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

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

*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-contain-

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

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

nucleosome is present in the phylum with same PTMs and similar biological role.

clustered sites could inhibit nucleosome condensation, facilitating transcription [86].

ing proteins that can recognize some of the acetylated lysine [92].

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

104 Chromatin and Epigenetics

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

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 downstream TARE 6, and is characterized by members of multiple antigen gene families including *var*, *rif*, *stevor*, and *pfmc-2tm* genes [94, 95].

chromosome loop formation. In addition, an association between PfSir2 and H3K9me3 was found, since the lack of the sirtuin deacetylases causes changes in H3K9me3 localization at the chromosome and generates disruption of the monoallelic transcription of *var* genes, suggesting the existence of perinuclear repressive centers associated with control of expression of

Flueck et al. [120] described the presence of an ApiAP2 family member in *P. falciparum*, designated as SIP2 that binds to TARE-2 and TARE-3 regions and the upstream regions of *var* upsB *in vivo*. Immunofluorescence and genome-wide high-resolution ChIP analyses demonstrated that *P. falciparum* SIP2 and HP1 proteins co-localize and associate with the same subtelomeric region, suggesting that both proteins participate in the assembly of telomeric heterochromatin. A recent report from Gupta et al. [121] has demonstrated that the protein CAF-1, a chaperone that loads the H3-H4 to the nucleosome assembly after DDR, co-localizes with PfHP1

**Yeast Mammals** *T. gondii* **(ToxoDB number)** *P. falciparum* **(PlasmoDB PF3D7** 

Sir2 (P06700) Sir2 (Q8IXJ6) Sir2A (227020) Sir2A (1328800)

NF HP1 (Q13185) Chromo1 (268280) HP1 (1220900)

Stn1 (P38960) NF NF NF Ten1 (Q07921) NF NF NF Cdc13 (P32797) NF NF NF NF TPP1 (Q96AP0) NF NF NF POT1 (Q9NUX5) NF NF Pif1 (P07271) Pif1 (Q9H611) NF NF

NF: Not found.\*

**Table 1.** Telomeric proteins.

ToxoDB with PfOrc1 amino acidic sequence by BlastP.

(283900)\*

Sir3 (P06701) Sir3 (Q9NTG7) ATPase, AAA family protein

Sir4 (P11978) Sir4 (Q9Y6E7) NF NF RAP1 (P11938) RAP1 (Q9NYB0) NF NF RIF1 (P29539) RIF1 (Q5UIP0) NF NF RIF2 (Q06208) NF NF NF Ku70 (P32807) XRCC6 (B1AHC9) Ku70 (248160) NF Ku80 (Q04437) XRCC5 (P13010) Ku80 (312510) NF Taz1 (P79005) TRF1 (P54274) NF NF NF TRF2 (Q15554) NF NF NF TIN2 (Q9BSI4) NF NF

**number)**

Apicomplexa and Histone Variants: What's New? http://dx.doi.org/10.5772/intechopen.81409 107

Orc1/Sir3 like activity (1203000)\*

Sir2B (267360) Sir2B (1451400)

P. falciparum Orc1 complement yeast Sir3 activity [113]. T. gondii counterpart was detected by searching

malaria parasite genes involved in phenotypic variation and pathogenesis [119].

The telomeres and TAS regions are dynamic structures associated to a plethora of specific factors that not only give it structure, but also configures all the regions as constitutive heterochromatin that participates in an epigenetic way to regulate subtelomeric genes expression (**Figure 3**). This epigenetic mechanism is carried out by proteins that introduce, recognize, and implement a repressive state over the gene expression under normal environmental conditions. It has been reported that under nutritional or environmental stress, the repressed subtelomeric genes activate their expression in response to events promoting growth and survival [87, 106–109].

It is important to highlight that the *T. gondii* TAS regions show a nucleosome composition enriched in H2A.X and heterochromatin markers [100]. An *in silico* analysis using the *Plasmodium* and *Toxoplasma* databases reveals the presence of only some orthologs to the yeast and mammal's telomeric-subtelomeric proteins as TRF1-2, HP1, KU70/KU80, and Sir2 proteins (**Table 1**). But interestingly, the principal actor in this scenario would be the histone deacetylase type III -Sir2. This NAD+ deacetylase-dependent has also been implicated in different signaling pathways. *P. falciparum* has two Sir2 paralogues, Sir2A and B; with overlapping but distinct roles that regulate different subsets of *var* genes[110], binding reversibly with the promoter regions of silent but not active subtelomeric *var* genes [111]. PfSir2A is implicated in telomere length regulation [112]. In *T. gondii*, two deacetylases containing the Sir2-domain were identified: TgSir2A and TgSir2B, but their function has not been characterized yet. Another protein that had been described in *Plasmodium* is PfOrc1 (origin recognition complex 1), which together with Sir2 promotes the epigenetic silencing in *P. falciparum* TAS [113]. PfOrc1 has a role in DNA replication but also cooperates with Sir2 to coordinate the spreading of heterochromatin and regulation of *var* gene expression [114]. In general, Sir2 proteins act by removing acetyl groups in cytosolic targets and at the nuclear level at H3K9, K14 and K56, but it also was described to act on the histone mark H4K16 promoting the deposition of methyl groups on H4K20, H4K20me3 being a chromatin mark associated with heterochromatin [115]. Thus, Sir2 seems to play a very important role in linking signaling processes to gene expression and chromosome architecture.

Additionally, a member of the Alba protein family (PfAlba3) was demonstrated via ChIP assays to bind to telomeric and subtelomeric regions co-localizing with Sir2A in the periphery of the nucleus. PfAlba3 inhibits transcription *in vitro* by binding to DNA. PfSir2A was shown to interact with PfAlba3 deacetylating the lysine residue of N-terminal peptide of PfAlba3 specific for DNA binding [116] (**Figure 3**). In archaea, this interaction had been reported, in which Sir2 regulates silencing through deacetylation of the major archaeal chromatin protein Alba, highlighting an ancestrally conserved mechanism of gene regulation [117].

As stated above, heterochromatin protein 1 (HP1) is a very important protein that has been described to recognize the trimethylation on H3K9, a critical mark for the establishment, maintenance and silencing of centromeric and telomeric heterochromatic regions in various model organisms. In *P. falciparum*, it has been identified as PfHP1 [118] and the H3K9me3 mark was mainly associated with *var* genes at TAS regions, as said before [119]. Moreover, high levels of H3K9me3 correlate with genes localized to the nuclear periphery, implying chromosome loop formation. In addition, an association between PfSir2 and H3K9me3 was found, since the lack of the sirtuin deacetylases causes changes in H3K9me3 localization at the chromosome and generates disruption of the monoallelic transcription of *var* genes, suggesting the existence of perinuclear repressive centers associated with control of expression of malaria parasite genes involved in phenotypic variation and pathogenesis [119].

Flueck et al. [120] described the presence of an ApiAP2 family member in *P. falciparum*, designated as SIP2 that binds to TARE-2 and TARE-3 regions and the upstream regions of *var* upsB *in vivo*. Immunofluorescence and genome-wide high-resolution ChIP analyses demonstrated that *P. falciparum* SIP2 and HP1 proteins co-localize and associate with the same subtelomeric region, suggesting that both proteins participate in the assembly of telomeric heterochromatin. A recent report from Gupta et al. [121] has demonstrated that the protein CAF-1, a chaperone that loads the H3-H4 to the nucleosome assembly after DDR, co-localizes with PfHP1


NF: Not found.\* P. falciparum Orc1 complement yeast Sir3 activity [113]. T. gondii counterpart was detected by searching ToxoDB with PfOrc1 amino acidic sequence by BlastP.

**Table 1.** Telomeric proteins.

downstream TARE 6, and is characterized by members of multiple antigen gene families

The telomeres and TAS regions are dynamic structures associated to a plethora of specific factors that not only give it structure, but also configures all the regions as constitutive heterochromatin that participates in an epigenetic way to regulate subtelomeric genes expression (**Figure 3**). This epigenetic mechanism is carried out by proteins that introduce, recognize, and implement a repressive state over the gene expression under normal environmental conditions. It has been reported that under nutritional or environmental stress, the repressed subtelomeric genes activate their expression in response to events promoting growth and survival [87, 106–109].

It is important to highlight that the *T. gondii* TAS regions show a nucleosome composition enriched in H2A.X and heterochromatin markers [100]. An *in silico* analysis using the *Plasmodium* and *Toxoplasma* databases reveals the presence of only some orthologs to the yeast and mammal's telomeric-subtelomeric proteins as TRF1-2, HP1, KU70/KU80, and Sir2 proteins (**Table 1**). But interestingly, the principal actor in this scenario would be the histone deacetylase type III -Sir2. This NAD+ deacetylase-dependent has also been implicated in different signaling pathways. *P. falciparum* has two Sir2 paralogues, Sir2A and B; with overlapping but distinct roles that regulate different subsets of *var* genes[110], binding reversibly with the promoter regions of silent but not active subtelomeric *var* genes [111]. PfSir2A is implicated in telomere length regulation [112]. In *T. gondii*, two deacetylases containing the Sir2-domain were identified: TgSir2A and TgSir2B, but their function has not been characterized yet. Another protein that had been described in *Plasmodium* is PfOrc1 (origin recognition complex 1), which together with Sir2 promotes the epigenetic silencing in *P. falciparum* TAS [113]. PfOrc1 has a role in DNA replication but also cooperates with Sir2 to coordinate the spreading of heterochromatin and regulation of *var* gene expression [114]. In general, Sir2 proteins act by removing acetyl groups in cytosolic targets and at the nuclear level at H3K9, K14 and K56, but it also was described to act on the histone mark H4K16 promoting the deposition of methyl groups on H4K20, H4K20me3 being a chromatin mark associated with heterochromatin [115]. Thus, Sir2 seems to play a very important role in linking signaling

Additionally, a member of the Alba protein family (PfAlba3) was demonstrated via ChIP assays to bind to telomeric and subtelomeric regions co-localizing with Sir2A in the periphery of the nucleus. PfAlba3 inhibits transcription *in vitro* by binding to DNA. PfSir2A was shown to interact with PfAlba3 deacetylating the lysine residue of N-terminal peptide of PfAlba3 specific for DNA binding [116] (**Figure 3**). In archaea, this interaction had been reported, in which Sir2 regulates silencing through deacetylation of the major archaeal chromatin protein

As stated above, heterochromatin protein 1 (HP1) is a very important protein that has been described to recognize the trimethylation on H3K9, a critical mark for the establishment, maintenance and silencing of centromeric and telomeric heterochromatic regions in various model organisms. In *P. falciparum*, it has been identified as PfHP1 [118] and the H3K9me3 mark was mainly associated with *var* genes at TAS regions, as said before [119]. Moreover, high levels of H3K9me3 correlate with genes localized to the nuclear periphery, implying

Alba, highlighting an ancestrally conserved mechanism of gene regulation [117].

including *var*, *rif*, *stevor*, and *pfmc-2tm* genes [94, 95].

106 Chromatin and Epigenetics

processes to gene expression and chromosome architecture.

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 but also with PfAlba3 among others [121].

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

Apicomplexa and Histone Variants: What's New? http://dx.doi.org/10.5772/intechopen.81409 109

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

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

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

blot, but it was detected by tachyzoites overexpressing MYST-B HAT [140].

the connections with DNA repair remain to be elucidated.

KAP-1 protein at ToxoDB and PlasmoDB databases [151].

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 [62, 63, 100, 123].
