**7. Double-strand break repair: H2A.X and chromatin**

Cells are exposed to DNA lesions produced by exogenous (e.g., chemicals, UV-irradiation, and ionization) or endogenous factors (e.g., DNA replication stress, meiotic recombination). One of the most deleterious forms of DNA damage is the double-strand break (DSB) [124]. DSBs activate the signal transduction pathway to induce DNA damage checkpoints that delay cell cycle progression, which allows the cell to activate DNA repair mechanism [125]. The phosphorylation of SQE/DФ motif (where Ф represents a hydrophobic residue) on histone H2A.X (referred to as γH2A.X) is one of the earliest responses to DSB [126, 127]. H2A.X seems to be incorporated randomly in the genome of resting cells [128], whereas γH2A.X is clearly observed forming foci, labeling the DSB and replication fork sites, spreading along the chromosome up to 2 Mb from the damaged site. In addition, chromatin is subjected to several changes at damage sites playing an important role in regulating DNA repair [129]. DSB can be produced by various events, either external as ionizing and UV radiations or internal such as collapse of replication forks and transcription-associated damage, among others [130, 131]. DSB can be repaired by two main mechanisms: nonhomologous end joining (NHEJ) and homologous recombination repair (HRR); the first is an error-prone mechanism available along the cell cycle, and the second is an error-free mechanism active at S/G2 phases of cell cycle because of the requirement of sister chromatid as template [131–134]. Both mechanisms were described in *T. gondii* [14, 135], but *Plasmodium* genus is thought to rely only on HRR [136–138].

Before the election of NHEJ or HRR mechanism, DSB triggers a cascade of events that starts with Mre11-RAD50-Nbs1/Xrs2 (MRN in mammals and MRX in yeast) complex binding to the damaged site, which recruits and activates ATM kinase (**Figure 4A**) [139]. ATM is able to phosphorylate H2A.X at SQE motif as well as other DSB repair enzymes allowing the spreading of γH2A.X and a correct DNA damage response (DDR) at DSB site (**Figure 4A**). ATM kinase is present in *T. gondii* and *P. falciparum* [14]. In *T. gondii*, the MYST family lysine acetyltransferase TgMYST-B has shown to mediate DDR induced by methyl methanesulfonate (MMS) and to stimulate the ATM expression at gene level [140]. In addition to this finding, histone acetyltransferases (HATs) have a predominant role in DDR on the basis of chromatin modulation. Chromatin responds to DSB first by increasing the compaction stage by replacing H2A/H2A.X with the H2A.Z variant and by methylating H3K9 by suv39h1 methyltransferase, which is recruited after spreading the DDR response at both sides of DSB sites (**Figure 4B**) [141, 142]. The arrival of H3K9me3 allows its interaction with the HAT Tip60 and the acetylation of H4 on K16 together with the acetylation of ATM kinase, an important PTM for the activation of autophosphorylation and subsequent activation of ATM (**Figure 4**) [143, 144]. The H3K9me3 and H4K16ac marks were identified in *T. gondii* and *P. falciparum* by mass spectrometry analysis [48–51]. However, in the case of *T. gondii*, an acetylated residue was also detected in H3K9 in a more frequent fashion than H3K9me1,2,3, suggesting that chromatin is preferentially in an open state and that this lysine PTM can be regulated [51]. As it was stated before, H3K9me2/3 is also enriched in centromeres in *T. gondii* [21]. In addition, *T. gondii* H4K16ac was one of the most abundant PTMs found in the mass spectrometry analysis [51]. In the case of *P. falciparum*, the treatment with MMS has increased the level of H4K8ac and H4K16ac and reduction of H3K9ac [145]. Both, *T. gondii* and *P. falciparum* present H3K9me1,2,3 and H3K9ac in normal conditions suggesting a conserved mechanism of chromatin modulation [51]. The role of these histone marks on Apicomplexan histones and the connections with DNA repair remain to be elucidated.

at the same subtelomeric localization, in the nuclear periphery, and also demonstrated its binding to TARE1-3 and co-localization with H3K56ac, a signal of completion on chromatin reassembly after DDR [122]. Interestingly, immunoprecipitation with PfCAF1 followed by LC-MS/MS analysis demonstrated that this protein would be interacting not only with PfHP1

In *T. gondii*, an HP1 protein was identified as TgChromo1, linked to the sequestration of chromosomes at the nuclear periphery and the process of cell division of the parasite [22]. TgChromo1 has shown to localize at *T. gondii* telomeres but not subtelomeres. However, by that time, subtelomeric regions had not yet been described and, in some cases, the sequences in these regions were not correctly assembled. Also, the presence of H4K20me3 and H2A.X at some TARE sequences and a region near *tsf* gene, previously named TgIRE, was observed

Cells are exposed to DNA lesions produced by exogenous (e.g., chemicals, UV-irradiation, and ionization) or endogenous factors (e.g., DNA replication stress, meiotic recombination). One of the most deleterious forms of DNA damage is the double-strand break (DSB) [124]. DSBs activate the signal transduction pathway to induce DNA damage checkpoints that delay cell cycle progression, which allows the cell to activate DNA repair mechanism [125]. The phosphorylation of SQE/DФ motif (where Ф represents a hydrophobic residue) on histone H2A.X (referred to as γH2A.X) is one of the earliest responses to DSB [126, 127]. H2A.X seems to be incorporated randomly in the genome of resting cells [128], whereas γH2A.X is clearly observed forming foci, labeling the DSB and replication fork sites, spreading along the chromosome up to 2 Mb from the damaged site. In addition, chromatin is subjected to several changes at damage sites playing an important role in regulating DNA repair [129]. DSB can be produced by various events, either external as ionizing and UV radiations or internal such as collapse of replication forks and transcription-associated damage, among others [130, 131]. DSB can be repaired by two main mechanisms: nonhomologous end joining (NHEJ) and homologous recombination repair (HRR); the first is an error-prone mechanism available along the cell cycle, and the second is an error-free mechanism active at S/G2 phases of cell cycle because of the requirement of sister chromatid as template [131–134]. Both mechanisms were described in

*T. gondii* [14, 135], but *Plasmodium* genus is thought to rely only on HRR [136–138].

Before the election of NHEJ or HRR mechanism, DSB triggers a cascade of events that starts with Mre11-RAD50-Nbs1/Xrs2 (MRN in mammals and MRX in yeast) complex binding to the damaged site, which recruits and activates ATM kinase (**Figure 4A**) [139]. ATM is able to phosphorylate H2A.X at SQE motif as well as other DSB repair enzymes allowing the spreading of γH2A.X and a correct DNA damage response (DDR) at DSB site (**Figure 4A**). ATM kinase is present in *T. gondii* and *P. falciparum* [14]. In *T. gondii*, the MYST family lysine acetyltransferase TgMYST-B has shown to mediate DDR induced by methyl methanesulfonate (MMS) and to stimulate the ATM expression at gene level [140]. In addition to this

but also with PfAlba3 among others [121].

**7. Double-strand break repair: H2A.X and chromatin**

[62, 63, 100, 123].

108 Chromatin and Epigenetics

As mentioned above, γH2A.X spreading is a crucial step to initiate a correct DDR at DSB sites. In *T. gondii*, this PTM mark is accompanied by other DDR marks such as H3K9me2,3 and H4K16 in normal conditions of growth, opening the question whether DSBs are being produced during parasite replication [51, 135]. The *T. gondii* tachyzoite replicates at high rates, in a range of 5–9 hours [146]. So, a putative collapse of replication fork could be occurring in this stage. However, *T. gondii* ATM kinase could not be detected in normal conditions by Western blot, but it was detected by tachyzoites overexpressing MYST-B HAT [140].

The chromatin compaction that occurs early during DDR includes the remodeling of chromatin at DSB sites in which the H2A-H2B dimer is replaced by H2A.Z-H2B [142, 147]. This event is transient, allowing the recruitment of repressive kap-1(TRIM28)/HP1/suv39h1 complex that can be important to inhibit transcription. The presence of H2A-H2B dimer in the nucleosomal core particle produces a unique negatively charged region on the surface of the nucleosome, called the "acidic patch," which is extended in H2A.Z (**Figure 4B**) [148–150]. The acidic patch favors the binding of H4 N-tail, resulting in an increase in the interaction between nucleosomes and chromatin compaction [150]. Interestingly, this seems a necessary step to continue with a relaxed chromatin state, since this compaction and recruitment of kap-1(TRIM28)/HP1/ suv39h1 complex lead to methylation of H3K9 and phosphorylation of KAP-1 by ATM kinase, which in turn promote H4K16 acetylation by Tip60 and release kap-1(TRIM28)/HP1/suv39h1 (**Figure 4B**) (see [142]). *T. gondii* and *P. falciparum* have the novel H2A.Z-H2B.Z double-variant nucleosome (see Section 5). However, *T. gondii* and *P. falciparum* H2A-H2B and variants conserve the acidic patch (**Figure 4B**). To note, *T. gondii* and *P. falciparum* do not appear to have KAP-1 protein at ToxoDB and PlasmoDB databases [151].

In higher eukaryotes, another important PTM mark associated to DDR is ubiquitination by E3 ubiquitin ligases RNF168 and RNF8 at DSB site after γH2A.X and MDC1 protein foci

above, in *T. gondii* and *P. falciparum*, the mark H4K20me1,2,3 was found [48, 51, 53]. However, *T. gondii* and *P. falciparum* H2As did not contain H2AK15ub and lysine 13 ubiquitylation was not detected either [51]. In addition, *T. gondii* and *P. falciparum* did not show the presence of orthologs of BRCA1 and/or 53BP1, though *T. gondii* presents three different BRCT domain

Apicomplexa and Histone Variants: What's New? http://dx.doi.org/10.5772/intechopen.81409 111

*T. gondii* and *P. falciparum* conserve several histone marks present in chromatin-associated DDR to DBS, as well as histone variants—in the case of *T. gondii*, the DDR, well studied H2A.X, is present, whereas *Plasmodium* has only canonical H2A [55], involved in the recruitment of several factors that spread and choose the DDR pathway in higher eukaryotes. Although, *T. gondii* and *P. falciparum* lack some key DDR regulators such as KAP-1, 53BP1, BRCA1, MDC1, RNF168 and RNF8 [14], both parasites present the HRR mechanism of DNA repair, whereas NHEJ is present only in *T. gondii*. So, the modulation of both DDR pathways is still an intriguing issue.

In protozoan parasites, the modulation of chromatin seems to be a key biological process to regulate gene expression, pathogenicity and DNA repair, the latter probably associated to DNA replication, ergo, the cell cycle. In Apicomplexa, highly evident in *Plasmodium* genus, the TAS or subtelomeric regions play an important role in the control of a group of genes essential in parasite pathogenicity. This fact suggests that subtelomeres have not a trivial impact in the evolution of these organisms, and their structure can influence the features of the cell. How this genomic domain has evolved within the Apicomplexa phylum remains to be elucidated. *T. gondii*, in which to date a scenario of variant antigenicity was not detected, has shown a someway conserved structure with the presence of tandem repeated boxes and a gene family of unknown function (*tsf*). Different from *Plasmodium*, which variant antigenassociated gene is represented by hundreds of members, *T. gondii* has only one gene per TAS. However, the predicted protein sequences show conserved N-tail and middle regions, with highly variable C-terminal ends. We believe that the elucidation of the localization, role, and antigenic potential of these gene family proteins will be of high impact in our knowledge of this parasite. Also, it could be interesting to know if the members of this gene family show

a regulation of gene expression similar to *Plasmodium* variable antigen gene family.

In addition to the presence of PTM marks similar to other organisms but with currently lesswell characterized readers and erasers, Apicomplexa chromatin presents a double-variant nucleosome based on the new histone variant H2B.Z. If considering the partitioned knowledge in these parasites, specially *P. falciparum*, where H3.3 variant has been found in the same regions as this double-variant nucleosome, but in different studies, it would be possible that a triple-variant nucleosome exists in Apicomplexa. Since the presence of H2B.Z arose early in Apicomplexa evolution, it is expected that the double-variant nucleosome could have been important in the expansion of the phylum, maybe modulating chromatin structure during the execution of different biological processes. Interestingly, the genome-wide analyses seem to indicate that *Plasmodium* and *Toxoplasma* double-variant nucleosomes do not have the same behavior. In *T. gondii*, it is enriched in active and poised genes, whereas in *P. falciparum*, it is

containing proteins [14].

**8. Concluding remarks**

**Figure 4.** (A) Recognition of DSB and initial steps of DDR pathways. MRN complex and ATM kinase are recruited to a DSB. ATM phosphorylates several DDR proteins and checkpoint kinases. Phosphorylation by ATM allows the spreading of γH2A.X and DDR foci. RNF8 E3 ubiquitin ligase complexes ubiquitinate H2A and H1 histones to compact and generate histone marks, which will be read by other DDR factors such as MDC1 and E3 ubiquitin ligase RNF168. (B) Model of chromatin modulation after DSB. During PARylation, the p400 ATPase (NuA4-Tip60 complex) replaces H2A-H2B dimers by H2A.Z containing nucleosomes. Since T. gondii and P. falciparum present a double-variant nucleosome, we speculate that H2A-H2B or H2A.X-H2Ba dimers may be replaced by H2A.Z-H2B.Z dimers. This exchange might increase the interaction of the acidic patch with the N-tail of H4 increasing the chromatin compaction (see the sequence alignment: in red letters are written the acidic residues involved in generating the acidic patch). PARylation also produces an increase in H3K9me2/3 and histone deacetylation repressive marks. After a short time, the H2A.Z containing nucleosome is replaced by H2A-H2B dimer and N-tail of H4 is acetylated leading to a relaxed chromatin and recruitment of different DDR factors, among them those related to the DDR pathway choice, such as 53BP1 or BRCA1.

spreading (**Figure 4A**). MDC1 is also phosphorylated by ATM kinase allowing the recruitment of RNF168 and RNF8 [152]. Ubiquitination on H1 and H2A recruits several BRCT domain containing proteins such as BRCA1 and 53BP1 [129]. In the case of 53BP1, its binding requires the H2AK13/15ub and H4K20me2 and addresses the DDR to NHEJ pathway (**Figure 4B**). By contrast, the presence of H4K16ac impairs the 53BP1 binding to the nucleosome allowing the recruitment of BRCA, which addresses the DDR to HRR (**Figure 4B**) (see [141, 142]). As stated above, in *T. gondii* and *P. falciparum*, the mark H4K20me1,2,3 was found [48, 51, 53]. However, *T. gondii* and *P. falciparum* H2As did not contain H2AK15ub and lysine 13 ubiquitylation was not detected either [51]. In addition, *T. gondii* and *P. falciparum* did not show the presence of orthologs of BRCA1 and/or 53BP1, though *T. gondii* presents three different BRCT domain containing proteins [14].

*T. gondii* and *P. falciparum* conserve several histone marks present in chromatin-associated DDR to DBS, as well as histone variants—in the case of *T. gondii*, the DDR, well studied H2A.X, is present, whereas *Plasmodium* has only canonical H2A [55], involved in the recruitment of several factors that spread and choose the DDR pathway in higher eukaryotes. Although, *T. gondii* and *P. falciparum* lack some key DDR regulators such as KAP-1, 53BP1, BRCA1, MDC1, RNF168 and RNF8 [14], both parasites present the HRR mechanism of DNA repair, whereas NHEJ is present only in *T. gondii*. So, the modulation of both DDR pathways is still an intriguing issue.
