**8. Concluding remarks**

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

DDR factors, among them those related to the DDR pathway choice, such as 53BP1 or BRCA1.

110 Chromatin and Epigenetics

**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 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 localized in active and silent promoters, excepting the *var* genes, in which the presence of the double-variant nucleosome is associated to active promoters. The analysis of this novel nucleosome in the different genera of the phylum can give more information to elucidate the reason of the presence of this H2B variant.

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Chromatin is also important to the DDR and has an important role in determining the different pathways of DNA repair after DSB. *T. gondii* seems to have every histone variant and histone mark as well as important proteins associated to every DDR pathway to repair a DSB: NHEJ (e.g., Ku70/Ku80) and HRR (RAD51). Different from *T. gondii*, *Plasmodium* does not present the histone variant H2A.X, whose phosphorylation (γH2A.X) is linked to the localization of DSB on DNA. Moreover, *T. gondii* has shown a basal level of γH2A.X, even without damage. Not expected, the proteins associated to the DDR pathway choice (NHEJ or HRR), which read the chromatin, were not detected in *T. gondii* nor in *Plasmodium*. So, it is unknown if these marks are associated to other proteins (*T. gondii* has three BRCT domain containing proteins) with similar roles and/or chromatin modulates DDR in another way.

Taken all together, these differences are not only interesting at the light of evolution but also can be analyzed in the context of the identification of new parasite-specific drug targets. Gene regulation, DNA replication, pathogenicity, and DNA repair are crucial biological processes, and all of them may offer new targets to exploit as future treatments against Apicomplexan pathogens.
