Epigenetic and Genetic Factors that Regulate Gene Expression in Toxoplasma gondii

C H A P T E R

18 Epigenetic and Genetic Factors that Regulate Gene Expression in Toxoplasma gondii William J. Sullivan, Jr. *, Joshua B. Radkey, Kami Kim**, Michael W. Whitey *Department of Pharmacology & Toxicology, and Microbiology & Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA yDepartments of Molecular Medicine and Global Health and the Florida Center for Drug Discovery and Innovation, University of South Florida, Tampa, Florida, USA **Departments of Medicine Pathology, and Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, New York, USA

O U T L I N E 18.1 Introduction

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18.2 Transcription in Toxoplasma 18.2.1 The Parasite Transcriptome and Transcriptional Regulation 18.2.2 Gene-Specific Cis-Elements 18.2.3 The Evolution of APETALA-2Related Proteins 18.2.4 ApiAP2 Structure Determination and DNA Binding 18.2.5 The Function of ApiAP2 Factors 18.2.6 Other Factors that Regulate Gene Expression

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18.3 Epigenetics in Toxoplasma

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Toxoplasma gondii, Second Edition http://dx.doi.org/10.1016/B978-0-12-396481-6.00018-0

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18.3.1 Chromatin and Chromatin Remodelling 18.3.2 Mapping the Toxoplasma Epigenome 18.3.2.1 Chromatin Signatures in Toxoplasma Biology 18.3.3 Histone Modifying Enzymes 18.3.3.1 Histone Acetylation 18.3.3.2 Histone Methylation 18.3.3.3 Other Histone Covalent Modifications 18.3.3.4 SWI2/SNF2 ATPases 18.3.4 Epigenetic Mechanisms as Drug Targets

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Copyright Ó 2014 Elsevier Ltd. All rights reserved.

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18.4 Post-Transcriptional Mechanisms in Toxoplasma 18.4.1 Translational Control 18.4.2 Noncoding and Small RNA 18.4.3 Other Post-Transcriptional Mechanisms

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18.5 Conclusions and Future Directions

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Acknowledgements

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References

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18.1 INTRODUCTION Toxoplasma gondii is distinct from nearly all other members of the large coccidian family (phylum Apicomplexa) owing to the exceptional range of animals (virtually all warm-blooded animals) that serve as host for its intermediate life cycle. Like other coccidians, Toxoplasma completes its definitive life cycle in a single animal host (Dubey et al., 1970); however, the ability of oocysts (shed from the feline definitive host) as well as tissue cysts produced in intermediate hosts to infect either host type (Dubey, 1988) has enabled Toxoplasma to increase its host range (Su et al., 2003). Sexual stages in the feline host lead to the development of oocysts that are shed into the environment (Long, 1982) where contamination of soil or water has led to epidemics of human toxoplasmosis (IsaacRenton et al., 1998; Choi et al., 1997; Bowie et al., 1997; Konishi and Takahashi, 1987; StrayPedersen and Lorentzen-Styr, 1980). Moreover, oocysts are the primary source of Toxoplasma infections of livestock destined for slaughter and human consumption (Mateus-Pinilla et al., 1999; Andrews et al., 1997). Given the importance of Toxoplasma infections to human populations, understanding developmental mechanisms initiated by sporozoites or bradyzoites leading to tissue cyst formation will be central to ultimately controlling transmission and chronic disease. Studies of Toxoplasma primary infections in animals and of sporozoiteand bradyzoite-infected cultures in vitro (Dubey and Frenkel, 1976; Dubey, 1998; Jerome et al., 1998; Radke et al., 2003) indicate that

development initiated by either the sporozoite or bradyzoite stage is similar and likely the consequence of a unified genetic programme (Radke et al., 2003). Thus, defining the changes in gene expression that accompany this development pathway will be important to understand the underlying mechanisms responsible for toxoplasmosis caused by either route of infection. In the following sections, we review our current understanding of Toxoplasma transcription, which undergoes dramatic changes during the parasite intermediate life cycle. Studies have shown that mRNA pools are dynamic and indicate that transcriptional control is a major mechanism employed to regulate developmental transitions in this parasite. It is in this context that we also discuss the evidence that Toxoplasma possesses a similar repertoire of epigenetic-based mechanisms to modulate transcription, as observed in other well-studied eukaryotes from yeast to multicellular animals. Finally, we discuss the emerging role of post-transcriptional regulation, which also appears to be active in this parasite.

18.2 TRANSCRIPTION IN TOXOPLASMA Apicomplexan parasites exhibit complicated, multi-stage life cycles that involve a variety of hosts. Coincident with their complex life cycles are wholesale changes in gene expression associated with each developmental stage or host, yet the mechanisms that control gene expression remain elusive.

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Early efforts to accelerate gene discovery in Toxoplasma led to the sequence for more than 120,000 ESTs from RH and ME49 strain tachyzoites as well as ME49 strain bradyzoites and VEG strain oocysts (Ajioka et al., 1998; Manger et al., 1998; Li et al., 2003, 2004a). A Toxoplasma SAGE project and a 10X-whole genome project for the Type II-Me49B7 followed by further genome sequence to provide 5X coverage of GT-1 (Type I) and VEG (Type III) and whole genome microarrays based on the Type II-Me49B7 reference strains were developed and made available to the Toxoplasma research community. The most recent release of http:// www.toxodb.org features a revised resequenced and re-annotated genome (Version 8), incorporating additional data including expression data derived from microarrays and next generation derived RNA-seq, genomewide chromatin immunoprecipitation studies (ChIP), as well as proteomics studies. Since the first edition of this book both microarrays and next generation sequencing have been used to develop a comprehensive view of the T. gondii transcriptome and permit the characterization of candidate factors that regulate gene expression (Hassan et al., 2012; Minot et al., 2012; Rosowski and Saeij, 2012; Reid et al., 2012; Bahl et al., 2010; Behnke et al., 2010). Much of these data are available on http:// www.toxodb.org and more will be available as the results and analysis of the Toxoplasma white paper project become available throughout 2013. This community effort resulted in the resequencing and re-annotation of the ME49 reference genome, RNA-seq transcriptome studies of ME49, as well as genome sequencing and tachyzoite RNA-seq for the 16 major genetic lineages of T. gondii (see Chapter 3 for discussion of the population biology of T. gondii and Chapter 16 for an overview of classical genetics). An additional set of representative strains has been sequenced at lower coverage. In addition, many groups have submitted RNA-seq data to http://www.toxodb.org, complementing

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numerous Affymetrix gene expression datasets already displayed.

18.2.1 The Parasite Transcriptome and Transcriptional Regulation The first microarray transcriptome studies of the Apicomplexa in Plasmodium illustrated that more than 80% of the transcripts were regulated, with most having a peak expression within a single timeframe in the sexual stages or intraerythrocytic cycle. The proper timing of mRNA accumulation applies not only to genes associated with parasite cell cycle that might be expected to have similar kinetics (Bozdech and Ginsburg, 2005), but also for genes encoding protein components of subcellular structures as the narrow window when these structures form during the division cycle may also require coordinated gene expression (Triglia et al., 2000). The large changes in transcript levels in Plasmodium suggested mRNA expression is governed by ‘just in time’ mechanisms, and the relatively low proportion of constitutive mRNAs in these parasites may reflect this concept (Llinas and DeRisi, 2004). Taken together, these results demonstrate that transcription is a major mechanism controlling gene expression in these parasites and this view is supported by comparison of the changes in the Plasmodium transcriptome and proteome that indicates alterations in mRNA levels have a higher correlation to protein changes in this parasite than is observed in yeast or higher eukaryotes (Le Roch et al., 2004). Initial transcriptome studies in T. gondii relied upon cDNA derived from EST projects for apicomplexan protozoa that were subsequently used to construct a limited Toxoplasma cDNA microarray that focused on tachyzoiteebradyzoite transitions in cell culture models of bradyzoite differentiation (Cleary et al., 2002) and explored gene expression in mutants that were unable to differentiate (Singh et al., 2002; Matrajt et al., 2002). These studies supported a role for

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transcriptional mechanisms in determining developmental stage characteristics in Toxoplasma and evidence for co-regulation of transcription in this parasite (Singh et al., 2002). In the transcripts of four mutants generated by chemical mutagenesis, and selected against the ability to differentiate, a common set of mRNAs was affected and unable to be induced, while other affected mRNA groups appeared to cluster with two or three mutants suggesting hierarchical gene expression may direct bradyzoite development (Singh et al., 2002). Subsequent SAGE and microarray projects supported the concept of co-regulated transcription and demonstrate that some of the general concepts emerging from the Plasmodium studies apply to gene expression in Toxoplasma. A community effort led to the fabrication of an Affymetrix gene array that has now become the platform of choice for transcriptome analysis (Bahl et al., 2010). Currently there are numerous published expression datasets from different strains of parasites exposed to different experimental conditions, including bradyzoite inducing conditions (Behnke et al., 2008; Lescault et al., 2010; Buchholz et al., 2011). Most of these datasets are summarized at http://www.toxodb. org and the primary data are usually accessible in the GEO or ArrayExpress databases. Unlike the high expression of metabolic or structural genes in animal cells, nearly one third of the Toxoplasma most abundant mRNA are Apicomplexa-specific genes that have simple genomic structures containing few, if any, introns (Radke et al., 2005). Metabolic or structural genes typically contain introns. Many transcripts encoding proteins of the basal metabolic machinery and subcellular structures appear to be transcribed in Toxoplasma only when needed during parasite growth and development (Behnke et al., 2010), consistent with the ‘just in time’ concept put forth from studies of Plasmodium (Llinas and DeRisi, 2004). Overall, development-specific genes (sporozoite, tachyzoite, bradyzoite), genes encoding proteins

from biochemical pathways and genes representing mRNA abundance classes are dispersed between all Toxoplasma chromosomes. Gene clusters rarely occur, and in those cases mRNA expression patterns do not appear to be strongly influenced by physical proximity. For example, genes encoding enolase 1 and 2 are less than 1500 bp apart on chromosome VIII, yet expressed exclusively in bradyzoite or tachyzoite stages, respectively (Lyons et al., 2002). These observations suggest that local changes in chromatin structure or the recruitment of RNA polymerase to promoters has little influence on nearby genes. The basal transcriptional complex that controls the expression of protein encoding genes (class II) in most eukaryotes is carried out by RNA polymerase II and its associated general transcription factors (GTF). Comparisons of these transcription factors, as well as the similarities in the three nuclear polymerases (Ranish and Hahn, 1996), have shown that these mechanisms are largely conserved throughout evolution, from the Archaea to mammals. In well-studied unicellular and multicellular eukaryotes, transcription involves a series of co-regulatory complexes that work in concert to control the synthesis of RNA from a particular genomic region. Activating transcription factors (ATF) bind to cis-acting promoter element(s) and recruit chromatin remodelling enzymes which relax the chromatin around the ciselement-containing region as well as recruit the multi-subunit Mediator complex that contacts the RNA polymerase II pre-initiation complex (PIC) directly (Blazek et al., 2005). The accessibility of the cis-element to ATF binding is dependent upon the interaction with these remodelling enzymes, but can also be influenced by other factors such as the chromatin state at the regulatory sequence and the phase of the cell cycle (Fry and Peterson, 2002). In turn, the relaxed chromatin state allows for the formation of the PIC at the core promoter elements through activities contained within the Mediator that facilitate

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recruitment of RNA polymerase II and the GTFs. Current models of ATFs suggest that activation of RNA polymerase II by these factors occurs indirectly through their recruitment of ATP-dependent chromatin remodelling complexes (Blazek et al., 2005; Li et al., 2004b; Featherstone, 2002). The analysis of protein encoding genes in the Apicomplexa indicates that conventional RNA polymerases with similarity to other crown eukaryotes are present. Homologues for all known required eukaryotic RNA polymerases have been found in the Toxoplasma genome: RNA polymerase I (transcribes ribosomal RNA), RNA polymerase II (transcribes protein encoding transcripts) and RNA polymerase III (transcribes small RNA) (Li et al., 1989, 1991; Fox et al., 1993; Meissner and Soldati, 2005). Thus it appears that Plasmodium and Toxoplasma possess the conserved eukaryotic machinery whereby RNA polymerase II transcribes protein encoding genes. The core elements of class II eukaryotic promoters include TATA box, Initiator (Inr), and downstream promoter elements (DPE) that are recognized and bound by several GTFs: TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH (Blazek et al., 2005; Featherstone, 2002; Ruvalcaba-Salazar et al., 2005; Ranish and Hahn, 1996). The core of the GTF family includes the TATA Binding Protein (TBP), TFIID and RNA polymerase II. Homologues for various subunits for the GTFs (TFIID, TFIIE, TFIIF and TFIIH) and subunits of Mediator have been found in the Apicomplexa, and while GTFs are less conserved in the Apicomplexa, much of the basal transcriptional machinery and chromatin remodelling factors required for cooperative control of gene transcription in eukaryotes are present in these pathogens.

18.2.2 Gene-Specific Cis-Elements Classical promoter mapping strategies utilizing conventional protein reporters, including

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chloramphenicol acetyltransferase (CAT), bgalactosidase (bgal), green fluorescent protein (GFP) or firefly/renilla luciferase (luc), have been employed to map regulatory sequences in several promoters. In Toxoplasma, deletion studies to identify promoter cis-elements have been reported for various constitutive genes (GRAs and DHFR-TS), tachyzoite-specific genes (SAG1 and enolase 2), and bradyzoite-specific genes (hsp30/BAG1, hsp70, LDH2 and enolase 1) and confirm that promoter elements are primarily located upstream from the translational start site (Nakaar et al., 1998; Kibe et al., 2005; Matrajt et al., 2004; Ma et al., 2004; Roos et al., 1997; Bohne et al., 1997; Mercier et al., 1996; Soldati and Boothroyd, 1995). Promoter elements were observed to be active in either DNA strand, but may have a limited working distance from the transcriptional start or lose their influence when located downstream of the coding region (Soldati and Boothroyd, 1995). The level of detail within these studies varies and minimal sequence elements were determined in only a few studies (Mercier et al., 1996; Matrajt et al., 2004); moreover, no published study has fully resolved the question of functional sufficiency for any putative ciselement. Nonetheless, it is evident that a 27 bp repeat sequence (6X repeat) in the SAG1 promoter is required for function and a sequence element (A/TGAGACG) found in the GRA promoters was demonstrated to be required for basal expression within the context of a 53 bp minimal promoter (Mercier et al., 1996). It is notable that the GAGACG present in the central core of the SAG1 27 bp repeats is also found in regions implicated to contain regulatory ciselements by deletion analysis of the NTPI/II and DHFR-TS promoters (Nakaar et al., 1998; Matrajt et al., 2004). Development-specific changes in mRNA levels are a dominant feature of the Apicomplexa transcriptome. Nearly one quarter of the transcripts detected in the Toxoplasma SAGE project were observed to be uniquely expressed during

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parasite development and similar observations have emerged from functional genomics studies of the Plasmodium intraerythrocytic cycle (Le Roch et al., 2004; Bozdech and Ginsburg, 2005). Promoters controlling bradyzoite-specific genes, BAG1, hsp70 and LDH2, have now been mapped using alkaline-stress induction at a similar resolution to (Ma et al., 2004; Yang and Parmley 1997; Bohne et al., 1997), while these studies support the role of promoter elements in regulating stress-response in Toxoplasma, their resolution is too low to allow for the identification of common cis-elements. The reciprocal regulation of enolase 1 (bradyzoite-specific) and 2 (tachyzoite-specific) is of particular interest given their close proximity in the genome (in an ordered tandem array of enolase 2e1). Repression of enolase 1 expression in tachyzoites appears to require a distal region more than 600 bp from the enolase 1 ATG and these elements are distinct from inductive elements that were mapped closer to the start of transcription (Kibe et al., 2005). Employing a dual luciferase model, we have mapped bradyzoite-specific cis-elements within a Toxoplasma gene encoding a novel NTPase (BradyeNTPase; chromosome X, TGME49_ 225290) (Behnke et al., 2008). A series of sequential and internal deletions followed by 6 bp substitution mutagenesis have identified a 15 bp cis-element that is responsible for induction of the BradyeNTPase promoter under a variety of drug and stress conditions that co-induce native bradyzoite gene expression. This element lies within the first 500 bp of the BradyeNTPase promoter and 90% of the induction is lost when the element is mutated in the context of the full length promoter fragment (1495 bp). Mutation of this element does not lead to increased expression in the tachyzoite stage indicating that it is a true inductive element. Approximately 2800 mRNAs have cyclical profiles during parasite division that cluster into two major transcriptional waves; genes with maximum expression in the G1 subtranscriptome encode well-conserved metabolic

and biosynthetic functions, while those mRNAs in the S/M subtranscriptome are enriched for genes encoding proteins involved in daughter budding and egress (Behnke, 2010). FIRE (Finding Important Regulatory Elements) analyses of the proximal promoter regions for all cyclical mRNAs were scanned for enrichment of possible DNA regulatory elements. Nine DNA motifs were identified by these analyses (Behnke, 2010) that were distributed in genes with peak transcription spanning the full tachyzoite cell cycle. DNA motifs that were overrepresented in the promoters flanking G1 genes were generally underrepresented in the promoters of S/M genes (and vice versa). One of the DNA motifs enriched in G1 promoters (50 TGCATGC-30 ) is identical to the TgTRP2 ciselement required for transcription of ribosomal proteins (Van Poppel et al., 2006; Mullapudi et al., 2009) and is also identical to the 6 bp core DNA binding motif recently determined by PBM for AP2XI-3 (Kim, unpublished; see below). The mRNA for this AP2 also peaks during the G1 period (Behnke et al., 2010).

18.2.3 The Evolution of APETALA-2Related Proteins The recent discovery of a class of DNA-binding proteins in the Apicomplexa that are related to the APETALA-2 (AP2) class of plant transcription factors (ApiAP2 proteins) has uncovered an important set of proteins that are likely to have critical roles in parasite gene expression (Balaji et al., 2005). Until recently, AP2 domain-containing proteins were thought to be a plant-specific family of DNA binding proteins (Riechmann and Meyerowitz, 1998; Krizek, 2003). AP2 homologues in non-plant species such as cyanobacteria, ciliates and viruses indicate AP2 DNA-binding domains are widely conserved (Wuitschick et al., 2004; Magnani et al., 2004). Many of these proteins contain a second domain encoding a homing endonuclease function that confers the ability to

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operate as mobile genetic elements with the capacity to transpose, invade, and self-replicate by exploiting genome repair mechanisms (Chevalier and Stoddard, 2001; Koufopanou et al., 2002). While most homing endonucleases generally have no core cellular function and are eventually discarded, the HO endonuclease in yeast was adapted to trigger mating type interconversion (Raveh et al., 1989) and other proteins in this family function to catalyse intron selfsplicing in yeast (Chevalier and Stoddard, 2001). Importantly, homing endonucleases are thought to have expanded through lateral gene transfer and are represented in all biological kingdoms, including mitochondrial and chloroplast genomes in eukaryotes (Chevalier and Stoddard, 2001). Magnani et al. (2004) hypothesized that the plant AP2/ERF (ethylene response factor) family of transcription factors arose from the HNH-AP2 family of homing endonucleases present in bacteria or viruses and was incorporated into plant genomes via horizontal gene transfer, or alternatively was acquired indirectly from an endosymbiotic event, likely with a cyanobacterium with an early plant progenitor. Regardless of the source, over time the homing endonuclease function has been lost in plants as the AP2 DNA-binding domain has taken on specific regulatory roles in gene expression associated with plant development and stress response (Magnani et al., 2004; Altschul et al., 2010). Apicomplexan genomes including Plasmodium spp., Toxoplasma gondii, Cryptosporidium parvum and Theileria spp. encode multiple ApiAP2 proteins that may have a similar endosymbiotic origin (Balaji et al., 2005; Altschul et al., 2010). Unlike plant AP2 factors, ApiAP2 proteins have undergone a lineage-specific expansion of a few progenitor genes leading to ApiAp2 factors carrying up to eight AP2 domains (Balaji et al., 2005; Altschul et al., 2010). Interestingly, phylogenetic analysis of ApiAP2 proteins from Plasmodium (27 total), Theileria (19 total) and Cryptosporidium (21 total)

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suggests the common ancestor of these three protozoa contained nine conserved ApiAP2 proteins, with a higher number of orthologous pairs found in Plasmodium spp. and Theileria spp. genomes (Balaji et al., 2005). It is notable that with the exception of the ApiAP2 domain, these proteins are otherwise not conserved and there are no other known protein domains (activation, localization, or proteineprotein interaction) found in any of the Plasmodium spp. or Toxoplasma ApiAP2 proteins (Lindner et al., 2010; Altschul et al., 2010), lending further support to lineage-specific expansion of these DNA binding proteins.

18.2.4 ApiAP2 Structure Determination and DNA Binding The AP2 domain consists of approximately 60 amino acids and was first shown to confer DNA-binding specificity to the AP2/ethylene response element binding family (EREBP) found in plants (Jofuku et al., 1994; Ohme-Takagi and Shinshi, 1995; Balaji et al., 2005). AP2/EREBP proteins represent the second largest class of transcription factors in Arabidopsis thaliana, consisting of 145 proteins that are subdivided further into five sub-families based on AP2 domain architecture (Sakuma et al., 2002). DREB (dehydration responsive element binding) and ERF (ERF is identical to EREBP) protein groups constitute the two largest sub-families (56 DREB compared to 65 ERF) and contain one AP2 domain and a conserved WLG motif (Sakuma et al., 2002). While identical in domain architecture, these groups are defined by single amino acid changes within the DNA-binding domain that alter DNA-binding specificity (Sakuma et al., 2002). Proteins that include two AP2 domains (14 total proteins) are further divided into two subfamilies based on the presence or absence of a second nonAP2 DNA binding domain (Sakuma et al., 2002; Magnani et al., 2004). Alignment of ApiAP2 domains from Plasmodium, Cryptosporidium and Theileria to the

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structure of the ERF1 from Arabidopsis indicates strong conservation of 12 residues that correspond to areas of hydrophobic interactions responsible for the backbone of the DNA binding domain rather than specifying DNA binding (Balaji et al., 2005) (Fig. 18.1). Campbell et al. (2010) determined the DNA-binding specificity of 20 of the 27 P. falciparum ApiAP2s, revealing an unusual diversity of binding motifs that are either classically palindromic or nucleotide biased, and similar results are seen in T. gondii ApiAP2s (Kim, Sullivan, White, Llinas, unpublished). The structural basis of DNA-binding is evident in ApiAP2 factors that

share sequence beyond these 12 core hydrophobic residues. For example, orthologues from P. falciparum (PF14_0633) and Cryptosporidium parvum (cgd2_3490) that share 68% similarity in the AP2 domain show nearly identical DNA-binding specificities, providing evidence that with respect to DNA recognition there is conservation of function across divergent apicomplexan species (De Silva et al., 2008). Whereas the majority of plant AP2 domains cluster tightly around DNA contact residues, ApiAP2 domains exhibit considerable flexibility within the domain, a feature that suggests a greater diversity in DNA-binding specificity

FIGURE 18.1 The AP2 family of Toxoplasma gondii. There are 68 members of the T. gondii ApiAP2 family as determined by a T. gondii community annotation effort (White, Sullivan, Kim, Croken and Wootton; see http://www.toxodb.org). A schematic of the consensus DNA binding domain of the T. gondii family is shown in the upper left (Altschul et al., 2010). The inferred protein size and AP2 domain location within each TgAP2 is heterogeneous as can be seen in the stick figures representing TgAP2 whose mRNA vary during the cell cycle (right). The peak timing of mRNA of the cell cycle regulated TgAP2 within the cell cycle is indicated, as determined by Affymetrix microarray analysis (Behnke et al., 2010). The mRNA expression of some additional Tg ApiAP2 members suggests that they are expressed in other developmental stages (Behnke et al., 2010).

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(Balaji et al., 2005). Supporting this hypothesis, individual PfAP2 domains showed affinity for more than one sequence motif implying a single ApiAP2 factor could indeed regulate multiple gene targets. If ApiAP2 factors operate under a relaxed DNA sequence recognition (De Silva et al., 2008; Campbell et al., 2010), then this feature could account for the smaller repertoire of other transcription factor orthologues encoded in Apicomplexa genomes. Interestingly, the limited domain architecture in plants (one or two domains) is not conserved among the Apicomplexa, where proteins with up to eight AP2 domains are found (Balaji et al., 2005; Altschul et al., 2010). The protein diversity in the ApiAP2 family of proteins is also large. ApiAP2 factors in P. falciparum and T. gondii range in size from 200 to greater than 4000 amino acids that are highly disordered outside the globular AP2 domain(s) (Campbell et al., 2010; V. Uversky, personal communication; see Fig. 18.1). Proteins of this secondary structure type are thought to undergo conformational changes upon interacting with other proteins (Xue et al., 2012), consistent with transcription factor functions. The high content of disordered proteins in parasitic eukaryotes (Xue et al., 2012) is thought to be a crucial adaptation to distinct environment niches. Structural determination of representatives of the Arabidopsis and Plasmodium AP2 families has provided valuable insight into the mechanism by which the AP2 domain binds target DNA sequences. The conserved secondary structure for the monomeric AP2 domain predicts three N-terminal anti-parallel b-sheets and a Cterminal a-helix (Allen et al., 1998; Lindner et al., 2010). The NMR structure of Arabidopsis ERF1 GCC box binding domain (GBD is AP2 domain, target sequence ¼ 50 -A/GCCGAC-30 ) in complex with DNA revealed a novel interaction. AtEFR1 binds DNA via interaction with the b-sheets at specific locations along the DNA backbone while being supported by the a-helix (Allen et al., 1998). This interaction, as depicted in the structure of the single AP2 domain

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containing ERF1, is based on 11 highly conserved residues, seven of which target specific interaction with the GCC box (50 AGCCGCC-30 ). These residues are located within the b-sheets that bind to the sugarephosphate backbone and comprise the framework for specific DNA interaction (Allen et al., 1998). The delineating feature of the DREB sub-family from the ERF sub-family of single domain AP2 proteins is a change in two conserved amino acids: V14 to A, E19 to D respectively. This alters the target DNA-binding sequence (50 -TACCGACAT-30 ) illustrating that the diversity of recognition sequence is dictated by a limited number of evolutionarily conserved residues (Sakuma et al., 2002). Interestingly, the dual AP2 domain containing AINTEGUMENTA contains the conserved arginine and tryptophan residues, but mutation of these residues has little to no effect on DNA binding, suggesting dual AP2 domain proteins exhibit a greater complexity in target DNA-binding sequences and each domain utilizes unique residues to facilitate binding (target sequence ¼ 50 -gCAC(A/G)N(A/T) TcCC(a/g)ANG(c/t)-30 ) (Krizek, 2003). These results from plants suggest dual AP2 domain proteins are more complex in their DNA binding and this may also apply to ApiAP2 factors. In Plasmodium, initial characterization of the dual ApiAP2 protein PFF0200c indicated only one AP2 domain actively bound to a specific 10 mer sequence (De Silva et al., 2008). However, studies of the full-length protein in parasites clearly demonstrate that PfSIP2 (PFF0200c) requires both AP2 domains in order to bind the 16 bp bipartite SPE2 sequence motif (Voss et al., 2003; Flueck et al., 2010). This highlights the limitations associated with using any single approach to determining ApiAP2 function. Solving the crystal structure of the prototypical ApiAP2 domain from P. falciparum (PF14_0633) provided further insight into AP2 function (Lindner et al., 2010). While ApiAP2 domains retain many of the canonical features

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previously described in the Arabidopsis thaliana (Allen et al., 1998), key differences have been identified. In contrast to the A. thaliana structure, which acts as a monomer, PfAP2s are thought to dimerize via a domain-swapping mechanism, with the a-helix of one promoter packed against a b-sheet of its partner (Lindner et al., 2010). This model suggests that DNA binding triggers stabilization of the homodimer or that an AP2 binds DNA as a monomer, elucidating a conformational change that then attracts the second monomer to bind (Lindner et al., 2010). In the case of Pf14_0633, dimerization is thought to be critical to combining distal regions of DNA to function in gene-specific transcription of sporozoite stage genes (Lindner et al., 2010). In addition, ApiAP2 may work in concert as proteomics studies of nuclear complexes have often identified more than one ApiAP2 in pull-downs of macromolecular complexes (Flueck et al., 2010; Sullivan, White, Kim, unpublished).

18.2.5 The Function of ApiAP2 Factors Studies in Arabidopsis and other plant species have described major roles for AP2 proteins in a wide variety of developmental and stress responses. These transcription factors systematically regulate a diverse set of plant processes, including meristem, flower and seed development and environmental responses to drought or attack from plant pathogens (Riechmann and Meyerowitz, 1998; Dietz et al., 2010). The number of AP2 domains contained within a plant transcription factor (one or two AP2 domains) predicts function: single AP2 domain containing factors regulate genes associated with pathogenesis and environmental response pathways whereas dual AP2 domain containing proteins regulate genes responsible for plant development (Riechmann and Meyerowitz, 1998). Much less is known concerning the role of ApiAP2 proteins, although the structuree function dichotomy of plant AP2s is not conserved in the Apicomplexa.

The Toxoplasma genome encodes 68 ApiAP2 domain-containing genes (Fig. 18.1; for product names see http://www.ToxoDB.org and Altschul et al., 2010), more than twice the number found in P. falciparum (27 in total) and other Apicomplexa (Balaji et al., 2005). ApiAP2s in Toxoplasma (TgAP2) are expressed during parasite development (Behnke et al., 2010; Buchholz et al., 2011). Roughly a third (24 in total) of TgAP2 genes are cell cycle regulated with mRNA expression profiles that span the tachyzoite division cycle (Behnke et al., 2010). Eleven TgAP2 mRNAs are induced during bradyzoite differentiation, suggesting a role in developmental gene expression. The remaining TgAP2 domain containing proteins are either constitutively expressed (27 in total) or are undetectable in tachyzoite and bradyzoites, indicating possible roles in the sporozoite or oocyst stages of development (six in total) (Behnke et al., 2010). Of the 68 ApiAP2 domain-containing genes, only about 50 have all structural features predicted to be necessary for DNA binding (Altschul et al., 2010). In most cases, there are no other obvious clues as to protein function, although all studies to date for TgAP2 have shown nuclear localization (Kim, White, Sullivan, unpublished). Yeast two hybrid studies in Plasmodium (LaCount et al., 2005) and proteomics studies in Plasmodium (Zhang et al., 2011; Flueck et al., 2010), as well as published (Saksouk et al., 2005a; Braun et al., 2010) and unpublished studies in Toxoplasma (Kim, White, Sullivan, unpublished), support a role for ApiAP2 in gene regulation. In the rodent malaria P. berghei, it was determined that an ookinete specific AP2 (AP2-O, PF11_0442 orthologue) is critical to mosquito mid-gut invasion. AP2-O was found to directly interact within the proximal promoter regions of 15 genes, including 10 that had previously been defined as ookinete specific or required for ookinete development within the mosquito mid-gut. AP2-O knock-out parasites exhibited

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normal gametogenesis; however, they lacked the ability to infect mosquitos (Yuda et al., 2009b). A second P. berghei AP2, AP2-Sporozoite (AP2Sp), is a trans-acting factor whose cognate binding site is enriched in proximal promoter regions of known sporozoite specific genes, likely interacting with cis-elements to promote stage-specific gene expression (Yuda et al., 2009a; Helm et al., 2010). The P. falciparum orthologue of this protein, PF14_0633, has a DNA binding domain that shared a conserved DNA binding motif (GCATGC) with both C. parvum (De Silva et al., 2008) as well as T. gondii orthologues (Kim, unpublished). In contrast to Plasmodium, the T. gondii gene appears to be essential and to date it has been refractory to disruption. In T. gondii, this motif is a cis-acting motif required to drive luciferase activity in reporter constructs (Kim, unpublished). While many of the DNA binding motifs of ApiAP2 appear to be phylogenetically conserved, there is no conclusive evidence as to whether or not these factors have orthologous functions. Further evidence for transcription factor activity for AP2 proteins comes from a promoter mapping study of a liver stage exclusive promoter. Four repeats of the ApiAP2 PB000252.02.0 (PF11_0404 orthologue) DNAbinding motif were found in the minimal promoter. Interestingly, mutation of a single copy of the binding site within the liver stage promoter increased promoter luciferase gene expression, suggesting PbAP2 PB000252.02.0 has a role as a transcriptional repressor (Helm et al., 2010), which is a common mechanism used by plant AP2 factors to regulate the timing of developmental gene expression (Song et al., 2005; Schmid et al., 2003; Andriankaja et al., 2007). Finally, the Bilker and Soldati groups used the DNA binding domains of ApiAP2 to develop regulated promoters (see description in Chapter 17) that work in both Plasmodium species as well as T. gondii, further bolstering the hypothesized role of these proteins in transcriptional regulation (Pino et al., 2012).

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Genetic studies suggest that Toxoplasma AP2 factor AP2IX-9, which is induced by alkaline stress, operates as a suppressor of bradyzoite differentiation through binding to specific bradyzoite gene promoters (Radke et al., 2013). Another AP2 factor, AP2XI-4, is up-regulated in bradyzoites and has been identified as an activator of bradyzoite differentiation (Walker et al., 2013). Thus, it is expected that ApiAP2 factors in Toxoplasma, much like plant and Plasmodium spp. factors, will have an important role in regulating developmental gene expression in the intermediate and definitive life cycles. Analysis of ApiAP2 mRNA expression patterns in P. falciparum reveals an ordered timing of expression for 22 of 26 ApiAP2 proteins during the intraerythrocytic development cycle (IDC) (Balaji et al., 2005; Campbell et al., 2010). The distribution of expression across the IDC suggests that ApiAP2 factors could be responsible for controlling the dynamic changes in gene expression during apicomplexan replication (Campbell et al., 2010). The enrichment of PfAP2 binding motifs in the promoters of cell cycle transcripts lends support to the idea that the trans-acting ApiAP2 regulator of groups of cell cycle genes will likely be co-expressed (Campbell et al., 2010). For example, the putative target genes for Pf13_0235, which includes ribosome function and heat shock genes, all have the same timing in the IDC. Also, enrichment of PfAP2 target sequences that are found in invasion and host cell entry genes may be regulated by Pf10_0075, and transcripts encoding DNA replication factors could be controlled by MAL8P1.153 (Campbell et al., 2010). In each case, the binding motif of the co-expressed PfAP2 was enriched in the promoters of the inclusive mRNA cluster. Thus far, there is limited experimental evidence available for a role of TgAP2 proteins in parasite replication. However, the sequential profiles of 24 cell cycle regulated TgAP2 factors now provides attractive candidates for an interacting network operating during Toxoplasma replication to coordinate a similar cell cycle

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transcription cascade. Like early work on cell cycle Plasmodium ApiAP2s, the study of Toxoplasma cell cycle ApiAP2s has focused on determining the DNA-binding specificity for selected proteins and the DNA binding preferences of a substantial fraction of TgAP2 have now been identified using the protein binding array technology previously adapted to Plasmodium AP2s (Campbell et al., 2010; De Silva et al., 2008; White, Sullivan, Llinas and Kim, unpublished). These motifs will be useful for genomewide computational searches for putative regulatory targets within proximal promoters as determined by epigenomics ChIPechip analysis (Gissot et al., 2007) and inferred transcription start sites (Yamagishi et al., 2010) . Efforts are also under way to sort essential versus non-essential TgAP2s, which will open the door to determining key regulatory mechanisms critical to cell cycle progression and possible interacting networks of AP2 proteins. While the cell cycle and developmental TgApiAP2s occupy largely unique groups, there are three TgAP2s (AP2VIIa-1, AP2VI-1, and AP2IX-4) that are periodically expressed in the tachyzoite cell cycle and are also elevated in late stage in vivo cysts (Behnke et al., 2010; Buchholz et al., 2011). These TgAP2 factors show peak mRNA levels in the S/M phase, which corresponds to a critical point in the tachyzoite cell cycle where the decision to continue replication as a tachyzoite or differentiate into a bradyzoite is made (Radke et al., 2003). It is well established that parasite replication is linked to development, making it possible that these TgAP2s have dual responsibilities in tachyzoite growth and bradyzoite developmental gene expression. TgAP2s have been identified as proteins that interact with chromatin remodellers HDAC3 (Saksouk et al., 2005a) and GCN5 (Kim and Sullivan, unpublished), as well as complexes implicated in RNA splicing (Kim, Sullivan and White, unpublished). A TgAP2 also was reported to be a component of the macromolecular complex that interacts with TgAgo, an essential

component of the RNA induced silencing complex (RISC) that mediates the activity of many small RNAs in gene expression (Braun et al., 2010). Thus TgAP2s are implicated in multiple critical processes in gene regulation. Studies in P. falciparum indicate ApiAP2 proteins may have non-transcriptional roles. Genome-wide interaction studies of PfSIP2 (ChIPechip), a dual domain PfAP2 protein, found this factor localized to sub-telomeric heterochromatin regions on all chromosomes (Flueck et al., 2010). After proteolytic processing, PfSIP-N exclusively localizes with the SPE2 DNA motif that is present in multiple copies in the promoter regions of upsB var genes associated with var gene silencing (Flueck et al., 2010; Voss et al., 2003). Over-expression of PfSIP2-N caused no changes in gene expression, lending support to a role in maintaining chromosome end biology (Flueck et al., 2010; Voss et al., 2003). This mechanism is supported by PfSIP2 orthologues that exist in other Plasmodium spp. that lack subtelomeric SPE2 motifs yet maintain the SPE2 motif in internal chromosome regions (Flueck et al., 2010). Taken together, PfSIP2 illustrates a novel function for ApiAP2 proteins in telomeric heterochromatin maintenance and gene silencing. A second PfAP2, Pf11_0091, binds to a motif within the var intron that is sufficient to localize DNA to a subnuclear compartment with silenced var genes in an actin-specific manner (Zhang et al., 2011). The var intron also has promoter activity (Calderwood et al., 2003), and as yet, the role of this ApiAP2 in transcriptional regulation has not been resolved. Evidence for non-transcriptional roles for Toxoplasma AP2 factors are starting to emerge. In genome-wide occupation studies of AP2VI-1 and AP2IV-4, which exhibit maximum expression coinciding with S/M phase, show localization to peri-centromeric regions on all chromosomes (Radke, Kim and White, unpublished). This pattern is reminiscent of the PfSIP2 localization to sub-telomeric heterochromatin and these AP2s could play critical roles in

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chromosome structure determination or chromatin remodelling.

18.2.6 Other Factors that Regulate Gene Expression Amongst the other factors that are developmentally regulated are the glycolytic enzymes, including ENO2 (tachyzoite) and ENO1 (bradyzoite). Intriguingly these proteins are localized to the nucleus suggesting that they might also play a role in gene regulation in T. gondii. Several glycolytic enzymes have been identified as components of transcriptional complexes including LDH and GAPDH in the OCA-S complex (Zheng et al., 2003). As the expression of the ENO genes is also regulated at the mRNA level, the ENO1 promoter was used as bait to identify nuclear factors that interact with this promoter (Olguin-Lamas et al., 2011). Amongst the 35 nuclear proteins identified, most are hypothetical proteins, but those with inferred function included two Alba family DNA/RNA binding proteins, a potential histone chaperone (NF3), and other proteins predicted to interact with RNA and DNA. NF3 is nucleolar protein whose overexpression alters nucleolar morphology and inhibits parasite virulence. In the bradyzoite stage NF3 is cytosolic. Chromatin immunoprecipitation studies have confirmed that NF3 (Olguin-Lamas et al., 2011) and Eno2 proteins (Tomavo and Kim, submitted) are associated with chromatin.

18.3 EPIGENETICS IN TOXOPLASMA Epigenetic gene regulation refers to heritable changes in gene expression that are not genetically encoded in the DNA sequence of an organism. Among the mechanisms of epigenetic regulation are those affecting accessibility of factors to chromatin such as DNA methylation, histone modification and nucleosome location. Noncoding RNA also affects a myriad of nuclear

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and cytoplasmic processes that regulate epigenetic gene regulation. Current models of eukaryotic transcriptional activation implicate a significantly greater number of co-factors than was appreciated more than a decade ago. The simple binding of gene-specific ATFs (activating transcription factors) to local sequences in nucleosomal DNA (Naar et al., 2001) is now recognized to be insufficient to recruit the RNA polymerase II-PIC. ATFs recruit chromatin remodellers to facilitate the assembly of the PIC on core promoter sequences (reviewed in Spector, 2003; Ehrenhofer-Murray, 2004; Li et al., 2004a). These findings bring chromatin dynamics to the forefront of gene expression research, and the discovery that histone proteins can be chemically modified in ways that enhance or inactivate transcription, along with ATPases capable of repositioning nucleosomes, has prompted intensive investigation into how these mechanisms act cooperatively to regulate gene expression. Although the order and assembly of transcriptional factors has not been demonstrated in any apicomplexan parasite, including Toxoplasma, the initial forays into understanding transcriptional regulation reveal these parasites possess essential features of the basal transcription machinery as well as a significant collection of chromatin remodelling machinery. Research into chromatin remodelling mechanisms for the purpose of new drug target discovery is an important area of investigation, first illustrated by the HDAC (histone deacetylase) inhibitor, apicidin, which has broad spectrum activity against a variety of apicomplexan parasites including human pathogens Toxoplasma and Plasmodium and veterinary pathogens from the Eimeria genera (Darkin-Rattray et al., 1996).

18.3.1 Chromatin and Chromatin Remodelling The fundamental building block of chromatin is the histone protein. Four canonical types of

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histones exist (H2A, H2B, H3 and H4) that form an octamer complexed with DNA (the nucleosome). Histone tails, particularly those of H3 and H4, are subject to a diverse array of covalent modifications that have different consequences on gene transcription (Peterson and Laniel, 2004). Like many other eukaryotes, Toxoplasma H3 and H4 are exceptionally well conserved with each residue in the N-terminal tail reported to be susceptible to chemical modification being present (Sullivan et al., 2003; Nardelli et al., 2013). Histone variants, which may be substituted for canonical histones to modulate DNA-driven processes, are also conserved. Toxoplasma contains a homologue of variant H3.3 in addition to the canonical H3, the former being associated with genes undergoing transcription in other species (Sullivan et al., 2003). An orthologue of the centromeric H3 variant (CenH3) has also been characterized and localized to an apical subnuclear compartment (Brooks et al., 2011). Beyond the well-conserved H3 and H4 classes, the complement of histone proteins in Toxoplasma exhibits a number of unusual features (Dalmasso et al., 2011). Like yeast, Toxoplasma may not possess H1, the extra-nucleosomal ‘linker’ histone involved in solenoid formation during chromatin condensation, although a small basic protein with homology to the H1 of kinetoplastids (Croken et al., 2012) is present in the genome. Two distinct lineages of H2B are present, including the constitutively expressed TgH2Bv1, a parasite-specific H2B variant, and potential stage-regulated TgH2Ba and TgH2Bb (Dalmasso et al., 2006). The canonical H2A protein is TgH2A1 and both H2AZ and H2AX variants exist (Dalmasso et al., 2009). TgH2A1 and TgH2AX both possess a C-terminal SQ motif, consistent with predicted roles in the DNA damage response. Coimmunoprecipitation experiments are beginning to reveal the composition of Toxoplasma nucleosomes (Dalmasso et al., 2009). TgH2AZ dimerizes with TgH2Bv1, but not TgH2AX. TgH2AZ and TgH2Bv1 localize with other acetylated

histones to actively transcribed genes (Dalmasso et al., 2009) whereas TgH2AX is present at repressed genes (Dalmasso et al., 2009). TgH2AZ also localizes to gene bodies of developmentally silenced genes (Nardelli and Kim, unpublished). Interestingly, TgH2AX expression increases during bradyzoite conversion, consistent with the increase in repressed genes during the latent stage. These studies are consistent with the hypothesis that TgH2AZ and TgH2Bv1 are involved in transcriptional activation while TgH2AX and TgH2A1 may populate chromatin during stress (Dalmasso et al., 2009). Histone N-terminal tails are generally rich in positively charged amino acids and interact tightly with negatively charged DNA, facilitating condensation. The assembly of genomic DNA into histone nucleosomes and then into higher order chromatin structure is associated with transcriptional repression and ‘silenced’ chromatin is thought to be the default mechanism guiding the formation of chromatin following DNA replication (Ehrenhofer-Murray, 2004). Thus, active steps must be taken to alter the normal state of chromatin in order to achieve stable transcriptional activation. A myriad of chemical modifications to histones are now known and are proposed to operate in combinatorial fashion, constituting a ‘histone code’ that reflects corresponding changes in the local activation (and inactivation) of specific genes (Strahl and Allis, 2000). The histone code of T. gondii has been characterized by mass spectrometry (Fig. 18.2; Nardelli et al., 2013) with T. gondii histones possessing novel modifications not previously described in protozoa. Similar to Plasmodium (Trelle et al., 2009) and in contrast to the metazoa, T. gondii nucleosomes consist primarily of euchromatic acetylated chromatin, consistent with chromatin that is accessible to the transcriptional machinery (Nardelli et al., 2013). Consistent with the histone code hypothesis is the discovery of protein motifs capable of binding specific histone modifications. Examples include

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FIGURE 18.2 The histone code of Toxoplasma gondii: a summary of post-translational modifications (PTM) identified on T. gondii canonical histones and histone variants by mass spectrometry. PTM on canonical histones are shown in comparison with P. falciparum. Circles in different colours represent the modifications. PTM on canonical and histone variants are shown in comparison with P. falciparum. Identical amino acids are represented in grey. Circles above the sequence represent histone PTM in T. gondii (Nardelli et al., 2013). Histones depicted are histone 3 (H3), histone 4 (H4), histone H2A (H2A), histone (H2B) and variant histones H2Bv, H2A.Z, H2A.X and CenH3. The grey cylinders indicate the approximate location of the histone globular domain. Numbers above the sequences represent the amino acid position in T. gondii while 1x, 2x and 3x above the red circles indicate mono-, di- or tri-methylation respectively. Figure courtesy of Sheila Nardelli.

bromodomains that interact with acetylated lysines, chromodomains that bind methylated lysines, and macrodomains that recognize ADPribose moieties (Dhalluin et al., 1999; Bannister et al., 2001; Karras et al., 2005). The chromodomain protein TgChromo1 binds H3K9me3 and localizes to centromeres as well as telomeres in T. gondii (Gissot et al., 2012), although only centromeres have been demonstrated to be enriched in

H3K9me2/3 (Brooks et al., 2011). In the following sections, we present a summary of histone modifications and discuss what is known about their occurrence in Toxoplasma.

18.3.2 Mapping the Toxoplasma Epigenome Genome-wide approaches have been used to illuminate the chromatin modifications

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associated with gene activation as well as define functional regions of the T. gondii genome (Fig. 18.3). The major technique used has been chromatin immunoprecipitation, which enriches for the DNAeprotein complexes of interest, followed by either hybridization of the enriched DNA to genome-wide arrays (Gissot et al., 2007) or high throughput sequencing (ChIP-seq). Because T. gondii histones and histone

modifications seen in model organisms are conserved (see Fig. 18.2), commercial antibodies specific for histone modifications can be used for this technique. In other species, direct modification of DNA, primarily cytosine methylation, also affects the accessibility of macromolecular complexes to chromatin, but it appears that T. gondii DNA is not cytosine methylated and there currently is no evidence for modification of DNA affecting gene

FIGURE 18.3 Chromatin modifications that define the epigenome of Toxoplasma gondii. Genome-wide chromatin immunoprecipitation microarray hybridization studies (ChIPechip) define the epigenome of T. gondii. Chromatin was harvested from intracellular tachyzoites and chromatin immunoprecipitations were performed with antibodies specific for the indicated histone post-translational modification. Enriched DNA was hybridized to a custom Nimblegen Toxoplasma genome tiled microarray and the results of hybridization are shown as log2 ratios of signal to input control DNA. cDNA was harvested in parallel and hybridized to the chip. H3K9ac, H3K4me3 are enriched at sites of active promoters, whereas H3K4me1 co-localizes with gene bodies of actively transcribed genes (cDNA track). Genes and exons are indicated with boxes above the baseline indicating genes predicted to be on the positive strand and boxes below the baseline indicating genes transcribed on the negative strand. The specialized centromeric histone CenH3 marks a gene-poor region of the chromosome and localizes to each chromosomal centromere with H3K9me2.

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expression in T. gondii or any Apicomplexa (Gissot et al., 2008; reviewed in Croken et al., 2012). 18.3.2.1 Chromatin Signatures in Toxoplasma Biology Through performing ChIP with antibodies to either acetylated H3 or H4, it was demonstrated that relative acetylation versus deacetylation can be correlated with specific gene activation or repression in Toxoplasma, respectively (Saksouk et al., 2005b). This was shown in the context of stage conversion to latent bradyzoites, demonstrating that in parasites cultured as tachyzoites, H3 and H4, were acetylated at tachyzoitespecific promoters like SAG1 and SAG2A, while no acetylation is detected at bradyzoite-specific promoters such as LDH2 and BAG1 (Saksouk et al., 2005b). Conversely, in a parasite population induced to enter the bradyzoite pathway, acetylation at tachyzoite promoters was diminished while acetylation at bradyzoite promoters increased. As expected, intergenic regions upstream of constitutively expressed genes were found in the acetylated state in either population in vitro. Confirmation of the differential state of histone acetylation that is associated with specific remodelling enzymes was also observed in parasite transgenic lines expressing epitope-tagged TgGCN5-A and TgHDAC3 proteins. Lysine acetyltransferase (KAT) TgGCN5-A was present at tachyzoite promoters in tachyzoites, but absent at bradyzoite promoters, whereas TgHDAC3 was associated with promoters that were down-regulated in each respective developmental stage (Saksouk et al., 2005b). Studies have also shown that the TgCARM1-mediated methylation of H3R17 is another signature of gene expression in Toxoplasma, with the presence of this protein at active genes in either the tachyzoite or bradyzoite stage (Saksouk et al., 2005b). Interestingly, genes marked with methylated H3R17 also displayed enrichment of acetylated

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H3K18, a potentially synergistic signature for gene activation that relies on crosstalk between acetyl- and methyltransferase complexes. ChIP and mass spectrometry have shown that H3K4 can be mono-, di- or tri-methylated in Toxoplasma. More specifically, tri-methylated H3K4 is enriched at tachyzoite promoters during the tachyzoite stage (Gissot et al., 2007) and becomes enriched at bradyzoite promoters following differentiation, representing another mark of gene activation in this parasite (Saksouk et al., 2005b). More recent genome-wide ChIP studies have established that acetylation of H3K9, H4 and tri-methylation of H3K4 occur at promoters of actively expressed genes (Gissot et al., 2007). Tri-methylation of H3K9 and H4K20 occur at repressed genes in heterochromatic territories (Sautel et al., 2007), with the H3K9me2/3 marks enriched in centromeres (Brooks et al., 2011). Monomethylation of H3K4 is associated with gene bodies of actively transcribed genes (Fig. 18.3). In contrast to Plasmodium, T. gondii does not encode major antigenic variant gene families whose silencing is associated with deposition of the heterochromatic histone marks H3K9me2/3 (Lopez-Rubio et al., 2007, 2009). As observed in other species, phosphorylation of TgH2AX has been linked to the parasite DNA damage response. H2AX is phosphorylated in response to double-stranded breaks at its C-terminal SQ(E/D)F motif (F denoting a hydrophobic residue) (Escargueil et al., 2008). Treatment of Toxoplasma with DNA-damaging agents MMS (methyl methanesulphonate) or H2O2 led to increased phosphorylation of TgH2AX, as detected by immunoblotting with monoclonal antibody (Vonlaufen et al., 2010; Dalmasso et al., 2009). These studies indicate that phosphorylated TgH2AX can be used as a chromatin biomarker for DNA injury. Phosphorylation of H3S10 has also been reported in Toxoplasma, a mark that peaks during mitosis with monomethylation of H4K20 (Sautel et al., 2007). The function of H3S10 phosphorylation

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has been linked to chromosome condensation in fellow alveolate protozoan Tetrahymena (Wei et al., 1998).

18.3.3 Histone Modifying Enzymes Chromatin remodellers generally fall into two distinct classes: those capable of covalently modifying histones or those that use ATP to reposition nucleosomes (SWI2/SNF2 family ATPases). Both types of chromatin remodelling machinery can be found in Toxoplasma and other protozoa as well (Sullivan et al., 2006; Dixon et al., 2010). Apicomplexan histones are subject to a wide variety of covalent modifications that include acetylation, methylation, phosphorylation, ubiquitination, and sumoylation (see Fig. 18.2 for a comparison of T. gondii and Plasmodium falciparum). In the following sections, we will discuss histone modifying enzymes found in Toxoplasma and what we have learned to date about their role in parasite biology. 18.3.3.1 Histone Acetylation In other eukaryotes, acetylation of lysine residues in the N-terminal histone tails is linked to gene activation. Conversely, the removal of acetyl groups is associated with transcriptional repression. A wide variety of HATs (histone acetyltransferases) and HDACs (histone deacetylases) have been characterized among eukaryotes that control the acetylation status of nucleosomal histones, and hence play an important role in the regulation of gene expression (Sterner and Berger, 2000; Thiagalingam et al., 2003). The Toxoplasma genome predicts at least seven HATs and seven HDACs present in the parasite. Recently, it has been proposed that proteins historically referred to as HATs and HDACs be renamed KATs and KDACs (lysine acetyltransferases and deacetylases, respectively) since many of them also act on non-histone substrates (Allis et al., 2007). Given the discovery of widespread lysine acetylation in nonnuclear compartments

of Toxoplasma, we propose this change in nomenclature be adopted for the parasite (Jeffers and Sullivan, 2012). Two MYST family KAT proteins (MOZ, Ybf2/Sas3, Sas2, Tip60) exist in Toxoplasma, each possessing a chromodomain and the atypical C2HC zinc finger domain upstream of the KAT domain (Smith et al., 2005; Vonlaufen et al., 2010). The predicted proteins, named TgMYST-A (AY578183) and -B (DQ104220), have features consistent with the ‘MYST þ CHD’ subclass, homologous to yeast Esa1, human Tip60, and MOF (Utley and Cote, 2003). Previous studies demonstrate that this type of KAT has a preference for acetylating lysines in H4 and the observation that recombinant TgMYSTs also prefer H4 as substrate in assays using free core histones functionally validates this classification (Smith et al., 2005). Given this similarity, it was not surprising that genes encoding TgMYST-A and -B could not be disrupted by homologous recombination as the Esa1 homologue in yeast is also an essential gene (Smith et al., 1998). TgMYST-A is not amendable to stable over-expression unless the recombinant protein is mutated to nullify its KAT activity, suggesting a delicate balance of TgMYST-Amediated acetylation exists in Toxoplasma (Smith et al., 2005). Despite their histone acetylation abilities, both TgMYST KATs are predominantly cytoplasmic, suggesting they may act on nonhistone substrates (Jeffers and Sullivan, 2012). Over-expression of TgMYST-B is tolerated, but results in a significantly reduced proliferation rate unless enzymatic activity is ablated (Vonlaufen et al., 2010). Interestingly, the delayed replication may be connected to the dramatic resistance to DNA-damage observed for TgMYST-B overexpressing parasites. Consistent with heightened protection from DNA-damaging agents, parasites over-expressing TgMYST-B have increased levels of ataxia telangiectasia mutated (ATM) kinase and phosphorylated H2AX (Vonlaufen et al., 2010). Increased gH2AX leads to cell cycle arrest and a decrease in the number of cells in mitosis,

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likely explaining why parasites over-expressing TgMYST-B exhibit delayed replication (Vonlaufen et al., 2010; Rios-Doria et al., 2009). The connection between TgMYST-B and the ATM kinase-mediated DNA damage response was further supported when pharmacological inhibitors of ATM kinase or KATs rescued parasites over-expressing TgMYST-B from slowed replication (Vonlaufen et al., 2010). Two KAT proteins of the GCN5 class also exist in Toxoplasma designated TgGCN5-A (AAF29981) and TgGCN5-B (AAW72884), which is a highly unusual arrangement in a lower eukaryote. Aside from the close relative Neospora caninum, the presence of two GCN5 KATs in a single cell has not been documented for any other invertebrate. In contrast, mammalian species have two GCN5 KAT enzymes referred to as GCN5 and PCAF (p300/CBP Associating Factor). Deletion of mouse GCN5 is embryonic lethal while the loss of PCAF has no discernible phenotype (Xu et al., 2000; Yamauchi et al., 2000). There is a striking parallel in Toxoplasma, in which TgGCN5-A is dispensable in tachyzoites yet TgGCN5-B appears to be essential (Bhatti et al., 2006). The two TgGCN5s differ in other ways as well. GCN5 family members show a strong preference to acetylate H3, particularly lysine 14 (K14). Recombinant TgGCN5-A was found to have an exquisite selectivity to acetylate H3K18 whereas TgGCN5-B was more prototypical and capable of targeting H3K9, H3K14, and H3K18 in vitro (Bhatti et al., 2006; Saksouk et al., 2005a). Another difference between the TgGCN5 KATs is their ability to bind with the ADA2 co-activator, for which two homologues have been identified in the Toxoplasma (TgADA-A, DQ112184; TgADA2-B, DQ112185). By yeast two-hybrid assay, TgGCN5-B has been shown to interact with either TgADA2 homologue, while TgGCN5-A can only associate with TgADA2-B (Bhatti et al., 2006). It has been proposed that TgGCN5-B may be required for tachyzoite replication and

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TgGCN5-A may be required only for specific circumstances, such as the stress response. Indeed, Saccharomyces cerevisiae remains viable without GCN5, but is impaired when grown on minimal media (Marcus et al., 1994). Support for this idea was recently obtained in studies of the TgGCN5-A knock-out that showed a significant recovery defect following exposure to alkaline pH stress, a condition commonly used to induce bradyzoite development in vitro. Microarray analyses revealed that parasites lacking TgGCN5-A fail to up-regulate w75% of the genes normally induced during alkaline stress, including bradyzoite-specific induction markers BAG1 and LDH2 (Naguleswaran et al., 2010). While repeated attempts to knock out TgGCN5-B have failed, even in Dku80 parasites, an inducible dominant-negative strategy supports that TgGCN5-B is essential in tachyzoites (Sullivan, unpublished results). Apicomplexan GCN5 KATs contain an unusual N-terminal extension upstream of the well-conserved catalytic and bromodomains. Curiously, the length and amino acid composition varies greatly among the Apicomplexa, and even among the pair of GCN5s in Toxoplasma. Most GCN5s from early eukaryotes do not have appreciable sequence upstream of the KAT domain. In contrast, mammalian GCN5 and PCAF have N-terminal extensions, but they are very similar to each other. The function of the N-terminal extension may be to mediate proteineprotein interactions (e.g. the binding of CBP) and/or substrate recognition, as GCN5 lacking the N-terminal extension can only acetylate free histones and not nucleosomal histones (Xu et al., 1998). The N-terminal extensions of TgGCN5-A and -B are required for nuclear localization, but dispensable for enzyme activity on free histones (Bhatti and Sullivan, 2005; Dixon et al., 2011). A six amino acid, basic-rich motif in the N-terminal extension of TgGCN5-A has been mapped as a necessary and sufficient nuclear localization signal (NLS) that interacts with the nuclear chaperone importin alpha

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(Bhatti and Sullivan, 2005). The NLS for TgGCN5-B is also rich in basic residues and found in the N-terminal extension, although its sequence is distinct from the NLS of TgGCN5A (Dixon et al., 2011). Previous work has also noted that KDAC proteins exist in Plasmodium (Joshi et al., 1999; Freitas-Junior et al., 2005) and analysis of Toxoplasma genomic sequence indicates there are seven potential KDAC genes with one experimentally characterized to date (TgHDAC3). Recombinant TgHDAC3 exhibits histone deacetylase activity that is inhibited by butyrate, aroyl-pyrrole-hydroxy-amides, and trichostatin A and a native TgHDAC3-containing complex has been purified (TgCRC for CoRepressor Complex) (Saksouk et al., 2005b). The TgCRC contains several protein components that are homologous to subunits found in the human N-CoR and SMRT complexes as well as two large parasite-specific proteins of unknown function (Saksouk et al., 2005b). Pull-down studies suggest HDAC3 may interact with both TgAgo1 (Braun et al., 2010) as well as TgAP2 (Saksouk et al., 2005a; Kim, unpublished), supporting a critical role of TgHDAC3 in gene regulation in T. gondii. With the exception of TgSIR2a, the TgKDACs have been refractory to disruption, suggesting that they have essential functions in the biology of T. gondii (Kim, unpublished). 18.3.3.2 Histone Methylation The addition of methyl groups occurs on lysine and arginine residues of histones and can lead to gene activation or silencing (Zhang and Reinberg, 2001). There is an added layer of complexity in methyl-modifications as residues can be mono-, di- or tri-methylated. Toxoplasma possesses five protein arginine methyltransferase (PRMT) homologues, designated TgPRMT1e5. Recombinant TgPRMT1 (AY820756) is capable of methylating H4R3, while TgPRMT4 (referred to as TgCARM1, co-activator associated arginine methyltransferase, AY820755) methylates H3R17

(Saksouk et al., 2005b), which parallels the substrate specificity of their human homologues. The importance of TgPRMT1 for histone methylation is not yet clear, as the phenotype of parasites lacking TgPRMT1 is a cell cycle phenotype that affects daughter cell counting (El Bissati et al., submitted). Human CARM1 has been associated with SWI2/SNF2 ATPases, including the Snf2Related CBP Activator Protein, or SRCAP (Xu et al., 2004; Monroy et al., 2003). Recombinant TgCARM1 incubated with parasite extract enriched for ATP-dependent nucleosome disruption activity indicated that TgCARM1 is likely to interact with a Toxoplasma SWI2/SNF2 member (Saksouk et al., 2005b), and an SRCAP SWI2/ SNF2 homologue has been characterized in Toxoplasma (see section below and Sullivan et al., 2003). Together, these data suggest a conserved connection between CARM1 and SRCAP complexes. Lysine methyltransferases share a common feature known as the SET (Suv(39)-E(z)-TRX) domain (Dillon et al., 2005). Searching for this domain in the predicted proteins contained in the Toxoplasma genome reveals at least 19 candidates and a surprising amount of duplication and divergence within this protein family (Bougdour et al., 2010a). As has been observed with acetyl transferases, novel non-histone substrates for methyltransferases have been described in many systems including T. gondii. Commercial methyl lysine antibodies often cross react with cytosolic or cytoskeletal structures in T. gondii (Sautel et al., 2007; Xiao et al., 2010) and one SET protein, AKMT or apical lysine methyltransferase, is implicated in methylation of apical cytoskeletal structures (Xiao et al., 2010). In Plasmodium, PfSET10, a methylase with H3K4me specificity, has been implicated in maintaining the active var gene in a poised state during the cell cycle (Volz et al., 2012). It is likely that some of the TgSET will have analogous functions in lysine methylation of histones, particularly since histone lysine methylation is a common histone post-translational modification

18.3 EPIGENETICS IN TOXOPLASMA

(Fig. 18.2). Although bioinformatics studies have inferred specificity (Bougdour et al., 2010b; Sullivan et al., 2006), in most cases these predictions have not yet been validated experimentally, with the exception of the studies cited below. ChIPechip studies localize KMTox (formerly TgSET13) to genes related to antioxidant defences, heat-shock proteins/chaperones, and genes involved in translation and carbohydrate metabolism. KMTox also protects against H2O2 exposure, and was found to associate with 2cys peroxiredoxin-1 (TgPrx1) under oxidative conditions, bolstering the idea that KMTox contributes to the parasite’s antioxidant defence system (Sautel et al., 2009). Unlike its monomethylating human counterpart, biochemical and structural modelling analyses show that TgSET8 is capable of mono-, di- and tri-methylation of H4K20 (Sautel et al., 2007). Tachyzoites expressing a mutated version of TgSET8 (F1808Y) that abolishes the monomethylation of H4K20 are not able to progress through the cell cycle, suggesting that monomethyl H4K20 is required for parasite division (Bougdour et al., 2010a). TgSET8 may also play important roles during the latent cyst stage, as suggested by high levels of monomethylated H4K20 in bradyzoites (Sautel et al., 2007). With regard to the removal of methyl groups, Toxoplasma appears to encode seven JmjC (Jumonji) domain demethylating proteins. Only one has both the Jumonji N and C domains characteristic of JARID-like H3K4 and JMJD1like H3K9 demethylases and preliminary data suggest that this may be a dual function histone demethylase (Kim, unpublished). The other members of this family belong to the JMJD6 family that demethylate H3R2 and H4R3 (Bougdour et al., 2010a). More recent studies have questioned the exclusive role of the JmjC (Jumonji) domain proteins in protein demethylation e with new functions reported in RNA processing (Hong et al., 2010), so further studies will be needed to determine the function of these

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putative demethylases. Toxoplasma also appears to encode homologues of lysine-specific demethylases, but the function of these proteins has not been characterized. This atypical expansion of demethylases in Toxoplasma may counter the extensive number of methyltransferases and the expansion of the methyltransferase and demethylase families is an aspect of T. gondii biology that differs from Plasmodium. 18.3.3.3 Other Histone Covalent Modifications Considerably less work has been done to dissect the roles of histone phosphorylation, ADP-ribosylation and ubiquitylation in Toxoplasma. As mentioned, H3S10 phosphorylation has been reported, and Toxoplasma possesses two predicted proteins with strong similarity to histone kinase Snf1. Toxoplasma also appears to possess proteins containing PARP and PARG domains, required for the addition or removal (respectively) of ADP-ribose subunits (Dixon et al., 2010). There is also no shortage of ubiquitin-conjugating enzymes in this organism, including Ubc9, which is implicated in gene repression via the sumoylation of H4 (Shiio and Eisenman, 2003). H3 ubiquitination has been confirmed by mass spectrometry (Nardelli, Che et al., submitted and Fig. 18.2). A small ubiquitin-like modifier (SUMO)-conjugating system has been characterized in Toxoplasma (Braun et al., 2009), and sumoylation has been reported on Plasmodium H2A and H2AZ. While sumoylation of T. gondii histones is detectable by immunoblot (Nardelli et al., 2013), the exact modified residues have not yet been mapped. A number of novel histone modifications of unknown function have been reported in the metazoa including propionylation, O-GlcNAcylation and succinylation. These modifications are also present on T. gondii histones (Fig. 18.2) and based upon conjectures in other organisms, these histone modifications may provide a mechanism by which changes in metabolism are sensed and can impact epigenetic gene regulation.

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In summary, previous reports coupled with bioinformatic analyses of the completed genome demonstrate that Toxoplasma is capable of mediating most known histone modifications. The extensive array of chromatin remodelling machinery suggests that histone modifications and epigenetics are likely to be instrumental during progression of the parasite life cycle. These observations underscore the antiquity of epigenetics in the evolution of the eukaryotic cell and indicate that much of this machinery has evolved along parasite-specific trajectories. 18.3.3.4 SWI2/SNF2 ATPases The second broad class of chromatin remodelling complexes in eukaryotes is comprised of the SWI2/SNF2 DNA-dependent ATPases that have roles in both transcriptional repression as well as activation (Mohrmann and Verrijzer, 2005). While the mechanism of action of these factors is incompletely understood, it is believed the energy of ATP hydrolysis is used to reposition or relocate the nucleosome (Johnson et al., 2005). All members of the SWI2/SNF2 family contain a distinctive ATPase domain consisting of an N-terminal DEXDc portion and a C-terminal HELICc portion. Further classification based on sequence homology and additional structural features leads to four separate types: Snf2 members (contain a bromodomain), ISWI (contain a SANT domain), Mi-2 (contain a chromodomain), and Ino80/SRCAP/p400 (contain a lengthy insert between the DEXDc and HELICc domains). Previous reports have described SWI2/SNF2 factors in Apicomplexa, including an ISWI homologue in Plasmodium and an SRCAP homologue in Toxoplasma (TgSRCAP, AAL29689), Cryptosporidium, and Plasmodium (Ji and Arnot, 1997; Sullivan et al., 2003). Only TgSRCAP has been studied in any great detail. Like human SRCAP, TgSRCAP can function to enhance CREB-mediated transcription in the presence of the HAT CBP in vitro (Sullivan et al., 2003). However, a protein with similarity to CBP or CREB has not been found, therefore,

its role in Toxoplasma remains to be elucidated. To facilitate a better understanding of what TgSRCAP may do, a yeast two-hybrid screen was conducted using the lengthy ‘spacer’ region separating the DEXDc and HELICc domains as ‘bait’ (Nallani and Sullivan, 2005). The corresponding region in human SRCAP binds CBP (Johnston et al., 1999). Most of the strongest interacting proteins isolated and confirmed by in vitro co-immunoprecipitation are novel parasitespecific proteins having no homologues in other eukaryotes. A few of these are from genes predicted to encode domains suggestive of a role in DNA processes e including transcription. Of particular interest is the first protein described in Toxoplasma to contain Kelch repeats and a BTB/POZ domain (Nallani and Sullivan, 2005). POZ domains from several zinc finger proteins have been shown to mediate transcriptional repression and to interact with components of histone deacetylase co-repressor complexes. The gene has subsequently been cloned (DQ174778) and termed TgLZTR since it is most similar to human Leucine-Zipper-like Transcriptional Regulator, a gene deleted in people with DiGeorge syndrome (Kurahashi et al., 1995). Future studies should elucidate the role of TgLZTR and whether it associates with TgSRCAP in vivo. In addition to TgSRCAP, the Toxoplasma genome contains at least 17 possible SWI2/ SNF2 homologues. Two bear high sequence similarity to Snf2 subclass members, with one harbouring a bromodomain downstream of the ATPase domains. Another SWI2/SNF2 protein has a SANT domain, making it a likely orthologue of ISWI. A second SWI2/SNF2 has strong ISWI homology, but possesses an AT-hook domain instead of a SANT domain. There is also a predicted SWI2/SNF2 family member in Toxoplasma that contains a chromodomain, making it a probable Mi-2 orthologue. This protein was identified as part of the TgCRC (Saksouk et al., 2005a). How this extensive family of SWI2/SNF2 ATPases contributes to gene

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expression in parasites remains an open area for future investigation.

18.3.4 Epigenetic Mechanisms as Drug Targets It has now been established that chromatin remodelling plays critical roles in various aspects of parasite physiology, prompting discussions about targeting this machinery for novel drug design. Histone acetylation has been validated as a drug target as early as 1996, when it was discovered that a fungal metabolite (now called apicidin) with potent antiprotozoal activity inhibited apicomplexan KDACs (Darkin-Rattray et al., 1996). Given the conservation of histone modifying enzymes in human, and the application of KDAC inhibitors now in cancer, legitimate questions regarding selective toxicity arise. Selective toxicity may be achievable simply because the parasites replicate rapidly, requiring therapeutic levels of drug that have minimal effect on host cells and systems. Second, parasite chromatin remodelling enzymes have significant divergent sequence outside of the catalytic domain that could be targeted selectively by small molecule inhibitors. There are also recent examples that show small divergence within catalytic domains could be sufficient to achieve selective toxicity. Apicomplexan HDAC3 is more susceptible to the KDAC inhibitor FR235222 due to the presence of a unique 2-amino acid insertion in the catalytic domain (Bougdour et al., 2009). In contrast to KDAC inhibitors, surprisingly few specific KAT inhibitors are available. Anacardic acid and curcumin inhibit Plasmodium replication and can inhibit PfGCN5 in vitro, but these compounds are believed to have many offtarget effects (Cui et al., 2007, 2008). As epigenetic mechanisms contribute to bradyzoite conversion, it may be possible to short-circuit this important pathogenic process with small molecules. Tachyzoites treated with the KDAC inhibitor FR235222 convert into bradyzoites and

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this compound may have activity against ex vivo Toxoplasma tissue cysts (Bougdour et al., 2009; Maubon et al., 2010). Genetic studies suggest that pharmacological interference of TgGCN5-A might be able to thwart bradyzoite cyst gene expression (Naguleswaran et al., 2010). Interference with parasite histone methylation may also disrupt control of differentiation, and inhibitors specific for Plasmodium methyltransferases have recently been reported to alter expression of variant genes (Malmquist et al., 2012). While the reasons remain unclear, pre-treating tachyzoites with a CARM1 (PRMT4) inhibitor leads to a higher frequency of bradyzoite development upon infecting host cells in vitro (Saksouk et al., 2005a). An important consideration with respect to targeting histone modification enzymes is the recent set of studies demonstrating the abundance of non-histone substrates for these enzymes (Smith and Workman, 2009). Several Toxoplasma KATs, namely the MYST KATs, are found predominantly outside of the parasite nucleus. An acetylome has been published for Toxoplasma tachyzoites, revealing that lysine acetylation is widespread across proteins of diverse function and location within the parasite (Jeffers and Sullivan, 2012). The activity of KDAC inhibitors, therefore, may not be limited to dysregulation of gene expression, but could exert their antiparasitic effect through inhibition of cytosolic substrate acetylation (Jeffers and Sullivan, 2012). While less extensively studied than lysine acetylation, it appears that methylation of T. gondii proteins of diverse function also occurs (Heaslip et al., 2011; Xiao et al., 2010) and inhibitors of these enzymes may also have specific antiparasitic activities.

18.4 POST-TRANSCRIPTIONAL MECHANISMS IN TOXOPLASMA 18.4.1 Translational Control Examples of post-transcriptional mechanisms that regulate the level of specific proteins among

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protozoa have been described (Rochette et al., 2005; Larreta et al., 2004; Chow and Wirth, 2003; Garcia-Salcedo et al., 2002; Shapira et al., 2001). Transcription in the kinetoplastidae such as Leishmania and Trypanosoma is polycistronic and post-transcriptional trans-splicing mechanisms are required to achieve mature mRNA (Campbell et al., 2003; Shapira et al., 2001). Thus, in Leishmania and Trypanosoma species, mRNA levels are dictated mostly by posttranscriptional processing and the stability of the mRNA itself (Purdy et al., 2005a, b; Webb et al., 2005b; Cevallos et al., 2005; Webb et al., 2005a; Haile et al., 2003). RNA-binding proteins with demonstrated roles in the regulation of translation and/or RNA stability have been found in Plasmodium, including Puf2 (Miao et al., 2010; Muller et al., 2011; Gomes-Santos et al., 2011), the DDX6-class RNA helicase, DOZI (development of zygote inhibited) (Mair et al., 2006), and Alba proteins (Chene et al., 2012; Goyal et al., 2012). Other transcriptionassociated proteins with known roles in modulating mRNA decay and translation were also found in the genome (Coulson, 2004). The formation of stress-induced RNA granules has been reported for a number of parasite species, including Toxoplasma (Cassola, 2011; Lirussi and Matrajt, 2011). Such RNA granules are proposed to be holding areas for translationally regulated mRNAs. As such, indications of posttranscriptional control have been described for protozoal genes with defined roles in differentiation (Vervenne et al., 1994; Dechering et al., 1997; Sullivan et al., 2004; Narasimhan et al., 2008; Miao et al., 2010), mitochondrial RNA processing (Rehkopf et al., 2000), and surface antigens (Lanzer et al., 1993; Levitt et al., 1993; Spano et al., 2002). In Toxoplasma, unbalanced ratios of mRNA and protein have been observed for SAG-related Toxoplasma surface proteins, designated SAG5A, SAG5B and SAG5C (Spano et al., 2002), and for mRNAs encoding the proliferating cell nuclear antigens, TgPCNA1 and TgPCNA2 (Guerini et al., 2000). TgPCNA1

mRNA was found to be 7-fold higher than that of TgPCNA2, yet TgPCNA1 and -2 on Western blots were expressed at nearly equally levels in all strains examined (Guerini et al., 2000). In the context of stage-specific gene expression in Toxoplasma, mRNAs encoding bradyzoite-specific proteins G6-PI and MAG1 can also be detected during the tachyzoite stage, and mRNA encoding tachyzoite-specific proteins like LDH1 can also be detected in bradyzoites (Yang and Parmley, 1997; Dzierszinski et al., 1999; Weiss and Kim, 2000). These discrepancies in the levels of mRNA and protein indicate that post-transcriptional events occur in apicomplexan parasites, although the mechanisms of translational control are just beginning to be elucidated. Significant advancements have taken place in the past decade particularly in the area of translational control through the phosphorylation of eukaryotic initiation factor-2 alpha subunit, eIF2a. The eIF2 complex governs the rate-limiting step in the initiation of protein synthesis. In response to a wide variety of cellular stresses, eIF2a becomes phosphorylated, leading to a global cessation in translation except for a subset of mRNAs encoding transcription factors that reprogramme the expressed genome to enable cell survival mechanisms (Wek et al., 2006). The eIF2a translational control pathway was first characterized in Toxoplasma and linked to stress-induced bradyzoite development; moreover, TgIF2a remains in its phosphorylated state during the latent cyst stage (Sullivan et al., 2004; Narasimhan et al., 2008). A specific inhibitor of TgIF2a dephosphorylation was also found to trigger bradyzoite differentiation, supporting the idea that translational control is a major contributor to the development and maintenance of microbial latency (Joyce et al., 2011; Narasimhan et al., 2008). Studies in Plasmodium as well as kinetoplastid parasites lend further support to this model (Zhang et al., 2010; Chow et al., 2011). Translational control through the phosphorylation of parasite eIF2a has significant roles

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beyond the modulation of latency and appears to be required even for normal progression through lytic cycles. Allelic replacement of the endogenous TgIF2a gene with a version incapable of being phosphorylated on the regulatory serine residue (Ser-71) exhibits fitness defects in vitro and in vivo that have been linked to decreased viability following egress from its host cell (Joyce et al., 2010). A similar allelic replacement produces nonviable parasites in Plasmodium, demonstrating that PfIF2a phosphorylation is essential during the erythrocytic cycle (Zhang et al., 2012) Four eIF2a kinases have been identified in the Toxoplasma genome, designated TgIF2K-A through -D. TgIF2K-A localizes to the parasite endoplasmic reticulum (ER) and interacts with GRP/BiP in a stress-dependent manner, making it a likely orthologue of PERK, an eIF2a kinase in higher eukaryotes that contributes to the unfolded protein response (UPR) that allows cells to adapt to ER stress (Narasimhan et al., 2008). PERK homologues have also been found to be critical in Plasmodium and Leishmania (Chow et al., 2011; Zhang et al., 2012). TgIF2K-B appears to be a cytoplasmic eIF2a kinase that is specific to Toxoplasma and its function has yet to be resolved. TgIF2K-C and -D are two GCN2-like kinases, which in other species are well-characterized responders to nutritional stress. To date, it has been determined using knock-out strategies that TgIF2K-D is the primary kinase phosphorylating TgIF2a upon egress from the host cell, and its loss phenocopies the non-phosphorylatable TgIF2a mutant (Konrad et al., 2011). A GCN2-like kinase in Plasmodium called PfeIK1 has been found to regulate the parasite’s response to amino acid starvation (Fennell et al., 2009). A key area of current investigation is linking translational control to transcriptional control. In other eukaryotes, following eIF2a phosphorylation, a select group of mRNAs is preferentially translated due to the presence of upstream open reading frames in the 50 UTR (untranslated

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region) (Vattem and Wek, 2004). These messages tend to encode basic-leucine zipper transcription factors such as GCN4/ATF4, which activate genes that facilitate the cellular adaptive response. Such transcription factors are not present in Apicomplexa, which probably use a subset of AP2 factors to regulate transcription. Employing the ability to generate polyribosome profiles in Toxoplasma (Narasimhan et al., 2008), it has been shown that several messages encoding AP2 factors are preferentially translated during stress in these parasites (Sullivan, unpublished). Future studies are required to determine if the mechanism of translational control is analogous to other species.

18.4.2 Noncoding and Small RNA One of the most exciting discoveries over the past two decades has been the role of small RNAs and longer RNAs in regulation of gene expression. T. gondii small RNAs have been catalogued (Wang et al., 2012; Braun et al., 2010) and RNA-seq studies have also identified long ncRNA (lncRNA) (Hassan et al., 2012; Kim, unpublished). Many parasitic species use small RNAs to regulate gene expression. Unlike Plasmodium species, T. gondii encodes the essential components of the RNA induced silencing complex (RISC), including a single Argonaute protein, a Dicer protein (with RNAseIII catalytic domains) and an RNA-dependent RNA polymerase, suggesting that the RNA silencing pathway is fully functional (Braun et al., 2010; Al Riyahi et al., 2006). The single T. gondii Argonaute protein is not predicted to have slicer activity and thus the RISC complex is proposed to regulate gene expression by translation repression rather than RNA degradation (Braun et al., 2010). The mechanistic details of the RISC complex activity are not yet completely clear because methylation of TgAgo1 results in recruitment of a staphylococcal endonuclease (Musiyenko et al., 2012) that alters the activity of the RNA slicing complex as well as changing

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the specificity of the slicing from mismatches to perfect match RNA targets. Characterization of the small RNAome of T. gondii by RNA-seq led to the identification of 14 miRNA families that were unique without significant homology to known miRNA of plants and metazoan, but most were conserved in Neospora (Braun et al., 2010; Reid et al., 2012). A second study identified 17 conserved miRNA and 339 novel miRNA (Wang et al., 2012). Many miRNAs are recruited to the Ago complex to affect RNA turnover or translation. Some of the miRNA families of T. gondii were associated with polysomes, consistent with a role in translational repression. In addition, several of the miRNA families were differentially expressed in the Type I, II and III lineages and differences were also seen in extracellular versus actively replicating parasites (Wang et al., 2012; Braun et al., 2010). The targets of these miRNA have been predicted, but not yet validated (Braun et al., 2010). A second group of small RNAs matched the repetitive elements REP1e3, mitochondrial-like sequences dispersed throughout the T. gondii genome (Braun et al., 2010). The dispersal of these elements has features suggestive of transposon dissemination and one proposed function of the RNAi pathway in T. gondii is to prevent expression of these elements (Braun et al., 2010). A third class of repeat associated small RNAs were identified that are proposed to maintain heterochromatin state at satellite DNA within the nucleus (Braun et al., 2010). At present the single Ago protein is proposed to mediate all the potential nuclear and cytoplasmic activities of the T. gondii RISC complex. Some of the miRNA families were associated with immunopurified TgAgo, as were proteins associated with RNA processing and the chromatin co-repressor complex (Braun et al., 2010). Despite reports of successful RNAi in T. gondii (Al-Anouti et al., 2003; Al Riyahi et al., 2006), widespread RNAi has not been documented and efforts by several groups to develop RNAi

methodology for T. gondii gene knock-down have failed (Matrajt, 2010) and reports of successful RNAi has been limited a few select genes (Al-Anouti et al., 2003, 2004; Ananvoranich et al., 2006). These observations suggest that the RNAi pathway has a specialized biological function in T. gondii. Consistent with this hypothesis, in the virulent RH strain, conditional disruption of the single Argonaute gene AGO1 has a very modest phenotype (Musiyenko et al., 2012). Future evaluation and functional validation of the catalogued miRNA of T. gondii should establish the function and importance of these RNA species. There is also circumstantial evidence that long non-coding RNA (ncRNA) will have important roles in the biology of T. gondii. While the functions of long ncRNA are only now emerging, Plasmodium falciparum has several long ncRNA of interest, including a non-coding transcript associated with silenced var genes that is transcribed from a promoter located in the conserved var intron (Calderwood et al., 2003). In addition, there are long ncRNA associated with telomeric regions of Plasmodium that have been hypothesized as important for chromosome stability (Calderwood et al., 2003; Sierra-Miranda et al., 2012; Broadbent et al., 2011). Long ncRNA are associated with developmental regulation in the metazoa (Braun et al., 2010). Several groups have discovered long ncRNA that are upregulated during the process of bradyzoite differentiation (Matrajt, 2010). Some of these long ncRNA are antisense to sense transcripts, raising the hypothesis that long ncRNA may play a role in translational regulation or mRNA stability during the stress response. Finally, inspection of RNA-seq studies reveals many instances of antisense transcripts that could potentially regulate gene expression of specific genes that are important for the host pathogen interaction (see http://www.toxodb. org for numerous RNA-seq datasets from various strains and developmental conditions). The antisense transcripts are often present in

18.5 CONCLUSIONS AND FUTURE DIRECTIONS

either 50 or 30 UTR of genes. At present the significance of these anti-sense RNAs is not known, but these could potentially act as an important regulator of cell cycle progression or developmental transitions.

18.4.3 Other Post-Transcriptional Mechanisms Another completely unexplored area is the role of RNA processing, RNA trafficking and RNA stability in T. gondii gene regulation. While cell cycle transcription is likely to explain the cell cycle periodicity of mRNA, mRNA degradation must also have a prominent role in regulation of steady state RNAs. As in other eukaryotes, T. gondii encodes numerous proteins with predicted RNA binding domains. One of these, RRM1, has been demonstrated to have an essential and conserved role in RNA splicing (Suvorova et al., 2013). In other systems, alternative splicing and RNA stability are mechanisms by which gene expression is regulated posttranscriptionally, and these events can be influenced by the metabolic state of the cell. Both chromatin remodellers and TgAP2 appear to interact with RNA binding proteins or the splicing machinery (Kim, White, Sullivan, unpublished) implicating regulation of RNA splicing as another area of potential importance in T. gondii. Long ncRNA have also been implicated in the regulation of splicing and RNA trafficking (Guttman and Rinn, 2012).

18.5 CONCLUSIONS AND FUTURE DIRECTIONS We now have sufficient knowledge of global mRNA expression in Plasmodium and Toxoplasma to conclude that transcriptional mechanisms play a major role in regulating the developmental programme of these parasites. The observations that co-regulated genes are dispersed across parasite chromosomes, along with the

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presence of much of the conventional eukaryotic transcriptional machinery in the Apicomplexa genomes including chromatin remodellers, is consistent with growing evidence that promoter structures in these parasites contain cis-elements that are regulated by trans-acting factors. The discovery and preliminary characterization of plant-like AP2 DNA-binding domains in Apicomplexa has been a major new advance towards completing our picture of transcriptional regulation in these parasites. ApiAP2 DNA binding domains represent the largest family of putative transcription factors discovered in Apicomplexa parasites. It is early in the study of ApiAP2 factors, yet already there are examples indicating that these factors operate as activators or suppressors of development and stress-responsive gene expression. It is expected that in the next few years, an inventory of sorts will be generated matching specific ApiAP2 factors to the mRNAs they regulate. The new roles for ApiAP2 factors in chromosome structure emerging from studies in Plasmodium and Toxoplasma are an exciting new development. There is no evidence that in plants AP2s act to maintain or modify chromosome structure, although the majority of these plant AP2s have not been characterized. AP2 proteins are not found in mammals. Thus, AP2 related mechanisms may be a novel invention in the Apicomplexa and a target for therapeutic development. Recent studies have also made it clear that epigenetic-based gene regulation provides an important contribution to Toxoplasma gene expression, with several links to stage-specific gene expression now well established for a variety of different types of histone modifying enzymes (Dixon et al., 2010). In higher eukaryotes, chromatin remodelling machinery and DNA-binding transcription factors work in concert to modulate gene expression, and emerging studies suggest that Toxoplasma is no exception. A great deal of work remains, however, in characterizing the large array of chromatin remodelling enzymes

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in Toxoplasma and understanding how these machineries are recruited to target promoters to work coordinately with TgAP2s or other possible DNA-binding factors. Equally important is the characterization of chromatin ‘reader’ proteins that harbour motifs that can bind specific histone modifications. As initial results with pharmacological agents that interfere with chromatin modifying enzymes have shown promise, epigenetic-based gene regulation remains a high priority for future investigation. It is also critical to characterize how cellular signals are interpreted by the parasite to result in a reprogramming of gene expression, particularly changes associated with stage conversion. These processes are likely to involve sensing metabolic fluxes and small metabolites, and integrating these changes to alter gene expression. Considerable preliminary data now suggest that mechanisms of translational control and other means of post-transcriptional gene regulation warrant more attention into how they interplay with more conventional components of transcriptional control.

Acknowledgements Research in our laboratories is supported by the following grants: NIH AI77502 (to WJS), AI087625 (to KK), AI077662 and AI089885 (to MWW) and RC4 AI092801 (to KK, WJS, MWW). We thank Sheila Nardelli for preparation of Fig. 18.2.

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