Malaria persists as a global burden to public health and in 2019, was responsible for 409000 deaths. Of the five Plasmodium species that infect humans, P. falciparum is the most likely to cause severe disease and accounts for the vast majority of deaths from malaria (WHO, 2020). The complexities of the P. falciparum life cycle are evident from the array of developmental stages and cycles that are compartmentalized into diverse host cell types within both the human and the mosquito vector (Sherman, 1979). The unique biology of each stage is underpinned by the expression of stage-specific gene sets, regulated at the epigenetic, transcriptional, post-transcriptional, and post-translational levels (Bozdech et al., 2003; Young et al., 2005; Lopez-Barragan et al., 2011; van Biljon et al., 2019). Recent studies involving a diverse set of chromatin-based technologies, including a combination of chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq), quantitative mass spectrometry (MS), fluorescence in situ hybridization (FISH), and histone mutagenesis and transcriptomics (microarrays and RNA-sequencing), have revealed that histone post-translational modifications (PTMs) are foundational in generating these stage-specific gene expression fingerprints and are thus proposed to be central to P. falciparum parasite life cycle regulation (Comeaux and Duraisingh, 2007; Issar et al., 2009; Trelle et al., 2009; Crowley et al., 2011; Kaur et al., 2016; Saraf et al., 2016; Coetzee et al., 2017; Gomez-Diaz et al., 2017; Gupta and Bozdech, 2017; Gupta et al., 2017; Ngwa et al., 2017; Herrera-Solorio et al., 2019; Witmer et al., 2020; Connacher et al., 2021; Kumar et al., 2021; Ngwa et al., 2021). Here, we review the current knowledge regarding epigenetic regulation in P. falciparum parasites with a specific focus on the recent developments in our understanding of how histone PTM landscapes orchestrate life cycle progression.

Malaria infections are initiated after sporozoite infection of liver cells. After exoerythrocytic development (EDC), merozoites are released into the peripheral circulation to invade erythrocytes, initiating a new asexual intraerythrocytic developmental cycle (IDC) (Figure 1) (Kappe et al., 2004). Here, ring, trophozoite and schizont development leads to population expansion and the release of new daughter merozoites to repeat the cycle (Dvorak et al., 1975). Parasite survival and transmission are ensured by the divergence of a small proportion (≤10%) of the parasites that commit to sexual differentiation. In P. falciparum, this process is uniquely associated with stage differentiation with stage I gametocytes sequestering in the bone marrow to maturate into stages II-IV (Figure 1) (Sinden et al., 1978; Carter and Graves, 1988; Aguilar et al., 2014; Joice et al., 2014). This process of gametocytogenesis yields mature stage V male and female gametocytes that re-enter the host’s circulatory system for transmission to female Anopheles mosquitoes during feeding (Smalley et al., 1981; Aguilar et al., 2014). Since stage V gametocytes are the only transmissible forms of the malaria parasite, they have become the focus of the discovery and development of transmission-blocking compounds to aid in malaria elimination strategies (Delves, 2012; Birkholtz et al., 2016). Following gametogenesis of micro- (male) and macro- (female) gametocytes in the mosquito midgut, haploid gametes are fertilized to produce a diploid zygote that transforms into a tetraploid motile ookinete (Figure 1). The maturation of ookinetes into oocysts results in the formation of sporozoites, which can be transmitted to a new host during feeding (Sinden et al., 1978; Billker et al., 1998).

The P. falciparum nucleosome consists of 147 bp of DNA associated with an octamer of four histone proteins, typically the H2A, H2B, H3, and H4 core histones, each of which display particular functional and stage-specific characteristics (Figure 2) (Miao et al., 2006; Trelle et al., 2009). Within certain nucleosomes, these core histones may be exchanged for their variants, namely, H2A.Z, H2B.Z, H3.3, and H3Cen, which may also be modified by an array of chemical groups. These variant histones are typically incorporated to demarcate or influence the properties of specific chromatin domains. For example, distinct intergenic regions of the P. falciparum genome are demarcated by double-variant nucleosomes containing H2A.Z and H2B.Z while H3.3 is incorporated to index euchromatic coding regions and subtelomeric repeat regions (Bartfai et al., 2010; Hoeijmakers et al., 2013; Petter et al., 2013; Fraschka et al., 2016). Despite sequence divergence, P. falciparum histones have not adapted increased binding affinity for the extremely A + T-rich (∼80%) genome (Gardner et al., 2002; Silberhorn et al., 2016). Combined with the apicomplexan-specific absence of the H1 linker histone (Gardner et al., 2002; Sullivan et al., 2006) and short nucleosome repeat length (155 bp) (Silberhorn et al., 2016), this evolution results in a general lack of stable nucleosome patterns in asexual P. falciparum parasites (Hoeijmakers et al., 2012). Except for a few internal gene clusters that are brought into proximity with subtelomeric heterochromatin via chromatin looping, typical eukaryotic topologically associating domains are therefore largely absent in P. falciparum parasites (Lemieux et al., 2013; Bunnik et al., 2014; Bunnik et al., 2018).

Three-dimensional chromatin structure influences the accessibility of DNA to chromatin-binding proteins and transcription factors, thereby enhancing or repressing gene expression (Zheng and Xie, 2019). As such, the rearrangement of genome spatial organization occurs throughout the parasite’s life cycle and corresponds with changes in transcriptional activity (Ay et al., 2014; Bunnik et al., 2014). A relatively simple and dichotomous nuclear organization characterizes the asexual parasite with the majority of the genome (∼90%) in a euchromatic state and with the remainder of the DNA present in perinuclear heterochromatic centers (Hoeijmakers et al., 2012; Brancucci et al., 2014; Coleman et al., 2014). Trophozoites exhibit the most euchromatic and accessible genome organization, in line with the increased level of transcriptional activity required in preparation of schizogony (Ay et al., 2014). Contrasting with this restricted distribution of heterochromatin in the asexual parasite stages, repressive factors become more prolific in the gametocyte stages (Brancucci et al., 2014; Coetzee et al., 2017; Bunnik et al., 2018; Fraschka et al., 2018), reflecting the specific transcriptional environment that underlies sexual differentiation and development (Poran et al., 2017; Walzer et al., 2018; van Biljon et al., 2019).

Chromatin status is largely influenced by post-translational modification of the N-terminal tails of histone proteins that make up the core of the nucleosome (Goll and Bestor, 2002). Quantitative mass spectrometry and antibody-based techniques such as Western blotting has been used to identify histone PTMs in P. falciparum parasites with acetylated, methylated, phosphorylated, ubiquitinated, and SUMOylated histones quantitatively detected throughout the life cycle (Miao et al., 2006; Salcedo-Amaya et al., 2009; Trelle et al., 2009; Treeck et al., 2011; Dastidar et al., 2013; Gupta et al., 2013; Ukaegbu et al., 2014; Cobbold et al., 2016; Kaur et al., 2016; Saraf et al., 2016; Coetzee et al., 2017; Gupta et al., 2017; Bui et al., 2019; Green et al., 2020). Acetylation and methylation marks indeed form the major component of histone PTMs in P. falciparum and have been accurately detected and quantified over multiple life cycle stages. This provides confidence as to their biological relevance, and indeed, a large number of these marks have been extensively functionally validated. The same is not true for the less prevalent marks, particularly for crotonylation and formylation. Quantitative detection of less abundant (or perhaps time-point specific) histone PTMs, including crotonylation, require antibody-based enrichment of histone PTMs prior to mass spectrometric analysis where, for example, H2AK3crK5cr, H2BK18cr, and H4K78cr was identified in P. falciparum asexual parasites (Wang and Zhang, 2020). However, besides detection, no information on the importance of crotonylation to parasite development is currently available and it remains to be seen if this histone PTM is functionally distinct from acetylation in the parasite. Remarkably, lysine crotonylation catalyzed by p300 increased transcriptional activation more potently than lysine acetylation in a cell-free system (Sabari et al., 2015) but such functional validation of a biological role for this histone PTM in Plasmodium is lacking.

In addition to individual PTMs, histones of mammalian cells for example are also readily modified with distinct patterns of co-existing PTMs at multiple sites, e.g., methylation of lysine 4 with lysine 9 acetylation of histone H3 (Muller and Muir, 2015). While the functional relevance of a relatively small number of single histone PTMs is well documented, evidence indicating that histone PTMs act in concert with one another to regulate transcriptional programs suggests that combinatorial histone PTMs landscapes contribute an additional layer of regulation in eukaryotes (Berger, 2002; Zhao and Garcia, 2015). The compendium of co-existing histone PTMs on specifically histone H3 and H3.3 in P. falciparum parasites was recently updated and revealed that PTM combinations are prevalent in asexual parasites and gametocytes and are considerably diverse and stage-stratified, as determined by quantitative mass spectrometric analysis of whole histone N-terminal tails (von Grüning et al., 2022). Histone PTM crosstalk influences the addition, removal, and binding of effector proteins between histone PTMs in combination (Hunter, 2007). While the enzymes responsible for depositing and removing these individual and combinatorial PTMs are somewhat divergent from other organisms based on homology (Miao et al., 2006; Cui et al., 2008a; Miao et al., 2010a), P. falciparum parasites employ a typical eukaryotic histone PTMs repertoire (Miao et al., 2006; Chung et al., 2009; Saraf et al., 2016; Coetzee et al., 2017), including functional acetylation, methylation and phosphorylation (Figure 3) to expand the regulatory capacity of nucleosomes using a limited set of effector proteins (summarized in Table 1).

The methylation of histones in P. falciparum is mediated by ten histone methyltransferases (HMTs), all of which belong to the evolutionarily conserved SET [Su (var)3-9, Enhancer of Zeste and Trithorax] domain-containing protein family (Cui et al., 2008a). The site specificities of all, except two (SET5 and SET9) of these HMTs have been determined through recombinant protein expression, pull-down proteomics, mutagenesis, and histone lysine methyltransferase (Cui et al., 2008a; Volz et al., 2012; Jiang et al., 2013; Chen et al., 2016; Ngwa et al., 2021) (Table 1). Less is known regarding the three Jumonji-C (JmjC) domain-containing histone demethylases (HDMs) and the one lysine-specific demethylase 1 (LSD1) that demethylate histones in P. falciparum, however, JMJC1 and JMJ3 have predicted and known H3K36-specificity (Cui et al., 2008a; Matthews et al., 2020). Although the precise mechanisms by which histone methyltransferases and demethylases modify histones remain relatively unclear, several studies have demonstrated their importance for asexual and sexual stage development through genetic disruption and chemical interrogation (Jiang et al., 2013; Ukaegbu et al., 2014; Zhang et al., 2018; Coetzee et al., 2020; Matthews et al., 2020; Connacher et al., 2021; Reader et al., 2021) (Table 1).

Histone acetylation is regulated by four histone acetyltransferases (HATs) including proteins from the MYST and GNAT families (Cui et al., 2008a). The acetyltransferase activities of GCN5 and MYST are specific to the lysine residues of H3 and H4, respectively (Cui et al., 2007b; Miao et al., 2010a), with the functional importance of these enzymes evident from the transcriptional deregulation arising from their genetic and chemical disruption (Cui et al., 2007b; Cui et al., 2008b; Chaal et al., 2010; Bhowmick et al., 2020; Miao et al., 2021) (Table 1). Acetyltransferase activities are antagonized by a repertoire of three histone deacetylases (HDACs) and two sirtuin histone deacetylase proteins in P. falciparum (Kanyal et al., 2018). Interestingly, the P. falciparum genome encodes for an essential YEATS-domain protein (PF3D7_0807000). Its orthologues in mammalians and yeast are readers of crotonylation. It would therefore be interesting to see if similar activity is present in Plasmodia and if so, crotonylation could be important to biological processes in the parasite despite it being a rarely identified PTM (Table 1).

The chromatin reorganization induced by these enzymes results either from a direct physical change in nucleosome structure or the recruitment of epigenetic “reader complexes” that mediate subsequent chromatin remodeling (Latchman, 2010; Abel and Le Roch, 2019) (Table 1). To date, five putative reader complexes have been identified in P. falciparum parasites, however, the vast majority remain to be functionally validated (Hoeijmakers et al., 2019). These P. falciparum readers largely exist in complexes with other reader proteins and/or transcription factors that would presumably require sequential binding occurrences for recruitment (Hoeijmakers et al., 2019). Such cooperation between epigenetic regulatory proteins is exemplified in the recruitment of the GCN5/ADA2 reader complex by H3K4me3 via interaction with PHD1 (Hoeijmakers et al., 2019). Additionally, evidence suggests that as in other organisms, epigenetic complexes in P falciparum may be recruited to or “flavored” to specific histone PTMs or combinations of PTMs, as determined by peptide pull-down coupled with mass spectrometry. For example, GCN5/ADA2 core reader complex has at least two distinct “flavors” with a unique composition of reader proteins, such as the presence or absence of PHD1 or PHD2 (Hoeijmakers et al., 2019; Miao et al., 2021). Additionally, the intricate nature of multiprotein reader complexes supports the existence of unique sets of functional histone PTMs combinations in P. falciparum parasites whereby different reader proteins bind to PTMs using reader domains. Whether these modifications directly influence chromatin structure via the recruitment of such readers or by virtue of their biochemical properties, HMs are undoubtedly at the core of epigenetic regulation.

Although up to 230 histone PTMs have been identified in various qualitative and quantitative proteomic based approaches P. falciparum parasites (Salcedo-Amaya et al., 2009; Trelle et al., 2009; Saraf et al., 2016), only 46 of these have been quantitatively validated (Coetzee et al., 2017) (Figure 3). The P. falciparum histone PTM repertoire expands beyond the typical eukaryotic histone modifications (e.g., H3K4me3, H3K9me3, and H3K36me3) and contains several less common modifications, including H4K20ac, H3.3K79me3, and H3K23me1 (Coetzee et al., 2017). Similar to the hierarchical cascades of gene expression that are associated with each asexual and sexual stage (Bozdech et al., 2003; van Biljon et al., 2019), strikingly unique and stage-specific histone PTM landscapes characterize each stage of the P. falciparum parasite life cycle (Figure 3) (Coetzee et al., 2017).

Given the central nature of histone PTMs for P. falciparum reviewed here, further understanding the mechanistic relevance of individual and combinatorial PTMs will help identification of the most suited epigenetic targets for drug-based intervention. Although the stage-specific functional relevance of certain individual histone PTMs has been elucidated, the remaining PTMs and combinations with stage-specific patterns should be investigated. Specifically, the influence of these PTMs on asexual and sexual development and the writers, erasers and readers of the histone PTM landscapes and thus insights into the direct or indirect consequences on transcription. In the future, we foresee the use of the combinatorial histone code as a “barcode” that could define parasite life cycle stages or ongoing biological processes. This barcode is dynamic and associated with remarkable changes to allow shifts in chromatin status in different developmental stages of the parasite. In-depth critical analyses of the available data clearly implicate this changing histone PTM landscape as higher-order mechanisms of regulation of gene expression, providing a clear blueprint associated with required phenotypic changes in the parasite to adapt to host responses (antigenic variation), allow massive population expansion in the host (asexual proliferation) and exploiting host environments to allow differentiation and species continuation (sexual development). However, as histone PTMs are not isolated but co-exist to fine-tune binding of effector proteins, the PTM landscape and combinatorial code may indeed guide a highly specific transcriptional program. Lastly, the cause and/or effect relationship between nutrient metabolism and available substrates to modify the histones requires deconvolution. Taken together, the findings discussed in this necessary update demonstrate that although many factors, including nucleosome positioning, chromatin structure and transcription factors participate in transcriptional regulation in P. falciparum parasites, the histone PTM landscapes represent the road maps that are central to the specific biology of each stage. The demonstration of histone PTMs as key determinants of both asexual proliferation and sexual differentiation of P. falciparum parasites highlights the individual factors as novel targets for disrupting malaria transmission and underscore the importance of a more thorough understanding of these critical regulators.