ac are not present [21, 52]. In contrast, H3K9me3 and heterochromatin protein 1 (HP1, chromodomain protein that binds to H3K9me3) were not associated with centromeres in *P. falciparum* [23], but rather found in islands of the genome that contain transcriptionally silent members of multigene families [56]. In this parasite, the enrichment of PfCenH3 on centromeres of all the chromosomes has also been demonstrated by genome-wide ChIP-seq analysis [23]. Besides, it has been characterized that a region within the carboxy-terminal histone fold domain, which is also named CENP-A targeting domain (CATD), is essential for mediating centromere targeting, while the

H2A family also has a canonical H2A and several variants: H2A.Z, H2A.X, both exchangeable by H2A.Z-H2B or H2A.X-H2B, allowing the modulation of gene transcription, DNA replication, and/or DNA damage repair [58, 59]. In vertebrates, the H2A family has two more

and inactive chromatin [54].

**4. Centromeric H3**

102 Chromatin and Epigenetics

N-terminus is not [57].

**5. H2A.Z-H2B.Z: the double variant nucleosome**

Different studies performed in *Toxoplasma* have shown a nucleosome composition in which H2A.Z, but not H2A.X, dimerizes with H2B.Z, while H2A.X dimerizes with canonical H2B (H2Ba in *T. gondii*), but never with H2B.Z [62, 63]. This fact is also seen in *P. falciparum*, although this parasite lacks H2A.X variant [41] and has driven the hypothesis of a new double variant nucleosome exclusive of parasites with particular characteristics that will be described in this section [64, 65] (**Figure 2A**). As it can be observed in the sequence alignment of H2B.Z in many Apicomplexan species, this histone variant is quite conserved (**Figure 2B**), suggesting that this histone, and likely the double variant nucleosome H2A.Z-H2B.Z, may have had an important role in the expansion of the phylum.

Since H2B.Z is not represented in yeast, insects, or mammals, almost all the current knowledge about the double-variant nucleosome relies on H2A.Z studies. H2A.Z is so widespread that has been catalogued as "universal" because of its origin before the divergence of eukaryotes [66]. The first observation that appears is the hyperacetylation of its N-terminal tail in most species [48–50, 67–69]. It is thought that this possibility gives H2A.Z the faculty of mediating responsiveness to the environmental changes, with so varied and seemingly contradictory effects as gene activation, heterochromatic silencing, transcriptional memory, and others, depending on the binding of activating or repressive complexes [66]. H2A.Z containing nucleosomes mark active and bivalent promoters as well as enhancers, correlating with open chromatin [70, 71]. However, acetylation of H2A.Z is necessary for gene induction and is most often associated with active gene transcription [67, 68, 70, 71], whereas ubiquitylation, which can occur at the C-terminal tail, is linked to transcriptional repression and polycomb silencing [72–75]. Acetylated H2A.Z composes nucleosomes flanking the nucleosome-depleted regions [76]. Regulation of gene expression by acetylation of H2A.Z histone tail may be a result of the participation of other proteins as "readers" in the histone code; for example, the SWR-C chromatin remodeling enzyme and related INO80 family are well characterized to catalyze chromatin incorporation of the histone variant from yeast to human, and the acetylation of histone H3 on lysine 56 (H3-K56Ac) was said to lead to promiscuous dimer exchange in which either H2A.Z or H2A can be exchanged from nucleosomes, although this is in discussion [77–82]. NuA4 acetylation activity, which is homologous to the TIP60/p400 complex, was found to be associated with SWR1-driven incorporation of H2A.Z into chromatin [83]. Besides, bromodomaincontaining proteins are known to be implicated in "reading" the acetylation patterns of H2A.Z: acetylated lysines in histones, and other proteins are recognized by this motif, common in remodelers [77, 78, 84, 85]. In fact, for SWR1, bromodomains have been studied to recognize a pattern of acetylation (including H3K14ac), which may influence the deposition of H2A.Z-H2B variant dimers into the appropriate nucleosome [77, 78]. By using *Tetrahymena* as a model, it could be observed that these protozoa cannot survive with all acetylatable lysines replaced by arginines, indicating that H2A.Z acetylation modulates a charge patch with an essential function in chromatin regulation [69, 75]. Unlike the histone code, these changes need not to be site-specific. If this hypothesis is true, modulation of the charge at any one of a number of clustered sites could inhibit nucleosome condensation, facilitating transcription [86].

as a similar physical switch to control gene expression in response to temperature change (for example, during fever or as *P. falciparum* is transmitted between its two hosts), as a thermosensory response that was seen in *Arabidopsis thaliana* and yeast [96]. This could be due to reduced DNA wrapping of H2A.Z containing nucleosomes at higher temperatures, resulting in a relaxed chromatin structure, although this variant histone has also been associated with a tighter relationship with DNA, especially in heterotypic H2A.Z/H2A nucleosomes [97]. While heterochromatic intergenic regions showed to contain low levels of histone variant H2B.Z [64], it is interesting that double-variant nucleosomes are depleted from silent *var* gene promoters but not from silent promoters of heterochromatic invasion gene families, which have similar patterns of variegated expression [65]. Besides, this correlation between double-variant nucleosome presence and expression was only seen in *var* genes, while this nucleosome was also found enriched in intergenic regions across the genome, associated with euchromatic histone modifications and not necessarily associated with transcription [64, 65, 86]. Moreover, long promoter containing intergenic regions that maintain higher variant histone levels as compared with 3'UTR containing regions, which are considerably shorter, presents higher AT content, so this correlation could simply be due to the minimal length of the AT-rich content in these short 3'UTR regions [64]. As it was previously observed for H3.3 variant histone (see Section 3), a correlation between nucleosome occupancy and GC/ AT content in the genome was observed, although contrary to H3.3 that was correlated with rich GC regions [42], here both H2B.Z and H2A.Z histone variant occupancy displayed a clear positive correlation toward genomic AT content [64]. In **Figure 2A**, a schematic representation

Apicomplexa and Histone Variants: What's New? http://dx.doi.org/10.5772/intechopen.81409 105

The telomere-associated sequences (TAS), also named subtelomeres, are heterochromatic regions adjacent to the telomeric-end looking toward the centromere. The telomeres and the TAS regions are the final structures at the chromosomes and integrate with the centromere the constitutive heterochromatin in the genome. These TAS regions have been described in *Plasmodium* and *Toxoplasma* with a size of 20–40 and near 30 Kpb, respectively (**Figure 3**) [98– 100]. In *T. gondii*, the structure contains three tandem repeated elements (TARE), separated by noncoding DNA and flanked at one end by the telomere and at the other, downstream TARE 3, by a *Toxoplasma*-specific gene family, the *tsf* gene, of unknown function [100]. In general, there is only one *tsf* gene per TAS. Interestingly, based on predicted amino acidic sequence, TSF proteins present a high degree of conservation in the N-tail and middle regions while being highly variable at the C-terminal end. Up to now, only few studies were per-

The TAS element in *Plasmodium*, instead, has been deeply studied because of the presence of different families of genes associated to virulence and pathogenicity with a clonal pattern of expression [101, 102]. Telomeres are spatially restricted to nuclear periphery, where they form clusters of three to seven heterologous chromosome ends [103–105]. *Plasmodium* TAS is composed of six different TAREs, and the coding part of the genome is localized directly

of *P. falciparum* and *T. gondii* nucleosome occupancy is proposed.

**6. Heterochromatin, telomeres, and subtelomeres**

formed on chromatin modulation of *T. gondii* TAS.

*T. gondii* H2A.Z, together with H2B.Z, was enriched in the promoters of active genes in tachyzoites, while repressed genes were enriched with H2A.X-H2Ba nucleosomes [63] (**Figure 2A**). In addition, H2A.Z-H2B.Z was also recruited within the coding region of silent bradyzoitespecific genes and within promoter regions but not coding regions of actively expressed genes [87]. It is tempting to speculate that the enrichment at active promoters or poised regions could be ruled by different PTM stages of these histone variants. In agreement with this, H2A.Z and H2B.Z have shown to be highly acetylated at the amino-terminal tail, in contrast to canonical H2A and H2B histones and the H2A.X variant [51]. Considering that H2A.Z has shown to be essential in regulating the changing gene expression program during differentiation [79–81, 88–90], and recently, it was observed that overexpression of mutated version of H2A.Z, where all five potential acetylatable lysines on H2A.Z-GFP (K4, 7, 11, 13, and 15) were mutated to arginines, blocked myoblast differentiation through disruption of myoD expression [91], it may be that the H2B variant is involved in the *T. gondii* cell differentiation process as part of H2A.Z-H2B.Z nucleosome. Whether through a patch charge modulation and/or histone code remains an open question, considering that *T. gondii* presents several bromodomain-containing proteins that can recognize some of the acetylated lysine [92].

As stated above, the sequence alignment of H2B.Z in many Apicomplexan species reveals a high degree of conservation for this histone variant (**Figure 2B**). Interestingly, every lysine that has been proved to be acetylated in *T. gondii* and *P. falciparum*, H2B.Z was detected in the other Apicomplexan species, which is also true for H2A.Z [50, 53]. Maybe, the double-variant nucleosome is present in the phylum with same PTMs and similar biological role.

#### **5.1. Double-variant nucleosome in var genes**

In *P. falciparum*, H2A.Z-containing nucleosomes were proposed to demarcate intergenic/regulatory regions of the genome, serving as a scaffold for stage specific as well as transcriptioncoupled recruitment of histone modifying enzymes [93]. H3K9ac and H3K4me3 were found preferentially placed/retained on or next to H2A.Z-containing nucleosomes [49]. However, it was observed that *P. falciparum* intergenic regions, including promoters, display a global nucleosome depletion, while telomeres harbored the highest nucleosomal occupancy, except for the *var* gene with the highest expression level, which again showed the lowest nucleosomal occupancy [94]. Apparently, the little amount of nucleosomes in these areas is composed largely of variant nucleosomes. Petter et al. [95] also showed an enrichment of PfH2A.Z in the promoter of a set of developmentally regulated genes in the euchromatin compartment, although not correlated with transcription levels nor with acetylation status. *P. falciparum* H2A.Z-H2B.Z promoter occupancy in *var* genes was found to be strongly associated with transcriptional activity, whereas silent or poised *var* genes would be depleted of doublevariant nucleosome (see **Figure 3**) [65, 86]. The authors have speculated that it may function as a similar physical switch to control gene expression in response to temperature change (for example, during fever or as *P. falciparum* is transmitted between its two hosts), as a thermosensory response that was seen in *Arabidopsis thaliana* and yeast [96]. This could be due to reduced DNA wrapping of H2A.Z containing nucleosomes at higher temperatures, resulting in a relaxed chromatin structure, although this variant histone has also been associated with a tighter relationship with DNA, especially in heterotypic H2A.Z/H2A nucleosomes [97]. While heterochromatic intergenic regions showed to contain low levels of histone variant H2B.Z [64], it is interesting that double-variant nucleosomes are depleted from silent *var* gene promoters but not from silent promoters of heterochromatic invasion gene families, which have similar patterns of variegated expression [65]. Besides, this correlation between double-variant nucleosome presence and expression was only seen in *var* genes, while this nucleosome was also found enriched in intergenic regions across the genome, associated with euchromatic histone modifications and not necessarily associated with transcription [64, 65, 86]. Moreover, long promoter containing intergenic regions that maintain higher variant histone levels as compared with 3'UTR containing regions, which are considerably shorter, presents higher AT content, so this correlation could simply be due to the minimal length of the AT-rich content in these short 3'UTR regions [64]. As it was previously observed for H3.3 variant histone (see Section 3), a correlation between nucleosome occupancy and GC/ AT content in the genome was observed, although contrary to H3.3 that was correlated with rich GC regions [42], here both H2B.Z and H2A.Z histone variant occupancy displayed a clear positive correlation toward genomic AT content [64]. In **Figure 2A**, a schematic representation of *P. falciparum* and *T. gondii* nucleosome occupancy is proposed.
