**2. Genome and nucleus**

Both *Plasmodium* protozoan parasites and *T. gondii* present a highly complex life cycle, involving several stages along the cycle (**Figure 1**). The genome sizes are 23.3 Mb for *Plasmodium* and 80 Mb for *Toxoplasma*, being haploid (1 N) almost all their life cycle but diploid during sexual replicative stages (2 N) (**Figure 1**). *Plasmodium* genus and *T. gondii* were the first apicomplexan parasites to be included in genome projects [9, 10]. Since then, several other apicomplexan parasites genome projects were taken forward and the data uploaded at EuPathDB (https:// eupathdb.org/eupathdb/). From these databases, it could be observed that *T. gondii* and *P. falciparum* present about 5300 (https://protists.ensembl.org/Plasmodium\_falciparum/Info/Index) and 8172 (http://protists.ensembl.org/Toxoplasma\_gondii/Info/Annotation/#assembly) gene transcripts, respectively, organized as single copy genes along the chromosomes, and in general, they are not clustered by function, pathway, or stage of expression. All of these data suggest that these genes are finely regulated throughout the cell cycle and the life cycle by transcriptional regulators and chromatin. Regarding transcription factors, they are poorly represented in apicomplexan, with only one large family of transcriptional regulators, with 24 AP2 family factors in *P. falciparum* and 68 in *T. gondii* [11, 12]. In this context, epigenetic

control was proposed as a key element to facilitate parasite gene expression, DNA replication,

of chromosomes and nuclear envelope interaction. IMC1: inner membrane complex 1.

**Figure 1.** (A) Life cycles of T. gondii and P. falciparum. Haploid and diploid DNA content is referred to as 1 and 2 N. Black arrows and letters represent common parasite stages, blue arrows and letters represent T. gondii specific stages, and red arrows and letters represent P. falciparum-specific stages. (B) Epichromatin labeling in T. gondii. Epichromatin is a conformational epitope formed by DNA and histones H2A and H2B localized only at the exterior chromatin surface. During tachyzoite replication, the 2 N nucleus is divided, entering one genome to each budding cell, forming a typical U shape. The nuclear envelope does not disappear, and chromosomes do not present high level of condensation. Epichromatin labels predominantly one side of the nucleus, suggesting a nonhomogeneous organization

Apicomplexa and Histone Variants: What's New? http://dx.doi.org/10.5772/intechopen.81409 97

An interesting aspect of apicomplexan parasites is that they never lose the nuclear envelope during cell division, and their chromosomes do not present the higher order level of condensation observed in metaphase chromosomes of higher eukaryotes [16]. So, the nucleus presents

and DNA repair [13–15].

as Malaria, a mosquito-borne infectious disease endemic in the tropical and subtropical zones of Asia, Africa, South, and Central America. Malaria also constitutes a serious problem for travelers as well as for people working in endemic regions. In 2016, an infection rate of 216 million cases was reported, causing some 445,000 deaths globally. Data show a stalling in declining burden of *Plasmodium* observed over the last decade (http://apps.who.int/iris/bits tream/10665/259492/1/9789241565523-eng.pdf?ua=1). In addition, the mass drug administration program has shown a limit due to the spreading of multidrug-resistant malaria [2]. The severe disease is mainly caused by *P. falciparum,* whereas *P. vivax* causes the majority of malaria morbidity outside Africa. The clinical manifestations of malaria include fever, shivering, arthralgia (joint pain), vomiting, jaundice, hemoglobinuria, convulsions, metabolic alterations, renal failure, liver and lung dysfunctions, anemia, and cerebral malaria (coma).

*Toxoplasma gondii* is the only one species of the *Toxoplasma* genus, and it is able to infect birds and mammals, including human, and cause toxoplasmosis. The infection occurs worldwide and the chronic stage reaches more than 500 million people [3]. During the first few weeks of infection, toxoplasmosis is either asymptomatic or causes a mild flu-like illness. However, those with a weakened immune system, such as AIDS patients, infected fetus during gestation or newborns with a congenital infection, may become seriously ill, and occasionally die. The parasite can cause encephalitis (inflammation of the brain) and neurologic diseases, and can affect the heart, liver, inner ears, and eyes (chorioretinitis). Recent research has also linked toxoplasmosis with neuropsychiatric symptoms such as attention-deficit hyperactivity disorder, obsessive compulsive disorder, bipolar disease, and schizophrenia [4–8]. The present chemotherapy for toxoplasmosis is efficient but, sometimes, it is not well tolerated by individuals with AIDS, and it is effective against the acute or active stage, but not against the

Both *Plasmodium* protozoan parasites and *T. gondii* present a highly complex life cycle, involving several stages along the cycle (**Figure 1**). The genome sizes are 23.3 Mb for *Plasmodium* and 80 Mb for *Toxoplasma*, being haploid (1 N) almost all their life cycle but diploid during sexual replicative stages (2 N) (**Figure 1**). *Plasmodium* genus and *T. gondii* were the first apicomplexan parasites to be included in genome projects [9, 10]. Since then, several other apicomplexan parasites genome projects were taken forward and the data uploaded at EuPathDB (https:// eupathdb.org/eupathdb/). From these databases, it could be observed that *T. gondii* and *P. falciparum* present about 5300 (https://protists.ensembl.org/Plasmodium\_falciparum/Info/Index) and 8172 (http://protists.ensembl.org/Toxoplasma\_gondii/Info/Annotation/#assembly) gene transcripts, respectively, organized as single copy genes along the chromosomes, and in general, they are not clustered by function, pathway, or stage of expression. All of these data suggest that these genes are finely regulated throughout the cell cycle and the life cycle by transcriptional regulators and chromatin. Regarding transcription factors, they are poorly represented in apicomplexan, with only one large family of transcriptional regulators, with 24 AP2 family factors in *P. falciparum* and 68 in *T. gondii* [11, 12]. In this context, epigenetic

chronic/latent stage.

96 Chromatin and Epigenetics

**2. Genome and nucleus**

**Figure 1.** (A) Life cycles of T. gondii and P. falciparum. Haploid and diploid DNA content is referred to as 1 and 2 N. Black arrows and letters represent common parasite stages, blue arrows and letters represent T. gondii specific stages, and red arrows and letters represent P. falciparum-specific stages. (B) Epichromatin labeling in T. gondii. Epichromatin is a conformational epitope formed by DNA and histones H2A and H2B localized only at the exterior chromatin surface. During tachyzoite replication, the 2 N nucleus is divided, entering one genome to each budding cell, forming a typical U shape. The nuclear envelope does not disappear, and chromosomes do not present high level of condensation. Epichromatin labels predominantly one side of the nucleus, suggesting a nonhomogeneous organization of chromosomes and nuclear envelope interaction. IMC1: inner membrane complex 1.

control was proposed as a key element to facilitate parasite gene expression, DNA replication, and DNA repair [13–15].

An interesting aspect of apicomplexan parasites is that they never lose the nuclear envelope during cell division, and their chromosomes do not present the higher order level of condensation observed in metaphase chromosomes of higher eukaryotes [16]. So, the nucleus presents 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 toward the cytoplasm of the incipient merozoite [20].

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

Apicomplexa and Histone Variants: What's New? http://dx.doi.org/10.5772/intechopen.81409 99

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

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

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

at position 54–55 in H3.3 [40].

*var* gene family members in any given parasite.

active one (see **Figure 3**) [47].

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