A genome-wide map of DNA replication at single-molecule resolution in the malaria parasite Plasmodium falciparum.

The malaria parasite Plasmodium falciparum replicates via schizogony: an unusual type of cell cycle involving asynchronous replication of multiple nuclei within the same cytoplasm. Here, we present the first comprehensive study of DNA replication origin specification and activation during Plasmodium schizogony. Potential replication origins were abundant, with ORC1-binding sites detected every ∼800 bp. In this extremely A/T-biased genome, the sites were biased towards areas of higher G/C content, and contained no specific sequence motif. Origin activation was then measured at single-molecule resolution using newly developed DNAscent technology: a powerful method of detecting replication fork movement via base analogues in DNA sequenced on the Oxford Nanopore platform. Unusually, origins were preferentially activated in areas of low transcriptional activity, and replication forks also moved fastest through lowly transcribed genes. This contrasts with the way that origin activation is organised in other systems, such as human cells, and suggests that P. falciparum has evolved its S-phase specifically to minimise conflicts between transcription and origin firing. This may be particularly important to maximise the efficiency and accuracy of schizogony, with its multiple rounds of DNA replication and its absence of canonical cell-cycle checkpoints.

Gene knockdown in malaria parasites via non-canonical RNAi.

The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called AgoshRNA. Using this strategy, we achieved robust and specific gene knockdown throughout the entire parasite life cycle. We also successfully silenced the endogenous gene perforin-like protein 2, phenocopying a full gene knockout. Transcriptionally restricting Ago2 expression to the liver stage further enabled us to perform a stage-specific gene knockout. The RNAi-competent Plasmodium lines reported here will be a valuable resource for loss-of-function phenotyping of the many uncharacterized genes of Plasmodium in low or high throughput, without the need to engineer the target gene locus. Thereby, our new strategy and transgenic Plasmodium lines will ultimately benefit the discovery of urgently needed antimalarial drug and vaccine candidates. Generally, the ability to render RNAi-negative organisms RNAi-competent by mere introduction of two components, Ago2 and AgoshRNA, is a unique paradigm that should find broad applicability in other species.

Epigenetic reader complexes of the human malaria parasite, Plasmodium falciparum.

Epigenetic regulatory mechanisms are central to the development and survival of all eukaryotic organisms. These mechanisms critically depend on the marking of chromatin domains with distinctive histone tail modifications (PTMs) and their recognition by effector protein complexes. Here we used quantitative proteomic approaches to unveil interactions between PTMs and associated reader protein complexes of Plasmodium falciparum, a unicellular parasite causing malaria. Histone peptide pull-downs with the most prominent and/or parasite-specific PTMs revealed the binding preference for 14 putative and novel reader proteins. Amongst others, they highlighted the acetylation-level-dependent recruitment of the BDP1/BDP2 complex and identified an PhD-finger protein (PHD 1, PF3D7_1008100) that could mediate a cross-talk between H3K4me2/3 and H3K9ac marks. Tagging and interaction proteomics of 12 identified proteins unveiled the composition of 5 major epigenetic complexes, including the elusive TBP-associated-factor complex as well as two distinct GCN5/ADA2 complexes. Furthermore, it has highlighted a remarkable degree of interaction between these five (sub)complexes. Collectively, this study provides an extensive inventory of PTM-reader interactions and composition of epigenetic complexes. It will not only fuel further explorations of gene regulation amongst ancient eukaryotes, but also provides a stepping stone for exploration of PTM-reader interactions for antimalarial drug development.

The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics of regulatory sequences.

In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-associated processes and it is central to the control of gene expression. For Plasmodium falciparum, a causative agent of human malaria, the nucleosome positioning profile of regulatory regions deserves particular attention because of their extreme AT-content. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit by demarcating landmark sites (transcription/translation start and end sites). In addition, our analysis provides strong indications for the function of positioned nucleosomes in splice site recognition. Transcription start sites (TSSs) are bordered by a small nucleosome-depleted region, but lack the stereotypic downstream nucleosome arrays, highlighting a key difference in chromatin organization compared to model organisms. Furthermore, we observe transcription-coupled eviction of nucleosomes on strong TSSs during intraerythrocytic development and demonstrate that nucleosome positioning and dynamics can be predictive for the functionality of regulatory DNA elements. Collectively, the strong nucleosome positioning over splice sites and surrounding putative transcription factor binding sites highlights the regulatory capacity of the nucleosome landscape in this deadly human pathogen.

Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: molecular insight into sex-specific processes and translational repression.

Sexual differentiation of malaria parasites into gametocytes in the vertebrate host and subsequent gamete fertilization in mosquitoes is essential for the spreading of the disease. The molecular processes orchestrating these transitions are far from fully understood. Here, we report the first transcriptome analysis of male and female Plasmodium falciparum gametocytes coupled with a comprehensive proteome analysis. In male gametocytes there is an enrichment of proteins involved in the formation of flagellated gametes; proteins involved in DNA replication, chromatin organization and axoneme formation. On the other hand, female gametocytes are enriched in proteins required for zygote formation and functions after fertilization; protein-, lipid- and energy-metabolism. Integration of transcriptome and proteome data revealed 512 highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time. Despite a high degree of conservation between Plasmodium species, 260 of these 'repressed transcripts' have not been previously described. Moreover, for some of these genes, protein expression is only reported in oocysts and sporozoites indicating that repressed transcripts can be partitioned into short- and long-term storage. Finally, these data sets provide an essential resource for identification of vaccine/drug targets and for further mechanistic studies.

H2A.Z/H2B.Z double-variant nucleosomes inhabit the AT-rich promoter regions of the Plasmodium falciparum genome.

Histone variants are key components of the epigenetic code and evolved to perform specific functions in transcriptional regulation, DNA repair, chromosome segregation and other fundamental processes. Although variants for histone H2A and H3 are found throughout the eukaryotic kingdom, variants of histone H2B and H4 are rarely encountered. H2B.Z is one of those rare H2B variants and is apicomplexan-specific. Here we show that in Plasmodium falciparum H2B.Z localizes to euchromatic intergenic regions throughout intraerythrocytic development and together with H2A.Z forms a double-variant nucleosome subtype. These nucleosomes are enriched in promoters over 3' intergenic regions and their occupancy generally correlates with the strength of the promoter, but not with its temporal activity. Remarkably, H2B.Z occupancy levels exhibit a clear correlation with the base-composition of the underlying DNA, raising the intriguing possibility that the extreme AT content of the intergenic regions within the Plasmodium genome might be instructive for histone variant deposition. In summary, our data show that the H2A.Z/H2B.Z double-variant nucleosome demarcates putative regulatory regions of the P. falciparum epigenome and likely provides a scaffold for dynamic regulation of gene expression in this deadly human pathogen.

Gonad differentiation in zebrafish is regulated by the canonical Wnt signaling pathway.

Zebrafish males undergo a "juvenile ovary-to-testis" gonadal transformation process. Several genes, including nuclear receptor subfamily 5, group A (nr5a) and anti-Müllerian hormone (amh), and pathways such as Tp53-mediated germ-cell apoptosis have been implicated in zebrafish testis formation. However, our knowledge of the regulation of this complex process is incomplete, and much remains to be investigated about the molecular pathways and network of genes that control it. Using a microarray-based analysis of transforming zebrafish male gonads, we demonstrated that their transcriptomes undergo transition from an ovary-like pattern to an ovotestis to a testis-like profile. Microarray results also validated the previous histological and immunohistochemical observation that there is high variation in the duration and extent of commitment to the juvenile ovary phase among individuals. Interestingly, global gene expression profiling of diverging zebrafish juvenile ovaries and transforming ovotestes revealed that some members of the canonical Wnt/beta-catenin signaling pathway were differentially expressed between these two phases. To investigate whether Wnt/beta-catenin signaling plays a role in zebrafish gonad differentiation, we used the Tg (hsp70l:dkk1b-GFP)w32 line to inhibit Wnt/beta-catenin signaling during gonad differentiation. Activation of dkk1b-GFP expression by heat shock resulted in an increased proportion of males and corresponding decrease in gonadal aromatase gene (cyp19a1a) expression. The Wnt target gene, lymphocyte enhancer binding factor 1 (lef1), was also down-regulated in the process. Together, these results provide the first functional evidence that, similarly to mammals, Wnt/beta-catenin signaling is a "pro-female" pathway that regulates gonad differentiation in zebrafish.

Plasmodium falciparum centromeres display a unique epigenetic makeup and cluster prior to and during schizogony.

Centromeres are essential for the faithful transmission of chromosomes to the next generation, therefore being essential in all eukaryotic organisms. The centromeres of Plasmodium falciparum, the causative agent of the most severe form of malaria, have been broadly mapped on most chromosomes, but their epigenetic composition remained undefined. Here, we reveal that the centromeric histone variant PfCENH3 occupies a 4-4.5 kb region on each P. falciparum chromosome, which is devoid of pericentric heterochromatin but harbours another histone variant, PfH2A.Z. These CENH3 covered regions pinpoint the exact position of the centromere on all chromosomes and revealed that all centromeric regions have similar size and sequence composition. Immunofluorescence assay of PfCENH3 strongly suggests that P. falciparum centromeres cluster to a single nuclear location prior to and during mitosis and cytokinesis but dissociate soon after invasion. In summary, we reveal a dynamic association of Plasmodium centromeres, which bear a unique epigenetic signature and conform to a strict structure. These findings suggest that DNA-associated and epigenetic elements play an important role in centromere establishment in this important human pathogen.

The troubled puberty of malaria parasites.

Sexual differentiation of malaria parasites is essential for transmission, yet the underlying mechanisms are poorly understood. Russell et al. elegantly combined a loss-of-function screen with single-cell RNA-sequencing to identify key factors in this process. Gomes et al. further characterized one of them, MD1, as a regulator contributing to male fate determination.

Epigenetically regulated RNA-binding proteins signify malaria hypnozoite dormancy.

Dormancy enables relapsing malaria parasites, such as Plasmodium vivax and cynomolgi, to survive unfavorable conditions. It is enabled by hypnozoites, parasites remaining quiescent inside hepatocytes before reactivating and establishing blood-stage infection. We integrate omics approaches to explore gene-regulatory mechanisms underlying hypnozoite dormancy. Genome-wide profiling of activating and repressing histone marks identifies a few genes that get silenced by heterochromatin during hepatic infection of relapsing parasites. By combining single-cell transcriptomics, chromatin accessibility profiling, and fluorescent in situ RNA hybridization, we show that these genes are expressed in hypnozoites and that their silencing precedes parasite development. Intriguingly, these hypnozoite-specific genes mainly encode proteins with RNA-binding domains. We hence hypothesize that these likely repressive RNA-binding proteins keep hypnozoites in a developmentally competent but dormant state and that heterochromatin-mediated silencing of the corresponding genes aids reactivation. Exploring the regulation and exact function of these proteins hence could provide clues for targeted reactivation and killing of these latent pathogens.

Gene-by-gene screen of the unknown proteins encoded on Plasmodium falciparum chromosome 3.

Taxon-specific proteins are key determinants defining the biology of all organisms and represent prime drug targets in pathogens. However, lacking comparability with proteins in other lineages makes them particularly difficult to study. In malaria parasites, this is exacerbated by technical limitations. Here, we analyzed the cellular location, essentiality, function, and, in selected cases, interactome of all unknown non-secretory proteins encoded on an entire P. falciparum chromosome. The nucleus was the most common localization, indicating that it is a hotspot of parasite-specific biology. More in-depth functional studies with four proteins revealed essential roles in DNA replication and mitosis. The mitosis proteins defined a possible orphan complex and a highly diverged complex needed for spindle-kinetochore connection. Structure-function comparisons indicated that the taxon-specific proteins evolved by different mechanisms. This work demonstrates the feasibility of gene-by-gene screens to elucidate the biology of malaria parasites and reveal critical parasite-specific processes of interest as drug targets.

A genome-wide chromatin-associated nuclear peroxiredoxin from the malaria parasite Plasmodium falciparum.

Malaria parasites are subjected to high levels of oxidative stress during their development inside erythrocytes and the ability of the parasite to defend itself against this assault is critical to its survival. Therefore, Plasmodium possesses an effective antioxidant defense system that could potentially be used as a target for the development of inhibitor-based therapy. We have identified an unusual peroxiredoxin protein that localizes to the nucleus of Plasmodium falciparum and have renamed it PfnPrx (PF10_0268, earlier called MCP1). Our work reveals that PfnPrx has a broad specificity of substrate being able to utilize thioredoxin and glutaredoxin as reductants and having the ability to reduce simple and complex peroxides. Intriguingly, chromatin immunoprecipitation followed by deep sequencing reveals that the enzyme associates with chromatin in a genome-wide manner with a slight enrichment in coding regions. Our results represent the first description of a dedicated chromatin-associated peroxiredoxin and potentially represent an ingenious way by which the parasite can survive the highly oxidative environment within its human host.

H2A.Z demarcates intergenic regions of the plasmodium falciparum epigenome that are dynamically marked by H3K9ac and H3K4me3.

Epigenetic regulatory mechanisms and their enzymes are promising targets for malaria therapeutic intervention; however, the epigenetic component of gene expression in P. falciparum is poorly understood. Dynamic or stable association of epigenetic marks with genomic features provides important clues about their function and helps to understand how histone variants/modifications are used for indexing the Plasmodium epigenome. We describe a novel, linear amplification method for next-generation sequencing (NGS) that allows unbiased analysis of the extremely AT-rich Plasmodium genome. We used this method for high resolution, genome-wide analysis of a histone H2A variant, H2A.Z and two histone H3 marks throughout parasite intraerythrocytic development. Unlike in other organisms, H2A.Z is a constant, ubiquitous feature of euchromatic intergenic regions throughout the intraerythrocytic cycle. The almost perfect colocalisation of H2A.Z with H3K9ac and H3K4me3 suggests that these marks are preferentially deposited on H2A.Z-containing nucleosomes. By performing RNA-seq on 8 time-points, we show that acetylation of H3K9 at promoter regions correlates very well with the transcriptional status whereas H3K4me3 appears to have stage-specific regulation, being low at early stages, peaking at trophozoite stage, but does not closely follow changes in gene expression. Our improved NGS library preparation procedure provides a foundation to exploit the malaria epigenome in detail. Furthermore, our findings place H2A.Z at the cradle of P. falciparum epigenetic regulation by stably defining intergenic regions and providing a platform for dynamic assembly of epigenetic and other transcription related complexes.

A major role for the Plasmodium falciparum ApiAP2 protein PfSIP2 in chromosome end biology.

The heterochromatic environment and physical clustering of chromosome ends at the nuclear periphery provide a functional and structural framework for antigenic variation and evolution of subtelomeric virulence gene families in the malaria parasite Plasmodium falciparum. While recent studies assigned important roles for reversible histone modifications, silent information regulator 2 and heterochromatin protein 1 (PfHP1) in epigenetic control of variegated expression, factors involved in the recruitment and organization of subtelomeric heterochromatin remain unknown. Here, we describe the purification and characterization of PfSIP2, a member of the ApiAP2 family of putative transcription factors, as the unknown nuclear factor interacting specifically with cis-acting SPE2 motif arrays in subtelomeric domains. Interestingly, SPE2 is not bound by the full-length protein but rather by a 60kDa N-terminal domain, PfSIP2-N, which is released during schizogony. Our experimental re-definition of the SPE2/PfSIP2-N interaction highlights the strict requirement of both adjacent AP2 domains and a conserved bipartite SPE2 consensus motif for high-affinity binding. Genome-wide in silico mapping identified 777 putative binding sites, 94% of which cluster in heterochromatic domains upstream of subtelomeric var genes and in telomere-associated repeat elements. Immunofluorescence and chromatin immunoprecipitation (ChIP) assays revealed co-localization of PfSIP2-N with PfHP1 at chromosome ends. Genome-wide ChIP demonstrated the exclusive binding of PfSIP2-N to subtelomeric SPE2 landmarks in vivo but not to single chromosome-internal sites. Consistent with this specialized distribution pattern, PfSIP2-N over-expression has no effect on global gene transcription. Hence, contrary to the previously proposed role for this factor in gene activation, our results provide strong evidence for the first time for the involvement of an ApiAP2 factor in heterochromatin formation and genome integrity. These findings are highly relevant for our understanding of chromosome end biology and variegated expression in P. falciparum and other eukaryotes, and for the future analysis of the role of ApiAP2-DNA interactions in parasite biology.

Experimentally controlled downregulation of the histone chaperone FACT in Plasmodium berghei reveals that it is critical to male gamete fertility.

Human FACT (facilitates chromatin transcription) consists of the proteins SPT16 and SSRP1 and acts as a histone chaperone in the (dis)assembly of nucleosome (and thereby chromatin) structure during transcription and DNA replication. We identified a Plasmodium berghei protein, termed FACT-L, with homology to the SPT16 subunit of FACT. Epitope tagging of FACT-L showed nuclear localization with high expression in the nuclei of (activated) male gametocytes. The gene encoding FACT-L could not be deleted indicating an essential role during blood-stage development. Using a 'promoter-swap' approach whereby the fact-l promoter was replaced by an 'asexual blood stage-specific' promoter that is silent in gametocytes, transcription of fact-l in promoter-swap mutant gametocytes was downregulated compared with wild-type gametocytes. These mutant male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility was strongly reduced while female gamete fertility was unaffected; residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony. Therefore FACT is critically involved in the formation of fertile male gametes and parasite transmission. 'Promoter swapping' is a powerful approach for the functional analysis of proteins in gametocytes (and beyond) that are essential during asexual blood-stage development.

Human branching cholangiocyte organoids recapitulate functional bile duct formation.

Human cholangiocyte organoids show great promise for regenerative therapies and in vitro modeling of bile duct development and diseases. However, the cystic organoids lack the branching morphology of intrahepatic bile ducts (IHBDs). Here, we report establishing human branching cholangiocyte organoid (BRCO) cultures. BRCOs self-organize into complex tubular structures resembling the IHBD architecture. Single-cell transcriptomics and functional analysis showed high similarity to primary cholangiocytes, and importantly, the branching growth mimics aspects of tubular development and is dependent on JAG1/NOTCH2 signaling. When applied to cholangiocarcinoma tumor organoids, the morphology changes to an in vitro morphology like primary tumors. Moreover, these branching cholangiocarcinoma organoids (BRCCAOs) better match the transcriptomic profile of primary tumors and showed increased chemoresistance to gemcitabine and cisplatin. In conclusion, BRCOs recapitulate a complex process of branching morphogenesis in vitro. This provides an improved model to study tubular formation, bile duct functionality, and associated biliary diseases.

Plasmodium falciparum heterochromatin protein 1 marks genomic loci linked to phenotypic variation of exported virulence factors.

Epigenetic processes are the main conductors of phenotypic variation in eukaryotes. The malaria parasite Plasmodium falciparum employs antigenic variation of the major surface antigen PfEMP1, encoded by 60 var genes, to evade acquired immune responses. Antigenic variation of PfEMP1 occurs through in situ switches in mono-allelic var gene transcription, which is PfSIR2-dependent and associated with the presence of repressive H3K9me3 marks at silenced loci. Here, we show that P. falciparum heterochromatin protein 1 (PfHP1) binds specifically to H3K9me3 but not to other repressive histone methyl marks. Based on nuclear fractionation and detailed immuno-localization assays, PfHP1 constitutes a major component of heterochromatin in perinuclear chromosome end clusters. High-resolution genome-wide chromatin immuno-precipitation demonstrates the striking association of PfHP1 with virulence gene arrays in subtelomeric and chromosome-internal islands and a high correlation with previously mapped H3K9me3 marks. These include not only var genes, but also the majority of P. falciparum lineage-specific gene families coding for exported proteins involved in host-parasite interactions. In addition, we identified a number of PfHP1-bound genes that were not enriched in H3K9me3, many of which code for proteins expressed during invasion or at different life cycle stages. Interestingly, PfHP1 is absent from centromeric regions, implying important differences in centromere biology between P. falciparum and its human host. Over-expression of PfHP1 results in an enhancement of variegated expression and highlights the presence of well-defined heterochromatic boundaries. In summary, we identify PfHP1 as a major effector of virulence gene silencing and phenotypic variation. Our results are instrumental for our understanding of this widely used survival strategy in unicellular pathogens.

The ApiAP2 factor PfAP2-HC is an integral component of heterochromatin in the malaria parasite Plasmodium falciparum.

Malaria parasites undergo a complex life cycle in the human host and the mosquito vector. The ApiAP2 family of DNA-binding proteins plays a dominant role in parasite development and life cycle progression. Most ApiAP2 factors studied to date act as transcription factors regulating stage-specific gene expression. Here, we characterized an ApiAP2 factor in Plasmodium falciparum that we termed PfAP2-HC. We demonstrate that PfAP2-HC specifically binds to heterochromatin throughout the genome. Intriguingly, PfAP2-HC does not bind DNA in vivo and recruitment of PfAP2-HC to heterochromatin is independent of its DNA-binding domain but strictly dependent on heterochromatin protein 1. Furthermore, our results suggest that PfAP2-HC functions neither in the regulation of gene expression nor in heterochromatin formation or maintenance. In summary, our findings reveal PfAP2-HC as a core component of heterochromatin in malaria parasites and identify unexpected properties and substantial functional divergence among the members of the ApiAP2 family of regulatory proteins.

Cholangiocyte organoids from human bile retain a local phenotype and can repopulate bile ducts in vitro.

The well-established 3D organoid culture method enabled efficient expansion of cholangiocyte-like cells from intrahepatic (IHBD) and extrahepatic bile duct (EHBD) tissue biopsies. The extensive expansion capacity of these organoids enables various applications, from cholangiocyte disease modelling to bile duct tissue engineering. Recent research demonstrated the feasibility of culturing cholangiocyte organoids from bile, which was minimal-invasive collected via endoscopic retrograde pancreaticography (ERCP). However, a detailed analysis of these bile cholangiocyte organoids (BCOs) and the cellular region of origin was not yet demonstrated. In this study, we characterize BCOs and mirror them to the already established organoids initiated from IHBD- and EHBD-tissue. We demonstrate successful organoid-initiation from extrahepatic bile collected from gallbladder after resection and by ERCP or percutaneous transhepatic cholangiopathy from a variety of patients. BCOs initiated from these three sources of bile all show features similar to in vivo cholangiocytes. The regional-specific characteristics of the BCOs are reflected by the exclusive expression of regional common bile duct genes (HOXB2 and HOXB3) by ERCP-derived BCOs and gallbladder-derived BCOs expressing gallbladder-specific genes. Moreover, BCOs have limited hepatocyte-fate differentiation potential compared to intrahepatic cholangiocyte organoids. These results indicate that organoid-initiating cells in bile are likely of local (extrahepatic) origin and are not of intrahepatic origin. Regarding the functionality of organoid initiating cells in bile, we demonstrate that BCOs efficiently repopulate decellularized EHBD scaffolds and restore the monolayer of cholangiocyte-like cells in vitro. Bile samples obtained through minimally invasive procedures provide a safe and effective alternative source of cholangiocyte organoids. The shedding of (organoid-initiating) cholangiocytes in bile provides a convenient source of organoids for regenerative medicine.

An epigenetic map of malaria parasite development from host to vector.

The malaria parasite replicates asexually in the red blood cells of its vertebrate host employing epigenetic mechanisms to regulate gene expression in response to changes in its environment. We used chromatin immunoprecipitation followed by sequencing in conjunction with RNA sequencing to create an epigenomic and transcriptomic map of the developmental transition from asexual blood stages to male and female gametocytes and to ookinetes in the rodent malaria parasite Plasmodium berghei. Across the developmental stages examined, heterochromatin protein 1 associates with variantly expressed gene families localised at subtelomeric regions and variant gene expression based on heterochromatic silencing is observed only in some genes. Conversely, the euchromatin mark histone 3 lysine 9 acetylation (H3K9ac) is abundant in non-heterochromatic regions across all developmental stages. H3K9ac presents a distinct pattern of enrichment around the start codon of ribosomal protein genes in all stages but male gametocytes. Additionally, H3K9ac occupancy positively correlates with transcript abundance in all stages but female gametocytes suggesting that transcription in this stage is independent of H3K9ac levels. This finding together with known mRNA repression in female gametocytes suggests a multilayered mechanism operating in female gametocytes in preparation for fertilization and zygote development, coinciding with parasite transition from host to vector.