**3. H3 histones: a multivariant family**

H3 histone family presents canonical forms: H3, H3.1, H3.2 and variants: H3.3 and cenH3 [24]. H3.3 differs from canonical H3s in various aspects. Canonical H3s are expressed and associate to chromatin during the S-phase of cell cycle. Canonical H3s and H3.3 are highly identical differing in only four to five amino acids. The CAF-1 complex is involved in the incorporation of canonical H3s whereas CHD1/ATRX remodelers as well as HIRA chaperone complex are involved in the incorporation of H3.3 [25–30]. In addition, H3.3 is enriched in transcribed genes, enhancers, regulatory elements, and also heterochromatic repeats, including telomeres and pericentromeric regions [31–34]. In general, H3.3 is linked to gene activation or open chromatin. Moreover, it has been found to be methylated at K4, K36, and K79 and acetylated at K9 and K14, all being marks of active chromatin [32, 35]. H3.3 and H2A.Z were detected at active promoters generating nucleosomes that promote gene transcription [36–38]. Recently, it was found that H3.3 plays an essential biological role during mammal development since mice that lack H3.3 presented developmental retardation and early embryonic lethality [39]. Rather than gene expression troubles, H3.3 depletion causes genome instability due to dysfunction of heterochromatin structures at telomeres, centromeres, and pericentromeric regions of chromosomes, leading to mitotic defects.

There is little information regarding H3 histone family and the variants H3.3 in Apicomplexan parasites. The first approach is from W.J Sullivan [40] who was able to clone the entire ORFs encoding H3 and H3.3 in *Toxoplasma gondii* and also in *Plasmodium falciparum*. In this work, it was confirmed that, like in most other organisms, there is not much difference between the two variants: only four amino acids in *T. gondii* and eight between the *P. falciparum* variants. In most other species, the critical residues that differ between H3 and H3.3, resulting in different roles of these histones, are a motif, which contains SAVM in H3 canonical histone, but changes to AAIG in H3.3 [24]. However, while PfH3 has the typical SAVM motif, it changes to QAVL in PfH3.3, whereas in TgH3, the motif is SAVL and changes to QAIL in TgH3.3 [40]. Besides, there is another difference in Apicomplexa, which seems to be exclusive: KF changes for RY at position 54–55 in H3.3 [40].

the same aspect along the cell cycle. However, it seems to be not homogenous: *Toxoplasma gondii* nuclear envelope and chromosomes seem to have a dynamic relocalization and/or rotation inside the nucleus during parasite budding as observed by epichromatin localization (**Figure 1B**). Epichromatin is a conformational epitope formed by DNA and histones H2A and H2B localized only at the exterior chromatin surface [17, 18]. More recently, it was observed that epichromatin forms superbead domains associated to DNA-A at the nuclear envelope [19]. A 3D analysis also shows that *P. falciparum* nucleus presents a polarization of the nuclear pore complex: in the early multinucleated schizont, it clusters in the nucleus region facing the mother plasma membrane, whereas in the late stages, when prepared for budding, it clusters

In addition to putative polarization of the genome inside the nucleus of Apicomplexan parasites, in *T. gondii*, it was observed that the centromeres (CenH3, see below) are localized at a single spot at the apical region of the nucleus, indicating that all of them are attached to the centrocone, a structure associated to the nuclear envelope, which is traversed by microtubules coordinating the cell division [21]. Similarly, Chromo1, a *T. gondii* protein that binds to the telomere, presents a focalized localization in the nucleus, also suggesting a certain degree of chromosome organization within the parasite nucleus [22]. In *P. falciparum* prior to replication, in late ring stages and young trophozoites, CenH3 localizes to a single nuclear focus suggesting that centromeres are clustered in a single spot that most likely continues to be attached to the mitotic spindle until the end of schizogony and the intraerythrocytic develop-

H3 histone family presents canonical forms: H3, H3.1, H3.2 and variants: H3.3 and cenH3 [24]. H3.3 differs from canonical H3s in various aspects. Canonical H3s are expressed and associate to chromatin during the S-phase of cell cycle. Canonical H3s and H3.3 are highly identical differing in only four to five amino acids. The CAF-1 complex is involved in the incorporation of canonical H3s whereas CHD1/ATRX remodelers as well as HIRA chaperone complex are involved in the incorporation of H3.3 [25–30]. In addition, H3.3 is enriched in transcribed genes, enhancers, regulatory elements, and also heterochromatic repeats, including telomeres and pericentromeric regions [31–34]. In general, H3.3 is linked to gene activation or open chromatin. Moreover, it has been found to be methylated at K4, K36, and K79 and acetylated at K9 and K14, all being marks of active chromatin [32, 35]. H3.3 and H2A.Z were detected at active promoters generating nucleosomes that promote gene transcription [36–38]. Recently, it was found that H3.3 plays an essential biological role during mammal development since mice that lack H3.3 presented developmental retardation and early embryonic lethality [39]. Rather than gene expression troubles, H3.3 depletion causes genome instability due to dysfunction of heterochromatin structures at telomeres, centromeres, and pericentro-

There is little information regarding H3 histone family and the variants H3.3 in Apicomplexan parasites. The first approach is from W.J Sullivan [40] who was able to clone the entire ORFs

toward the cytoplasm of the incipient merozoite [20].

98 Chromatin and Epigenetics

mental cycle, similar to that observed in *T. gondii* [23].

meric regions of chromosomes, leading to mitotic defects.

**3. H3 histones: a multivariant family**

In *Plasmodium*, H3.3 had a similar expression pattern to another important histone variant, H2A.Z, namely localization to active chromatin [41] (see **Figure 2**). As observed in other eukaryotic cells, it has been recently demonstrated by ChiP-seq experiments that euchromatic regions in the genome are demarcated by the presence of the H3.3 variant histone [42]. However, in *P. falciparum*, there is a particular AT versus GC content along the genome with euchromatic intergenic regions richer in AT-content compared to coding sequences with less AT content [23]. Fraschka et al. [42] have seen a particular correlation between enrichment in PfH3.3 histone variant and GC content, with this variant mainly located not only in euchromatic GC-rich sequences, but also in subtelomeric GC-rich repetitive regions. Interestingly, this correlation with the nucleotide composition is also observed with the double-variant nucleosome H2A.Z-H2B.Z (see below), but in this case, it is just the contrary: the regions with more AT content show abundance of this nucleosome [42]. However, GC-poor intergenic regions show the lowest H3.3 coverage, but the authors still argue that the incorporation of this variant to coding regions is more dependent on GC content than transcriptional activity.

It is well documented that *P. falciparum* depends on the *var* multigene family, encoding for a highly variable cytoadherence protein called *P. falciparum* erythrocyte membrane protein 1 (PfEMP1) to avoid host immunity [43–46]. This is due to the expression of only one of the ~60 *var* gene family members in any given parasite.

Regarding this important gene family, H3.3 stably occupies the promoter region and coding sequence of the active *var* gene but is evidently less incorporated into the promoter and coding sequence of silenced *var* genes [42] (see **Figure 3**). Additionally, it has been demonstrated that the PTMs affecting histone H3 are extremely important in the regulation of *var* expression. Data from fluorescence *in situ* hybridization (FISH) suggest that the *P. falciparum* SETvs (*P. falciparum variant-silencing SET* gene), which encodes an ortholog of *Drosophila melanogaster* ASH1 and controls histone H3 lysine 36 trimethylation (H3K36me3) on *var* genes, is specifically involved in *var* gene silencing, and its *knock-out* results in the transcription of virtually all *var* genes in the single parasite nuclei [47]. Besides, ChIP-qPCR analysis showed that the TSS occupancy of H3K36me3 is considerably higher in the silent *var* genes compared to the active one (see **Figure 3**) [47].

A detailed mass spectrometry study has been accomplished for *P. falciparum* histone PTMs by Trelle et al. [48], and it has been established that lysines 4, 9, 14, 18, 23, and 27 of both H3 and H3.3 are capable of being modified by acetylations and/or methylations. Also, arginine in position 17 may be mono or bimethylated. Some of these modifications had already been

**Figure 2.** (A) Schematic model for double-variant nucleosome in T. gondii and P. falciparum. Hypothetical active (or poised) and silenced genes are represented with the nucleosome composition in each promoter and also downstream the genes. PTM marks are represented as well and listed below with the differences found in the literature between both the parasites. Specific HATs and HDACs are speculated to acetylate/deacetylate the unique H2B.Z histone variant. (B) Sequence alignment of histone variant H2B.Z in Apicomplexa. Pf: Plasmodium falciparum, Tg: Toxoplasma gondii, Et: Eimeria tenella, Vb: Vitrella brassicaformis, Bv: Babesia bovis, Cf: Cytauxzoon felis, Gn: Gregarina niphandrodes and Cp: Cryptosporidium parvum. All sequences can be found in EuPathDB (https://eupathdb.org/eupathdb/).

identified for H3 and H3.3 also by Miao et al. [41]. More recently, a lysine residue in the core of H3, K56, was also indicated as a site of acetylation [49, 50]. In the same way, *T. gondii* histone H3 has many lysines and also arginines capable of being modified: lysines in the positions 4, 9, 14, 23, 27 and also 36, 37, 56, 115 and 122 can be acetylated, methylated and besides some of them receive formylation, ubiquitination, or succinylation [51]. Besides, arginines 2, 17, 26, 40, and 83 can be methylated [51].

most histones, including H3 and H3.3 [52]. These marks are frequently seen in combination with neighboring lysine acetylation (and methylation). In this work, they also described a

**Figure 3.** Hypothetical telomeric and subtelomeric structure in T. gondii (upper panel) and P. falciparum (lower panel). Here, we show the TgTAS and PfTAS composition and the possible protein interactors listed in **Table 1** and described in Section 6. In P. falciparum, we only illustrate three of the six TAREs associated. The punctuated lines define proteins described in other organisms, but some of them present an ortholog in any of those parasites. The proteins represented in continuous lines have already been described for those parasites. Proteins illustrated in grey are unknown proteins. We also show the possible histone variant exchange on silenced or active *tsf* or *var* genes. Specific PTMs on silenced or active genes are described in the figure. The questions are open because these proteins and PTMs have not been confirmed yet

Apicomplexa and Histone Variants: What's New? http://dx.doi.org/10.5772/intechopen.81409 101

In parasites, among the most conserved modifications is histone 3 trimethylation of lysine 4 (H3K4me3), a marker of potentially active promoters. Opposed to that is H3K9 methylation, associated with silent genes and densely packed heterochromatin, although protozoan parasite histones are more highly enriched in the activation marks associated with euchromatin with

Pf14-3-3 as a phosphohistone mark binding protein.

in these TAS regions.

But not only acetylations and methylations are marking histones; with the development of improved acid and high-salt purification methods for *P. falciparum* histone phosphoprotein analysis, multiple phosphorylation sites have been found mostly at the N-terminal region of

**Figure 3.** Hypothetical telomeric and subtelomeric structure in T. gondii (upper panel) and P. falciparum (lower panel). Here, we show the TgTAS and PfTAS composition and the possible protein interactors listed in **Table 1** and described in Section 6. In P. falciparum, we only illustrate three of the six TAREs associated. The punctuated lines define proteins described in other organisms, but some of them present an ortholog in any of those parasites. The proteins represented in continuous lines have already been described for those parasites. Proteins illustrated in grey are unknown proteins. We also show the possible histone variant exchange on silenced or active *tsf* or *var* genes. Specific PTMs on silenced or active genes are described in the figure. The questions are open because these proteins and PTMs have not been confirmed yet in these TAS regions.

most histones, including H3 and H3.3 [52]. These marks are frequently seen in combination with neighboring lysine acetylation (and methylation). In this work, they also described a Pf14-3-3 as a phosphohistone mark binding protein.

identified for H3 and H3.3 also by Miao et al. [41]. More recently, a lysine residue in the core of H3, K56, was also indicated as a site of acetylation [49, 50]. In the same way, *T. gondii* histone H3 has many lysines and also arginines capable of being modified: lysines in the positions 4, 9, 14, 23, 27 and also 36, 37, 56, 115 and 122 can be acetylated, methylated and besides some of them receive formylation, ubiquitination, or succinylation [51]. Besides, arginines 2,

Cp: Cryptosporidium parvum. All sequences can be found in EuPathDB (https://eupathdb.org/eupathdb/).

**Figure 2.** (A) Schematic model for double-variant nucleosome in T. gondii and P. falciparum. Hypothetical active (or poised) and silenced genes are represented with the nucleosome composition in each promoter and also downstream the genes. PTM marks are represented as well and listed below with the differences found in the literature between both the parasites. Specific HATs and HDACs are speculated to acetylate/deacetylate the unique H2B.Z histone variant. (B) Sequence alignment of histone variant H2B.Z in Apicomplexa. Pf: Plasmodium falciparum, Tg: Toxoplasma gondii, Et: Eimeria tenella, Vb: Vitrella brassicaformis, Bv: Babesia bovis, Cf: Cytauxzoon felis, Gn: Gregarina niphandrodes and

But not only acetylations and methylations are marking histones; with the development of improved acid and high-salt purification methods for *P. falciparum* histone phosphoprotein analysis, multiple phosphorylation sites have been found mostly at the N-terminal region of

17, 26, 40, and 83 can be methylated [51].

100 Chromatin and Epigenetics

In parasites, among the most conserved modifications is histone 3 trimethylation of lysine 4 (H3K4me3), a marker of potentially active promoters. Opposed to that is H3K9 methylation, associated with silent genes and densely packed heterochromatin, although protozoan parasite histones are more highly enriched in the activation marks associated with euchromatin with 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 and inactive chromatin [54].

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

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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

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

variants in human and other mammalian species (reviewed in [55]).

an important role in the expansion of the phylum.
