ApiAP2 Gene-Network Regulates Gametocytogenesis in Plasmodium Parasites

Malaria is a mosquito-borne infectious disease, caused by unicellular Apicomplexan protozoa of the genus Plasmodium . The sexual stage of Plasmodium is one of the most fascinating aspects of the Plasmodium life cycle, yet relatively less explored until now. The production of sexually ﬁ t gametocytes through gametocytogenesis is essential to the transmission of the Plasmodium parasite into an anopheline mosquito vector. Understanding how gametocytogenesis is regulated promotes the identi ﬁ cation of novel drug targets and also the development of transmission-blocking vaccines that would help reduce the disease burden in endemic areas. Transcriptional regulation in Plasmodium parasites is primarily controlled by a family of twenty-seven Apicomplexan Apetela 2 (ApiAP2) genes which act in a cascade to enable the parasite to progress through its asexual replication as well as gametocytogenesis. Here, we review the latest progress made on members of the ApiAP2 family characterized as key players of the transcriptional machinery of gametocytes. Further, we will highlight the transcriptional regulation network of ApiAP2 genes at each stage of gametocytogenesis.


Introduction
Malaria is considered as one of the diseases that has existed for centuries; it remains one of the devastating infectious diseases ever known. Its persistence till now is a reflection of the resilience of its etiological agent-Plasmodium-to crucially maintain transmission between mosquito vector and vertebrate hosts. The Plasmodium parasite has resisted eradication attempts in sub-Saharan Africa and persists in South-East Asia, Eastern Mediterranean, and the Americas [1]. From the latest world malaria report, a global estimate shows that there are about 241 million cases of malaria, and it accounts for an estimated 627,000 deaths worldwide [2].
The life cycle of Plasmodium is characterized by the transition of morphologically, metabolically, and molecularly unique life forms within a vertebrate host and anopheline mosquito vector. As shown in Figure 1, during a blood meal, a female anopheline mosquito inoculates sporozoites into the blood stream, which travel to the liver and undergo asexual replication to produce pockets of schizonts containing thousands of merozoites [3]. The schizonts rupture to release merozoites that enter the systemic circulation and subsequently invade red blood cells marking the initiation of an intraerythrocytic lytic cycle. The intraerythrocyte parasite forms are responsible for most malaria-associated pathologies. Within the erythrocytes, the parasites undergo asexual morphological transformations: early ring, trophozoite, and schizont. The schizonts eventually produce new asexual merozoites to continue invading new erythrocytes. Alternatively, the asexually replicating intraerythrocytic parasite forms differentiate into nonreplicative sexual gametocytes. The transition of intraerythrocytic parasites to gametocyte (gametocytogenesis) truncates the replication of the parasite within the vertebrate host; however, this process is crucial for transmission into a mosquito vector [4]. Only a limited proportion of the intraerythrocytic parasites complete the asexual-sexual switch [5][6][7]. For instance, less than 10% of Plasmodium falciparum (P. falciparum) intraerythrocytic parasites successfully differentiate into gametocytes in each 48 h cycle [5].
The search to uncover transcriptional regulators of gametocytogenesis is now a domain of intense focus due to their attractive prospects of serving as direct targets of transmission-blocking vaccines and drugs. A family of widely conserved DNA-binding proteins, Apicomplexan Apetela 2 (ApiAP2), have emerged as the key developmental regulators of transcription in Plasmodium species [8]. The ApiAP2 genes are homolog to AP2/ERF transcriptional factors from plant lineage and structurally possess one to three DNA-binding AP2 domains [9,10]. To date, a total of 27 annotated ApiAP2 genes have been described in P. falciparum with syntenic orthologs in Plasmodium berghei (P. berghei) and Plasmodium yoelii (P. yoelii) ( Table 1). Of interest, a subset of ApiAP2 genes, comprising AP2-G, AP2-G2, AP2-G3, and AP2-G5, are particularly integral to the transcriptional machinery of gametocytogenesis among Plasmodium species [11,12]. Functional genomic studies show that the transcriptional regulatory footprints of ApiAP2 genes putatively cover different stages of the entire life cycle of Plasmodium species (Table 1). Indeed, investigations into the transcriptional regulation of Plasmodium genes by ApiAP2 transcription factors have developed significantly over the last few years making it appropriate for a review. Here, we review the latest work on members of the ApiAP2 family that are essential to the transcriptional machinery of gametocytes. We will cover their involvement in each stage of gametocytogenesis spanning from sexual commitment to sexual maturation and highlight the ApiAP2 gene regulatory network that governs gametocytogenesis.

Regulation of Gametocytogenesis by AP2-G
3.1.1. AP2-G Phenotypes. In P. falciparum, the binding site of PfAP2-G is identified across three parasite stages: schizont, ring , and stage I gametocytes [24]. Electrophoretic mobility assay analysis reveals the binding motif of PfAP2-G as G × GTAC×G [25]. Transcriptional control by PfAP2-G is achieved by binding to (G/G) TAC and its reverse GTA (C/C) motifs, which were found upstream of hundreds of putative regulons including ap2-g itself [24,25]. In P. berghei, ChIP-seq analysis showed that the binding site of PbAP2-G is constituted of a pair of sixbase motifs, GTACTT and GTACAC [11].
Gametocytogenesis thrives under the control of different but functionally linked transcription factors. Of these, AP2-

Cellular Microbiology
G is a central factor that plays an indispensable role in ushering asexual Plasmodium parasites into the sexual cycle. AP2-G is dubbed the "master regulator" of gametocytogenesis. The central role of AP2-G required for the asexualsexual switch was first shown in P. falciparum and P. berghei which harbored mutations in ap2-g and had lost the capacity to produce gametocytes [25,26]. Recently, ChIP-seq analysis of anin vitro cultured transgenic P. berghei has substantiated the essentiality of AP2-G in gametocyte production and provided insight into its underpinning mechanism. This study posited that PbAP2-G is located at the summit of a gametocytogenesis-specific transcriptional cascade encompassing several ApiAP2 transcriptional factors [11]. The downstream transcription factors include AP2-G2, AP2-FG, and AP2-O3 [11]. As such, the expression of a fully functional AP2-G switches on the gametocytogenesisspecific transcriptional cascade [24,25,27] and thereby activates the regulatory control by downstream ApiAP2 transcription factors culminating in the production of a high number of gametocytes. On the opposites, AP2-G inactivation or disruption generates a negative ripple effect on downstream gametocyte-specific transcription factors resulting in a complete loss of capacity to produce gametocytes. Interestingly, the role of AP2-G as the "molecular switch" to gametocytogenesis is evolutionarily conserved across most Plasmodium species, highlighting the essentiality of AP2-G to the process ( Table 1).
As with all essential genes, stabilization of their expression is crucial to optimizing their function. Maintaining a highsteady concentration of mRNA transcript allows for an enhanced gene expression. The stabilization of AP2-G expression is thought to be in part promoted epigenetically by gametocyte development 1 (GDV1) and AP2-G in a positive autoregulatory feedback loop (Figure 3(c)) [28]. In sexually committed parasites, a stable expression of AP2-G not only facilitates the establishment of a gametocytogenesis-specific transcriptional program but also determines the route via which the parasite will be sexually converted. Thus, the time point at which AP2-G expression is successfully stabilized is critical. At one end, stabilization of PfAP2-G in early rings facilitates the conversion of committed parasites via the SCC pathway [29]. On the other end, stabilization of PfAP2-G in early rings favours the NCC pathway [18]. Intriguingly, the transcriptome analysis of gametocytes derived from these two pathways is demonstrated as light difference in their gene expression pattern. For instance, in P. falciparum, there were higher transcripts of pfg.14.748, epf1, and pf3d71476600 levels in gametocytes converted via the NCC pathway than the SCC pathway [14]. Furthermore, ChIP-seq analysis has revealed a difference in PfAP2-G occupancies between stage I gametocytes produced from SCC and NCC pathways [24]. Specifically, the PfAP2-G binding site appeared higher on AP2-G target genes of NCC-derived gametocytes compared with SCC-derived gametocytes [24]. Against this, PfAP2-G may induce different phenotypes from early gametocytes obtained via these two routes, but further studies are required to validate this posit.
Along the course of maturation, developing gametocytes diverge into specialized haploid male and female sexes. The fact that a haploid Plasmodium parasite gives rise to a haploid male and female sexual progenies suggests that sex determination does not arise from different genetic content but through differential gene expression [35]. Nonetheless, the genetic determinants underpinning this event are obscure. As established, sexual commitment precedes or is concurrent with sex determination [36] and as such the prominent role of AP2-G in initiating sexual commitment somewhat predicts that AP2-G may as well be involved in sex determination. Until recently, it remained a puzzle how AP2-G directs the differential expression of male and female-specific genes from a haploid precursor, but now, studies have begun to redress this process. Emerging evidence suggests that AP2-G orchestrates a sex-determining cascade that drives the bifurcation of gametocytes into distinct male and female sexes [37]. Earlier, a recombinant gene-based AP2-G overexpression system was used to demonstrate that overexpression of PbAP2-G coincides with the upregulation of zinc finger proteins, RNA binding proteins, and helicases [38], which are usually encoded by canonical sex-specific genes. Consistent with these findings, a global single-cell transcriptome analysis of mutagenized P. berghei confirmed that zinc finger proteins [PBANKA_0413400 (md3), PBANKA_0828000 (gd1) and PBANKA_1435200 (fd4)], RNA binding proteins [PBANKA_0716500 (md5), PBANKA_1454800 (fd1)], OST-HTS-associated domain protein [PBANKA_1302700 (md1)], AT-rich interactive domain-containing protein [PBANKA_0102400(md4)], ACDC domain-containing protein [PBANKA_ 1418100(fd3)], and two conserved proteins of unknown function [PBANKA_1447900 (md2), PBANKA_0902300 (fd2)] are crucial early sex-determining markers, putatively activated downstream by AP2-G [37]. Using uniform manifold approximation and projection plot, further evidence from Russell et al. [37] suggested that sexually committed parasites initially follow a common transcriptional pathway but bifurcate into male and female-specific transcriptional pathways of which the peaking of AP2-G was identified as the common diverging point [37]. Immediate to the induction of sex-determining cascade by AP2-G, md1, and md2 mediate upstream divergence of male gametocytes whereas gd1 and md3 mediate the divergence of female gametocytes. At one diverged end, male gametocytes undergo maturation by the expression of known male-specific proteins, such as gd1, md4 [37], alpha-tubulin 2 [39], P230p [40], and P48/ 45 [41]. At the other end, female gametocytes mature under the expression of female-specific proteins, such as fd1, fd2 [37], DOZI [42], p25, p28 [43], AP2-O3 [44], AP2-G3/FG [45], NEK2, NEK4 [46], CCP1, and CCP3 [47]. Once the sex-determining transcriptional cascade is activated, AP2-G likely directly augments the downstream transcriptional  8 Cellular Microbiology control of sex-specific genes by other ApiAP2 transcription factors. Evidently, ChIP-seq analysis has shown that the PbAP2-G binding site is highly enriched upstream of wellestablished sex-specific AP2-related transcription factors including AP2-FG and AP2-O3, which acts downstream of AP2-G [37]. Perhaps this provides a glimpse into the role of the PfAP2-G beyond stage I ( Figure 2). However, whether the sex-determining cascade proposed by Russell et al. [37] is adjusted or influenced by the genetic diversity of Plasmodium species has not been tested. Hence, it will be exciting to investigate the sex-determining transcriptional cascade in other Plasmodium species. Further, investigations into the involvement of epigenetic control at each point of the sexdetermining cascade will be crucial to a holistic understanding of the process. The asexual-sexual switch is marked by a repertoire of transcriptional changes, and this coincides with changes in host-immunological response [48][49][50][51][52]. As such, the sexually developing parasites are constantly faced with the prospect of elimination by host immune responses. In light of this, concurrent tight regulation of gametocytogenesis and surface antigen expression is crucial to ensure the survival of gametocytes. The highly polymorphic var genes encode the major antigenic/virulence protein, PfEMP1, which represents the prime immune targets of the human host [53]. As described previously, Plasmodium parasites adopt a facultative heterochromatin-mediated control of var genes, where only a singular variant antigenic gene is expressed at any given time to ensure a variegated gene expression, which is crucial to minimize host immune recognition [54]. Ap2-g locus is proximal to var genes, and both share a close association with HP1 and H3K9me3 repression domain suggesting the expression of both genes is under similar heterochromatin-mediated control [55]. This signifies a potential link between sexual commitment and variegated expression of PfEMP1. One postulate suggested that the expression of AP2-G is mutually exclusive with the expression of var genes [45]. This is argued for by the fact that AP2-G and PfEMP1 genes are flanked by a common silencing molecule, an insulator-like pairing element (PE), and thus, both are silenced in a similar manner [26,54]. Nonetheless, this hypothesis is challenged by the assertion that sexual commitment occurs before gametocytogenesis [16,36,56], and also, var gene and ap2-g are concurrently expressed, at least during the commitment cycle. At the committed trophozoite and schizont stages, Pfap2-g significantly interacts with the nearest var gene cluster demonstrating a direct genic interaction [57]. However, this interaction is lost around stages II-III as AP2-G dissociates from the H3K9me3 repressive complex [57]. The importance of this interaction is however unknown. Interestingly, emerging evidence suggests that upstream promoters of var genes that encode PfEMP1 proteins involved in immune evasion and cytoadhesion are associated with AP2-G [24]. This suggests that var genes that encode PfEMP1 proteins are potential direct targets of AP2-G, implying AP2-G possibly influences the variegated expression of PfEMP1. Against 9 Cellular Microbiology the backdrop that the promoters of var genes play a crucial role in maintaining epigenetic memory, AP2-G is more likely to crosstalk with the promoters of var genes to control the expression of PfEMP1 variants in the mitotic progeny [58,59]. However, further investigation is needed to fully delineate the relationship between AP2-G expression and variegated expression of PfEMP1 in the context of host immune evasion.
3.1.2. Regulation of AP2-G. Despite the role of AP2-G as the "master switch" to sexual differentiation, the molecular players that direct its transcription remain obscure. Emerging findings point to AP2-G3 as a putative candidate [28]. Among P. falciparum and P. yoelii parasites, AP2-G3 has been shown to act upstream of ap2-g and transduce cytosolic transcriptional signals into the nucleus to enhance the transcription of AP2-G (Figure 3(a)) [28,60]. This finding is based on the fact that AP2-G3 is expressed earlier during sexual commitment, and it is localized maximally in the cytoplasm compared to the nucleus. Evidently, ap2-g3 disruption reduces the expression of AP2-G but disruption in ap2-g has no bearing on AP2-G3 expression. Aside AP2-G3, AP2-G5 has also been implicated in the transcription regulation of ap2-g. A single-cell transcriptome analysis revealed that AP2-G5 is expressed following the expression of AP2-G during sexual commitment [61]. A follow-up study showed that disruption in or deletion of ap2-g5 binding domain in two P. falciparum parasite clones, NF54 and 3D7-G7, correlated with upregulation of AP2-G during sexual commitment, suggesting that AP2-G5 is a repressor of AP2-G. Further investigation using ChIP-seq analysis revealed that PfAP2-G5 directly binds to both the exogenic body and the upstream of Pfap2-g and represses its expression, thereby hampering the initiation of gametocyte commitment.
Aside it being transcriptionally regulated, ap2-g is also epigenetically regulated. The ap2-g promoter is identified with repressive histone marks, including Histone 3 lysine 9 trimethylation (H3K9me3), heterochromatin protein 1 (HP1), and histone deacetylase 2 (Hda2) [54,62]. H3K9me3 provides a docking site for HP1 [54]. As HP1 binds to methylated H3K9 and sustains repression, Hda2 regulates silencing at the ap2-locus [63,64]. In P. falciparum, gametocyte development 1 (GDV1) antisense RNA induces the expression GDV1 which consequently binds to the H3K9me3-HP1-ap2-g complex and thus facilitates the eviction of HP1 [65]. Eviction of HP1 induces posttranslational modification of the histone tail via acetylation [29]. This culminates in the conversion of heterochromatin to euchromatin leading to increased expression of AP2-G and thus functionally drives gametocyte commitment (Figure 3(c)). Recently, in trophozoites of P. falciparum, AP2-G5 has been shown to alter the GDV1-H3K9me3 interaction by reducing the occupancy of GDV1 via a mechanism independent of HP1 and Hda2 thereby maintaining a heterochromatin state [66]. Interestingly, AP2-G has sufficient power to drive gametocyte commitment even when GDV1 is defective or inhibited. This could result from other unknown auxiliary or complementary players that coregulate the activation of ap2-g beside GDV1. This is supported by the evidence that ap2-g is not entirely dependent on HP1 and Hda2 and there-fore might not necessarily require HP1 eviction to initiate sexual commitment [54]. This is strengthened by the fact that nonhuman Plasmodium parasites that lack syntenic GDV1 protein sufficiently undergo sexual commitment, which further suggests the functional redundancy of GDV1 in the sexual commitment process [65]. At the postcommitment stage, AP2-G is silenced by histone 3 lysine 36 dimethylation/tri-methylation (H3K36me2&3) and AP2-G2 once its function is no longer needed [67].

Regulation of Gametocytogenesis by AP2-G2
3.2.1. Phenotypes of AP2-G2. The study of AP2-G2 phenotypes is currently complicated probably due to its genomewide targets comprising a wide array of sexual and asexual stage specific genes. However, AP2-G2 is typically recognized as a global transcriptional repressor. In P. berghei, AP2-G2 binds to a predicted short five-base nucleotide GTTC (T/C), and its reverse complement (A/G) CAAC is found upstream of about 1500 genes [68]. Aberrant to this, a different study identified AGAA and ACCA as the predictive binding motifs of PfAP2-G2 for approximately 3000 putative genes [12]. Thus, despite their phenotypic similarities, target genes for AP2-G2 are heterogeneous among P. berghei and P. falciparum [69]. The difference in gene targets could be the consequence of posttranslational modifications that alters the binding specificity of AP2 proteins in vivo [70]. For instance, lysine acetylation, which is relatively prevalent in PfAP2 proteins, changes the binding recognition of AP2 proteins [70,71].
Based on gene targets of PbAP2-G2 obtained through ChIP-sequence analysis, one study speculated that AP2-G2 represses asexual genes required for asexual replication, and thereby ushers asexual parasites into sexual commitment [68]. A shred of different evidence from gametocyte transcriptomics demonstrated that, indeed, PfAP2-G2 represses asexual stage-specific genes including knobassociated histidine protein, hypothetical gene family protein, merozoite surface proteins, and serine repeat antigen 5 [12]. Yet, parallel to the earlier speculation, it appeared that the asexual stage repression effect of AP2-G2 in P. falciparum has no discernible impact on the replication rate of asexual stage parasites or sexual commitment [12].
A host of evidence indicates that aberration in or deletion of ap2-g2 substantially hampers the maturation of gametocytes [68,72]. Specifically, gametocytes with defective ap2-g2 seem not to mature beyond stage III [12,68,72]. Across three different studies, the expression of crucial late gametocyte markers has been reported to significantly decrease in the absence of AP2-G2 activities [12,48,68]. Of these, transcripts of dynein heavy chain, p25, and p28 have been consistently shown to be reduced across these three studies [12,48,68]. The disintegration of the gametocyte before stage III could be explained in two folds. First, the premature gametocyte disintegration could be the consequence of the reduced expression of the essential late gametocyte genes which are required to maintain the integrity of matured gametocytes [72][73][74]. Second, the untimely expression of stage-specific genes in the absence of AP2-G2 10 Cellular Microbiology repression may underscore the premature disintegration of the gametocytes. If the former hypothesis is true, it remains plausible that AP2-G2 is "bi-functional" acting as an activator (direct or indirect) of critical late gametocyte genes beyond stage III and also a repressor at the other life stages of the Plasmodium parasite. Interestingly, alteration in ap2-g2 of gametocytes already expressing sex-specific genes only resulted in reduced expression of the sex-specific genes and not an abrogation of the maturation process [68]. This somewhat supports the posit that AP2-G2 is a selective activator of a subset of essential sex-specific genes critically needed by gametocytes to develop beyond stage III-which is a deviation from its conventional repression function.
There are conflicting conclusions, nonetheless, about the effect of AP2-G2 on the gametocyte sexual dimorphism in the context of sex ratio. For instance, a gene knockout study in P. falciparum has demonstrated a disproportionate inhibition of male gametocyte divergence compared with their female gametocyte counterparts [72]. The skewed repression of male gametocytes production in AP2-G2 knockout mutants reflects the downregulation of PfMDV-1, an essential gene in the production of male gametocytes [72]. Paradox to this, other studies conducted in P. berghei observed no effects of AP2-G2 on sex ratio but observed increased expression levels of both male and female sex-specific genes [68,75]. Possibly, the specific sex-determining effect of AP2-G2 could be species-dependent or it could be dependent on AP2-G2 expression threshold. Hence, further investigation to ascertain the effect of AP2-G2 expression threshold on sexual dimorphism would be crucial.

3.2.2.
Regulation of AP2-G2. The understanding of the transcriptional regulation of AP2-G2 is complicated by the heterogeneity of Plasmodium species. In one study, the ChIPseq analysis demonstrated that the upstream of PbAP2-G2 possesses multiple binding motifs of PbAP2-G [11]. Further findings showed that the transcription of Pbap2-g2 is directly induced by PbAP2-G approximately around the time when sexually committed parasites are being converted into stage I gametocytes [11]. In contrast, RNA-seq and RT-qPCR analysis of schizont with disrupted Pfap2-g2 showed a reduced expression of PfAP2-G implying that PfAP2-G2 acts upstream of PfAP2-G at the schizont stage [72]. Nonetheless, it is not known whether the reduced expression of ap2-g in the schizonts was induced directly or indirectly by AP2-G2. Deductively, it seems that the effect of AP2-G on ap2-g2 transcription is somewhat dependent on the developmental stage of the Plasmodium parasite.
Given the immense role of histone posttranscriptional modifications (hPTM) in epigenetic control of ApiAP2 genes, there is a plausible prospect of their functional link to AP2-G2. Accordingly, recent studies have begun to explore the epigenetic relationship between AP2-G2 and hPTM variants. For instance, it has been demonstrated that AP2-G2 and H3K36me2&3 share 33% common regulons in stages II-III gametocytes [67], suggesting that AP2-G2 and H3K36me2&3 are more likely to form a complex to initiate a coordinated repression control at stages II-III. However, stages IV-V (matured) gametocytes seem to exhibit a differ-ent AP2-G2-hPTM interaction. A middle-down proteomic dataset revealed that a unique triple hPTM activating complex, H3R17me2K18acK23ac, exclusively recruits AP2-G2 [76]. The resulting complex coordinates the formation of Spt-Ada-Gcn5 acetyltransferase like complex (SAGA like complex) through an interaction with a host of malespecific effector proteins, such as Chromodomain helicase DNA binding protein 1 (CHD1), General control nonrepressed 5 protein (GCN5), Transcriptional adapter protein 2 (ADA2), Plant homodomain protein 2 (PHD2), Nucleosome assembly protein (NAPS), and Imitation Switch protein (ISWI) [76]. The interplay of AP2-G2 and the malespecific proteins alludes to the involvement of AP2-G2 in the regulation of male gametocytes as observed previously [72]. It is worthwhile to note that across eukaryotic cells, SAGA complexes are characterized as cellular coactivators that control transcription [77]. Against the interactions of AP2-G2 with H3R17me2K18acK23ac and the SAGA-like complex, it is however tempting to speculate that AP2-G2 is a transcriptional activator of sex-specific genes at the late gametocyte stages IV-V. This proposed activating function of AP2-G2 explains the reduced expression of essential late gametocyte markers in ap2-g2-disrupted phenotypes observed in previous studies [12,48,68]. This further strengthens the hypothesis that AP2-G2 is bifunctional and acts primarily as a repressor and also an activator of a subset of late gametocyte (stages IV-V) sex-specific genes. Nonetheless, further studies will be crucial to elucidate this hypothesis and also unravel the complexities that may be involved.

Regulation of Gametocytogenesis by Ap2-G3/FG
3.3.1. Phenotypes of AP2-G3/FG. Across available literature, the stage expression of AP2-G3 is marked by contradictions among different or same Plasmodium species. In P. falciparum, AP2-G3 appears not to be enriched in committed sexual rings or trophozoite [48,78,79]. However, Usui et al. [28] observed AP2-G3 enrichment in asexual rings. In addition, van Biljon et al. [48] observed a mild expression of AP2-G3 among stages II-IV gametocytes in P. falciparum ( Figure 2). Conversely, to what is generally observed in P. falciparum, AP2-G3 seems to be substantially expressed among asexual parasites of P. yoelii and P. berghei before sexual commitment. In both P. falciparum and P. yoelii, it is predicted that AP2-G3 acts to transduce cytoplasmic signals into the nucleus which subsequently augments the transcription of ap2-g (Figure 3(b)) [28,60].
AP2-G3, also described as AP2-FG in P. berghei by Yuda et al. [45], was putatively shown to activate the divergence of gametocytes into matured female gametocytes. Phenotypes of ap2-g3-disrupted parasites develop as immature female gametocytes, and this reflected a marked reduction of P47, NEK2, NEK3, P28, P25, putative ookinete proteins (PSOPs), CPW-WCP family proteins, and LAP family of proteins [45]. However, the development of ap2-g3-disrupted phenotypes into female gametocytes was not completely abrogated [45], which implies that AP2-G3 is not the molecular switch but rather a composite of a cascade of sex-specific gene 11 Cellular Microbiology regulators. Notably, no significant effects on matured male gametocytes were reported in this study [45]. Parallel to this, in P. yoelii, disruption in ap2-g3 substantially reduced the production of both matured male gametocytes and female gametocytes [60]. Thus, although there may be species differences, it seems that AP2-G3 has its regulatory mark on the commitment phase (trigger AP2-G expression) and sexual maturation/determination. Nonetheless, further studies are needed to unravel other functions of AP2-G3, owing to the scarcity of available studies.
3.3.2. Regulation of AP2-G3/FG. There is a paucity of data on the transcriptional regulation of AP2-G3. Using ChIP-seq, it was shown that AP2-G binding site was enriched upstream of AP2-G3 [11]. This suggests that AP2-G may directly regulate the expression of AP2-G3 at the transcription level.
Environmental cues such as high parasitaemia, anaemia, lysophosphatidylcholine (LysoPC), drug and immune pressure, and endoplasmic reticulum stress are associated with gametocyte commitment [80]. Against the model of AP2-G3/FG as a conduit for transmitting sexual commitment signals from the cytoplasm into the nucleus, AP2-G3 expression is likely influenced epigenetically by these environmental cues, which consequently impacts the expression of AP2-G. It is thought that LysoPC does not directly activate any molecular cascade, but rather, it causes a change within the intracellular space which consequently impacts epigenetic regulation of gametocytogenesis [81]. However, it remains to be elucidated how all known environmental cues of sexual commitment empirically influence AP2-G3 expression. Thus, a firm understanding of the potential mechanism(s) that underlines the activation of AP2-G3 by environmental cues seems lacking.

Regulation of Gametocytogenesis by AP2-G5
3.4.1. Phenotypes of AP2-G5. Recently, a comprehensive ChIP-seq analysis predicted the binding motif of PfAP2-G5 as a five-based genomic sequence, GAACA or AACAA [66]. A CRISPR-Cas9 mediated disruption or deletion of ap2-g5 in P. falciparum NF54 clones resulted in the upregulation of AP2-G suggesting that AP2-G5 is a repressor of ap2-g [66]. Comparative transcriptome analysis of a single ap2-g knockout and a dual ap2-g5-ap2-g knockout P. falciparum lines showed that AP2-G5 represses ap2-g, which consequently reduces the expression of essential AP2-G binding genes required for sexual commitment [66]. Thus, AP2-G5 prevents the initiation of sexual commitment. Further investigation showed that PfAP2-G5 suppresses and prevents the untimely expression of gametocyte maturation-essential genes at the sexual ring stage. Thus, it was thought that repression of early gametocyte genes by AP2-G5 at the sexual ring stage promotes the maturation of early gametocytes. Nonetheless, there is a paucity of studies on AP2-G5, and as such, future investigations are needed to unravel its other roles.

3.4.2.
Regulation of AP2-G5. An effort to delineate transcriptional regulation of AP2-G5 revealed that ap2-g5 is activated by AP2-G to express AP2-G5 which in turn represses ap2-g consequently reducing the expression of early gametocyte genes. Thus, AP2-G recruits AP2-G5 as a regulatory molecule to keep in check its activities [66]. Currently, there is no concrete data on how the expression of AP2-G5 is epigenetically regulated.

Regulation of Gametocytogenesis by Other ApiAP2
Genes. In P. yoelii, AP2-O3 has been demonstrated to be exclusively expressed in matured female gametocytes but not in male gametocytes [44]. Further finding from Li et al. [44] showed that the disruption of ap2-o3 in P. yoelii stalls the maturation of female gametocytes around stages II-III. The arrest of female gametocyte maturation in ap2-o3 mutants is attributed to the reduced expression of femalespecific genes (Figure 3(e)). Of notice, the downregulation of AP2-O3 in mutant P. yoelii parasite lines correlates with the upregulation of male-specific genes [20]. In addition, AP2-O in P. falciparum and AP2-SP/EXP in P. berghei are also implicated in gametocytogenesis (Table 1). For instance, phenotypes of AP2-O knockdown parasites have been shown to exhibit increased expression of many early gametocyte transcripts including Pfs16, suggesting that AP2-O perhaps is a repressor of these early gametocyte genes [82]. Also, in P. berghei, AP2-SP/EXP seems an indirect negative regulator of female-specific genes in gametocytes [75]. However, it remains to be elucidated how AP2-SP/EXP mediates the downregulation of female-specific genes. Further, in P. falciparum, stage I gametocytes are significantly enriched in AP2-SP2, AP2-O, and AP2-O3, but their functional relevance at this stage is unknown [48].

Conclusion
In the last decade, studies on functional genomes, transcriptomes, and proteomes have garnered impressive data on the transcriptional control of gametocytogenesis, and these have indeed illuminated in our understanding of a previously less-explored life stage of Plasmodium species. Keynotes from this review suggest that the role of AP2-G in sexual commitment and sexual conversion is conserved across different species and thus may present a more formidable target for transmission-blocking vaccines or drugs. Furthermore, the control of each step of gametocytogenesis by ApiAP2 genes is combinatorial and thus requires the effect of more than one ApiAP2 gene. For instance, the sexual commitment and sexual conversion stages are regulated primarily by AP2-G but do so alongside adjunct ApiAP2 players including AP2-G2, AP2-G3, and AP2-G5. The presence of the noncoding semiconserved intergenic region [83] within the ApiAP2 genome landscape likely provides avenues for the combinatorial control of a common phenotype by promoting the recruitment of other ApiAP2 genes. Nonetheless, there is very little homogeneity in the regulation of sexual maturation among different Plasmodium species, even by syntenic ortholog ApiAP2 genes. These observed discrepancies could be explained by (i) phenotypic plasticity that gives discrete genotypic clones the ability to express well-adapted phenotypes when exposed to different environments (different in vivo culturing environments); (ii) the 12 Cellular Microbiology subtle difference in epigenetic regulation of gametocyte maturation among different Plasmodium species yielding varied phenotypes [84]; and (iii) the different gene-disruption techniques (knockout, knockdown, and genome editing) employed by the different studies which may or may not completely abolish the function of the target gene and hence produce different phenotypes. It is important however to note that the hunt to unveil transcriptional roles of ApiAP2 genes, especially in gametocytogenesis, is still embryonic and has questions yet to be addressed. Importantly, a dataset obtained from a highresolution transcriptome of P. falciparum has shown that, of 27 members of the ApiAP2family, 15 are substantially expressed during gametocytogenesis (Figure 2) [48], suggesting they might play important roles in gametocytogenesis. Nonetheless, only five of these genes have extensively characterized roles in gametocytogenesis but the rest are not known. Future investigations could focus on characterizing the function of these genes in gametocytogenesis and the pathways in which they participate for a holistic understanding of how ApiAP2 genes regulate gametocytogenesis. Strikingly, there is no or very little exploration of the functional control of gametocytogenesis by ApiAP2 genes in P. vivax and P. malariae, which are important Plasmodium species that pose a considerable health threat in South Asia, Central America, and South America [85]. Therefore, future studies could explore the roles of ApiAP2 genes in P. vivax and P. malariae.
Interestingly, using PacBio sequencing, it has been shown that there are many splicing isoforms of PF3F7_0730300, PF3F7_0420300, and PF3F7_0613800_1239200 ApiAP2 genes [86], although the roles of these isoforms are not yet functionally characterized. Alternative, splicing produces distinct mRNA isoforms from a single gene and thereby alters the structure and function of the resulting expressed proteins [87] and thus may hold cues to the observed different phenotypes produced by syntenic orthologs of ApiAP2 genes. Considering the crucial role of alternative splicing in the regulation of essential genes [87], future research trajectory could be directed toward the explorations of its roles in gametocytogenesis using other long-read sequencing techniques such as nanopore sequencing [88].

Data Availability
All data used to support the findings of this study are included within the article.

Disclosure
The funders had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.

Conflicts of Interest
The authors declare no conflicts of interest.