Transcriptional Regulation and Epigenetic Mechanisms Underlying Host-Parasite Interactions in Human Malaria

TRANSCRIPTIONAL REGULATION AND EPIGENETIC MECHANISMS UNDERLYING HOST-PARASITE INTERACTIONS IN HUMAN MALARIA José Luis Ruiz Rodríguez PhD thesis 2022 Doctoral thesis in Molecular Biology, Biomedicine & Clinical Research 2 Doctoral thesis Molecular Biology, Biomedicine and Clinical Research TRANSCRIPTIONAL REGULATION AND EPIGENETIC MECHANISMS UNDERLYING HOST-PARASITE INTERACTIONS IN HUMAN MALARIA José Luis Ruiz Rodríguez Supervisor: Dra. Elena Gómez Díaz University of Seville (EIDUS) Spanish National Research Council (CSIC) 3 while(noSuccess){ tryAgain(); if(Dead) break; } Like chess, malaria is played with a few pieces, but it is capable of an infinite variety of situations L. W. Hackett, 1937 The measure of intelligence is the ability to change Albert Einstein, attributed to 4 5 6 Host-parasite interactions in human malaria: Transcriptional regulation and epigenetics 7 FIGURES INDEX 9 TABLES INDEX 12 ABBREVIATIONS 13 SUMMARY 15 INTRODUCTION 16 1. The human malaria disease 16 1.1. Historical and present context 16 1.2. Current strategies against malaria and gaps of knowledge 19 2. The life cycle of Plasmodium parasites 26 2.1. Development in human hosts. Pathogenic mechanisms 26 2.2. The development in the mosquito hosts 28 2.3. Anopheles mosquito responses during sporogony 30 3. General principles of transcriptional regulation in eukaryotes 33 3.1. Cis-regulatory elements and transcription factors 33 3.2. Epigenetic mechanisms controlling gene expression 35 4. Hallmarks of the Plasmodium genome and transcriptional regulation 41 4.1. The characteristics of the Plasmodium genome 41 4.2. Transcriptional regulation in Plasmodium 42 4.2.1. Transcription factors 45 4.2.2. Nucleosome landscape and chromatin accessibility 48 4.2.3. Post-translational modifications of histones 50 4.2.4. Other mechanisms of transcriptional regulation 53 4.3. Transcriptional variation 54 4.4. Post-transcriptional regulation in Plasmodium 58 5. Hallmarks of the mosquito genome and transcriptional regulation 60 5.1. The Anopheles genome 60 5.2. Transcriptional regulation in Anopheles 62 6. Next-generation sequencing techniques 64 6.1. Transcriptomics 65 6.2. Epigenomics 66 6.3. De novo genome assemblies 68 RESEARCH OBJECTIVES 72 SUMMARY OF RESULTS 73 Host-parasite interactions in human malaria: Transcriptional regulation and epigenetics 8 Section 1: The Plasmodium parasites 73 1. Chapter 1: Characterization of the accessible genome in the human malaria parasite Plasmodium falciparum 73 2. Chapter 2: The second life of Plasmodium in the mosquito host: gene regulation on the move 84 3. Chapter 3: From contigs to chromosomes: automatic Improvement of Long Read Assemblies (ILRA) 89 Section 2: The Anopheles mosquitoes 94 4. Chapter 4: Chromatin changes in Anopheles gambiae induced by a Plasmodium falciparum infection 94 5. Chapter 5: The regulatory genome of the malaria vector Anopheles gambiae: integrating chromatin accessibility and gene expression 98 DISCUSSION 105 CONCLUSIONS 115 REFERENCES 117 Host-parasite interactions in human malaria: Summary Transcriptional regulation and epigenetics 15 Summary Human malaria is one of the most important infectious diseases and a major cause of death and poverty worldwide. It is caused by protozoan parasites of the genus Plasmodium that are transmitted by the bites of mosquitoes of the genus Anopheles. The parasites Plasmodium falciparum and the mosquitoes Anopheles gambiae are the leading figures of this global burden, which disproportionally affects Africa and children under the age of five. To fulfill development and to achieve adaptation to changing environments in the human and mosquito hosts, Plasmodium parasites are capable of drastic transcriptional switches. The Anopheles mosquitoes are the main vectors for human malaria, and they can display phenotypic variability in life history traits, including vector competence or responses against Plasmodium. Yet, the transcriptional regulation underlying host parasite interactions in human malaria, particularly based on epigenetic mechanisms and regarding the life cycle in the mosquito, remain almost completely unknown. In this doctoral thesis, we have applied multi-omic approaches and bioinformatic analyses to investigate the regulatory genome of both P. falciparum and A. gambiae mosquitoes, associated with the Plasmodium development and interactions within hosts, and with the responses of Anopheles mosquitoes to the parasitic infection. We have integrated genomic, epigenomic and transcriptomic approach to unveil relevant cisregulatory elements and to assay the relationship between gene expression levels and chromatin-related mechanisms, such as histone marks or chromatin accessibility levels. We applied different techniques to these organisms, integrating RNA-seq, ChIP-seq and ATAC-seq for the first time. We reported the positive correlation between transcription and chromatin accessibility by ATAC-seq or active histone marks by ChIP-seq. We also identified thousands of active regulatory sequences, including enhancer candidates, that appeared to be linked to Plasmodium developmental transitions or clonally variant gene expression within humans, or that in the case of mosquitoes seemed to be specific to tissues or Plasmodium infection status. Ultimately, these allowed us to predict cognate transcription factors. Altogether, we provide evidence for genome-wide mechanisms and regulatory regions that may be involved in the dynamic transcriptional regulation underlying hostparasite interactions between malaria parasites and the human and mosquito hosts. This is much required in the context of current efforts against malaria, to inform existing and new mosquito-control and anti-malaria strategies. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 16 Introduction 1. The human malaria disease 1.1. Historical and present context Human malaria is an ancient disease that has accompanied human species since the very origin of hominids. The original transfer event from gorillas is relatively recent (10,000-100,000 years ago in Africa) 1–4, but there is evidence in Mesopotamian and Egyptian human remains dating up to 3,200 BC 5–10. The etymology of the word malaria is medieval from the Italian mala aria meaning ‘bad air’, which hints the impression that the symptoms of this invisible disease caused in our ancestors, who initially blamed foul air 11,12. Therefore, malaria has plagued humanity for millennia, becoming one of the deadliest diseases. In the 20th century alone, human malaria is estimated to have caused 2 to 5% of all deaths worldwide 13, and it has left unique trails through our evolution as species 13–16. These include protective red blood cell variants, such as sickle cell haemoglobin or Duffy-antigen negativity 17–23. Currently, we know that malaria is caused by protozoan parasites of the genus Plasmodium, which are transmitted by mosquitoes of the genus Anopheles. Human malaria is the deadliest mosquito-borne disease and an everlasting and leading public health problem that causes death, poverty, and suffering in dozens of tropical and subtropical countries around the world 24,25. It can range from asymptomatic or mild manifestations to a devastating disease with symptoms that include acute fevers, anaemia, coma, or death if not treated within 24 hours. In adults, multi-organ failure is also a frequent outcome. For pregnant women, malaria can cause anaemia and death before and after birth, as well as stillbirth, preterm birth, poor foetal growth, poor cognitive outcomes, mastitis, and increased risk of child death 24,26–28. Individuals older than 5 years who are continuously exposed to the disease may develop clinical immunity, and most adults in endemic regions can overcome infections without severe disease 29–32. Children often show higher levels of parasitaemia and exacerbated symptoms, including severe anaemia due to massive loss of red blood cells (RBCs), metabolic acidosis, respiratory distress, or cerebral malaria. Therefore, children younger than 5 years but older than 6 months, which is the threshold when loss of maternal antibodies occurs, are particularly vulnerable and account for most of the malaria burden 33. Globally, after the failure of the eradication campaign in the 1960s and decades of neglect, the international community embraced in the 1990s the still ongoing effort to Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 17 free the world from malaria 34–36. At the beginning of the 21st century, malaria transmission occurred in 107 countries across the five continents, comprising 80% of the global population 35. Today, around half of the global population is still at risk, particularly in tropical and subtropical countries (FIG. 1). In 2020 there were 241 million malaria cases in 85 malaria-endemic countries and 627,000 deaths, 77% of which were children under 5 years of age and ~50% of which occurred in 6 sub-Saharan African countries 25,35,37 (FIG. 1). Indeed, the malaria scourge is disproportionately distributed, with Africa accounting for 96% of all malaria deaths. In addition to its death toll, the disease is also responsible for a heavy economic burden in low-income countries, being intrinsically linked to socioeconomic development 38–40. In the last two decades, a momentum with steady and sustained commitment to malaria, international collaboration, and socioeconomic growth has led to unparalleled achievements. In the period 2000-2020 (the latest data available from the World Health Organization, WHO), it is estimated that 1,700 million malaria cases and 10,6 million malaria-related deaths have been averted. The global malaria mortality rate (i.e. deaths per 100,000 population at risk) has been reduced from 30 in the year 2000 to 13 in 2019 and 15 in 2020, the deaths among children under the age of 5 have decreased by 10%, no malaria-free country has been found to re-establish transmission, and 47 countries suffered fewer than 10,000 cases (up from 26 in 2000) 25 (FIG. 2). Dozens of countries have eliminated the disease and have been certified malaria-free or are on track (FIG. 3). However, the progress is uneven and fragile, and the resurgence of the disease a continuous threat 41. Additionally, the current and anticipated climate crisis in a context of globalization is also likely to increase the burden of mosquito-borne diseases, particularly malaria 42–50. The progress seems to be stalling when accounting for multi-year periods. The milestones and goals planned by WHO have not been met 51–53, with the target for the reduction of malaria cases missing by 40% (FIG. 4) and many countries not being able to eliminate malaria in 2020 54. This may reflect a change in the criteria for malaria statistics by WHO in 2021, but more importantly a worrying trend that has long been raising concerns 55. For example, global malaria cases were reduced by 27% between 2000-2015, while in the period 2015-2019 the reduction was less than 2%. This plateauing may be due to limited access to prophylaxis, treatment, or vector control approaches, as well as the emergence of insecticide-resistant mosquitoes and antimalarial-resistant parasites 56–58. In any case, the world is at a crucial juncture. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 18 Figure 1. Malaria by the numbers The global health burden of human malaria, with the focus on the African region and the trends in the period 2000-2020. Data has been extracted from the WHO Malaria Report 2020 and 2021 25,35 and Colón-González et al. 2021 42. Infographics have been adapted from material available at the Center for Disease Control. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 19 Furthermore, the COVID-19 pandemic declared in 2020 cannot be omitted. Most of the aspects surrounding COVID-19 and malaria remain to be explored 59–70, but at least the associated disruption of basic services has led to marked increases in malaria morbidity and mortality 25,71–75. In 2020, there were 14 million more malaria cases and 69,000 more deaths than in 2019, and it is estimated that 68% of the additional deaths were associated with the pandemic. Fortunately, despite the increases, updated reports point to efforts against malaria prevailing and to the setback being lower than anticipated 25,76–79. Overall, global malaria eradication may be within sight, but it is urgent to accelerate progress and to reverse current tendencies 80,81. 1.2. Current strategies against malaria and gaps of knowledge Human malaria parasites are constantly posing new challenges that oppose the eradication strategies being developed against the disease. For example, crucial biological threats include the deletion of HRP2/3 genes in parasites, which are the molecular basis for rapid diagnosis tests, so that many infections may remain undetected 82–84. The most important approach against the parasites is the use of antimalarial drugs, particularly front-line treatments based on artemisinin, which is the most recent and effective drug available 58,85. However, parasites are becoming increasingly resistant to antimalarial drugs. Resistance to artemisinin is widespread in Southeast Asia and has recently been reported in Africa for the first time, being associated with altered Figure 2. Number of malaria endemic countries The number of malaria endemic countries in the period 2000-2020 and trends of indigenous malaria cases. Adapted from Figure 4.1 in the WHO Malaria Report 2021 25. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 20 Figure 3. Dynamics of the disease in endemic countries The progress of the malaria endemic countries towards the WHO milestone in 2020 of at least 40% reduction from 2015. Adapted from Figure 8.2 in the WHO Malaria Report 2021 25. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 21 gene expression profiles 86–89 (FIG. 5). Given these obstacles, the transmissible stages and the development of malaria parasites in the mosquitoes have increasingly attracted attention as targets for new drugs and vaccines, but there are still many aspects and lifehistory traits that remain rather obscure. For example, recent evidence during development in the mosquito points to selective advantages for minority parasite genotypes related to drug resistance. This could explain the persistence of parasites with Figure 4. Dynamics and predictions of malaria risk The predictions of the progress of malaria cases (a) and mortality (b) if maintaining current trend (blue) or achieving WHO milestones (green). Adapted from Figure 8.1 in the WHO Malaria Report 2021 25. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 22 resistant alleles due to their preferred transmission 90. Finally, indirect exposure of parasites during development in the mosquitoes to other chemicals, such as insecticides, also has epidemiological significance, but this remains understudied 91. In particular, the adaptability potential displayed by the parasites is an outstanding challenge and many gaps of knowledge remain. Efforts against malaria must always be evidence-driven and accompanied by basic research, with the aim of informing and refining current strategies, together with developing new approaches to match the parasites 'arms race'. 92,93 Currently, vector control strategies are the main workhorse in the fight against malaria 25,94,95. There are more than 400 known Anopheles species, but only around 70 serve as vectors of importance for human malaria 96,97. Anopheles gambiae is Figure 5. Genetic distribution in Africa of K13 alleles linked to artemisinin resistance Genetic distribution in some African countries of alleles of the P. falciparum gene K13, known to be crucial for resistance to artemisinin 92,93. Extracted from Stokes et al. 2021 87. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 23 responsible for most of human malaria transmission on the African continent. The mosquito-human transmission is essential to the spread of the disease so, together with socio-economic reasons, the longer lifespan and strong human-biting habit of these mosquitoes are the main explanations for the disproportionately high incidence of malaria in Africa. Therefore, beyond parasite-related strategies, the mosquito is a very attractive intervention point. It is estimated that of the reduction in malaria incidence in the period 2000-2015 (~660 million malaria cases averted), 70% occurred due to the use of mass-distributed insecticide-treated bed nets and 10% due to indoor-residual spray 98. During the first Global Malaria Eradication program in the 1960s, large-scale campaigns using the insecticide DDT were effective to some extent, but had to be stopped due to the appearance of resistance, amongst other reasons 99. Since then, more selective vector control approaches have been implemented, but the increasing resistance of mosquito vectors to insecticides and behavioural adaptations are a pressing threat, and one of the pillars that sustain continued malaria transmission 100–108. According to WHO, 78 of the 88 countries that reported data in the period 2010-2020 identified resistance to at least one insecticide in one malaria vector species, and 29 countries reported resistance to all insecticide classes 25 (FIG. 6). Mechanisms remain not fully understood, although detoxification pathways are known to be relevant, such as those mediated by cytochrome P450s 109,110 or GST 111,112. However, up to this day, definitive malaria and mosquito control is yet to be achieved in the field, despite the continued development of novel approaches, such as insecticide chemistries or exposure of Anopheles to antimalarial compounds 113–115. Other aspects related to the biology of the vectors and the disease transmission may be of importance to the current malaria epidemiology, but have traditionally been overlooked. These include behavioural and morphological changes due to infection, control approaches, human habits, or anthropogenic environmental changes. Other understudied aspects affecting malaria transmission are the infection status by other Plasmodium strains or species, infection by other microbes, the role of the mosquito microbiome, or the importance of human attractive cues 116–123. Novel techniques for vector control include the manipulation of mosquito physiology to influence both the development of the parasites or the fecundity of mosquitoes 124, the targeting of Anopheles metabolic proteins 125,126, the targeting of male mosquitoes to impact mating or reproduction 127–129, or the manipulation of attractive cues 119,130. State-of-the-art genetic manipulation, for example to obtain transgenic mosquitoes affecting different stages of Plasmodium (FIG. 7) or gene drive techniques against mosquitoes are also Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 24 Figure 6. Global distribution of resistance to insecticides The number of insecticide classes for which resistance has been reported in at least one species of malaria vector in at least one site between 2010 and 2020. Adapted from Figure 9.4 in the WHO Malaria Report 2021 25. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 31 currently under debate and there is a large variability in the responses of the mosquitoes to Plasmodium infections, depending on both genetic and environmental factors. In this context, there is evidence that Anopheles mosquitoes have developed tolerance to Plasmodium infections and that the parasites have evolved lower virulence and mechanisms for immune evasion within mosquitoes 124. For example, there are not detrimental effects on mosquito survival, fertility, or fecundity when the parasites match their natural Anopheles vector, mosquitoes with increased egg development (i.e. more fertile) display higher parasite loads, and low-level infections are associated with increased mosquito fitness 124. However, more research is needed on the mechanisms underlying phenotypic responses against pathogens, particularly on the potential tolerance or fitness cost of Plasmodium infections, and their effect on mosquito survival, fecundity, or longevity 230,231.232 There is variability in mosquito traits related to Plasmodium infections, such as biting rate or immune responses (FIG. 13). To what extent this variability relies on mosquito genetics or dynamic transcriptional regulation remains poorly investigated, but both are important determinants of vector competence and one of the aspects underlying Figure 12. Non-genetic factors and vector competence The influence of non-genetic and environmental factors on the vector competence of Anopheles mosquitoes for Plasmodium parasites. Adapted from Lefèvre et al. 2013 232. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 32 the complex epidemiology of malaria. The precise molecular mechanisms underlying mosquito phenotypic responses to infections remain understudied, but some advances have been made. For example, regarding immunity, the A. gambiae response against pathogens is well characterized and happens mostly in the midguts. Consequently, P. falciparum is known to silence the nitration response and the complement-like TEP1 system in the mosquito midguts through the surface protein P47 233,234. However, P47 is not always sufficient, which hints at the existence of unknown factors and at the importance of genetic variability in both the parasites and the mosquitoes. Due to many different haplotypes and polymorphisms, survival would then depend on the precise mosquito-parasite combination 235. Figure 13. Feedback relationships between factors affecting Anopheles biology and vector competence Relationship and feedback between parameters related with the mosquito biology and vector competence to Plasmodium parasites. Red and blue arrows represent increase or decrease of malaria transmission, respectively. The parameters that are considered to be key are marked in gray, the parameters that may be adaptive are shaded in white and the potential benefits or costs to the vectors are depicted in yellow. Adapted from Cohuet et al. 2010 231. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 33 3. General principles of transcriptional regulation in eukaryotes In animals, the expression of protein-coding genes implies the transcription of messenger RNAs (mRNAs) from the DNA and the subsequent translation into proteins, which occurs via RNA polymerase and ribosomes. The most basic question regarding transcriptional regulation would be how the RNA polymerase II is recruited to the first position of the coding sequences (i.e. the transcription start site, TSS) to produce a precise level of mRNAs, which can be variable depending on the conditions, tissues, or cells assayed. There are many factors that contribute to the regulation of this process, such as specific noncoding regulatory DNA sequences (cis-regulatory elements, CREs), trans-acting proteins (transcription factors, TFs), proteins called chromatin remodelers, or epigenetic mechanisms, such as chemical modifications of the DNA or histone proteins, and changes in the 3D genomic structure. 3.1. Cis-regulatory elements and transcription factors In general, the transcription process in eukaryotic organisms is primarily regulated by the binding of TFs to CREs. As a principle, CREs should be accessible so that the TFs can reach the dynamic chromatin and exert their functions (i.e. activate or repress transcription) 236–238 (FIG. 14). DNA-binding proteins include TFs, but also other activators, coactivators, and enzymes that modify the chromatin (i.e. chromatin remodelers). These would all be recruited in a coordinated and combinatorial manner to bind the target CREs and consequently modulate transcription 239,240. Eukaryotic transcription factors can be classified into general TFs and specific TFs. The former are involved in the recruitment of the RNA polymerase II required for the transcription of protein-coding genes, while the latter are involved in the recruitment of chromatin remodelers that are required for the dynamic changes in the nucleosome landscape (the RNA polymerase II and the transcriptional machinery may access the chromatin). The categories of CREs include promoters, enhancers and insulators. Promoters are regions typically proximal and upstream to the genes, whereas enhancers may be distal or proximal to genes, and be located in intergenic, intronic and exonic regions 241– 243 (FIG. 14, 15). Regarding their function, promoters initiate transcription and contain the core promoters, which are the ~80 bp regions centered on the TSS. The core promoters guide the RNA polymerase II to the TSS and determine the direction of transcription, so expression is initiated at a basal level. This function is based on a 244 Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 34 multiprotein complex of TFs and the RNA polymerase (called preinitiation complex, PIC) that is assembled over the core promoters (FIG. 14) 245. Beyond basal transcription, enhancers modulate the transcription rate (i.e. gene activation or repression) by looping through the linear DNA sequence and contacting the promoters, regardless of orientation or distance 246–248 (FIG. 15). Enhancers are modular (i.e. the same gene can be targeted Figure 14. The eukaryotic transcriptional machinery Transcriptional regulators (transcription factors, TFs) and coregulators (CoR) bind to cisregulatory elements called enhancers, which are typically associated with transcription of regulatory enhancer RNAs (eRNAs). These modulate basal transcription levels by the RNA Pol II through the looping and interaction with the promoters, which are proximal to the target genes coding proteins or non-coding RNAs. General TFs (GTFs) are also regulating transcription through binding to the promoter. The long range interactions are mediated by protein complexes such as Mediator, Pre-Initiation (PIC) or Cohesin. Enhancers and promoters are associated with an open-chromatin environment and certain histone post-translational modifications (hPTMs) at adjacent nucleosomes. Adapted from Hou & Lee Kraus 2020 244. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 35 by different enhancers and the same enhancer can target multiple genes simultaneously, without a large degree of specificity), and they contain binding sites for TFs that may act in a combinatorial manner or be conditionally expressed (i.e. genes may be only expressed at certain times or conditions). Therefore, genetic variation in enhancers can lead to important phenotypic changes and disease 249. Finally, other CREs are insulators, which serve as barriers to isolate genes from neighbouring transcriptional activity or CREs, and segmentate the genome in different functional domains that typically match the 3D organization in topological domains 250. Insulators typically have long-range influence and binding sites for particular architectural proteins and TFs, such as CTCF. Research into the role of genome organization in transcriptional regulation is a hot issue that affects several areas in biology, but many questions remain to be answered.251 3.2. Epigenetic mechanisms controlling gene expression Globally, the term epigenetics refers to the changes in gene expression and consequent phenotype that do not involve changes in the underlying DNA sequences (i.e. the genotype), and that are also stable over time and heritable 252–254. A looser meaning includes any molecular factor or effector that contributes to an epigenetic phenotype and transcriptional status, but without necessarily being heritable 255,256. From a functional point of view, epigenetic mechanisms allow for fast phenotypic variation and provide the Figure 15. Transcriptional regulation of the A gambiae Hox complex An example of the landscape underlying four genes within the Hox complex in the A. gambiae mosquito. Transcriptional regulation occurs through chromatin loops, which allows the contact of enhancers and their target promoters, within compartments constrained by the chromatin accessibility levels and cis-regulatory elements that are called insulators and contain binding sites for architectural proteins. Adapted from Lezcano & Sánchez-Polo et al. 2020 251. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 36 means to integrate input signals from the environment and to mediate responses through transcriptional regulation 257. They shape the chromatin landscape by modulating the packaging and thus the accessibility of genes and CREs, which consequently impacts transcription 258,259. This way, the same gene can be expressed differently at different times or conditions (i.e. transcriptional heterogeneity), and this is key to adaptation to environmental constraints. In eukaryotes, the DNA that is ultimately condensed into chromosomes within the nucleus is first packaged into chromatin, which is composed of basic units termed nucleosomes 260 (FIG. 16). Canonically, a nucleosome comprises 147 bp of DNA wrapped in 1.65 turns around an octamer of four histone proteins (H2A, H2B, H3·and H4). These histones are packed into chromatin and stabilized by the linker histone H1 and other associated proteins (FIG. 17). Histone proteins are typically conserved through eukaryotes due to their central functions, but can also be replaced by variants with other properties 261–263. Given this structure, epigenetic regulation may first occur through a dynamic process of nucleosome repositioning and the subsequent changes in chromatin accessibility at CREs. Epigenetic factors modulate the accessibility of both the RNA polymerase and the TFs to the DNA, allowing or inhibiting binding and transcription 264,265 (FIG. 16). Therefore, an accessible CRE would be functional, whereas a CRE associated with a nucleosome would not be accessible and reachable by TFs, so it would not be exerting a regulatory function in transcription. This regulation layer is largely conserved through metazoans and beyond 266, but it also depends on species/specific nucleosome properties, such as the precise length of wrapped DNA, the evolution of divergent histone variants, or the repertoire of chromatin remodelers that alter chromatin structure (e.g. by and chemically modifying the DNA or histones) 267–269 (FIG. 17). A major epigenetic mechanism relies on a variety of chemical modifications of the aminoterminal domains of histone proteins, which protrude from the nucleosome and are referred to as histone tails 270,271 (FIG. 17). These are called post-translational modifications of histones (hPTMs), and they can alter the stability of the nucleosome and the underlying DNA accessibility and functionality. The hPTMs are also able to interact with protein complexes, to recruit DNA binding proteins, including TFs or chromatin remodelers 272, and they are the physical carriers of epigenetic memory 253,261,273. The combinatorial distribution of these epigenetic alterations, which is required for their functions, is known as the ‘histone code’ (i.e. a defined set of hPTMs would be required to perform a particular function or fulfilling development) 274–276.277 Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 37 Figure 16. The functional relevance of noncoding regulatory sequences Noncoding sequences may display different properties with relevant roles for transcriptional regulation of the coding genes. Examples are the different levels of chromatin accessibility or the repertoire of histone post-translational modifications (hPTMs) that are allowing the recruiting and binding of proteins, such as TFs. Adapted from Wong et al. 2021 265 and Lezcano & Sánchez-Polo et al. 2020 251. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 38 Globally, hPTMs can be classified into different categories that are a priori associated with different functions, such as DNA repair, chromosome condensation, or transcriptional regulation (Table 1). Finally, certain combinations of hPTMs define a regulatory landscape (termed ‘chromatin state’), which can be used to identify and categorize CREs, inferring their accessibility and their active/inactive state. In fact, the enrichment of certain hPTMs and chromatin states in CREs is highly predictive of the transcription of the target genes 278–281. For example, H3K27ac is an hPTM located at active promoters and active enhancers, H3K9me3 is linked to transcriptional repression, or H3K4me3 is a promoter-specific hPTM that displays a pattern considered to be conserved through all eukaryotes (i.e. enrichment in the two nucleosomes located immediately after and before an accessible/active promoter) 282. Apart from hPTMs, a related layer of epigenetic regulation are the chemical modifications of DNA sequences, such as the direct methylation of cytosines 283,284, which is associated with transcriptional repression when it occurs on enhancers and promoters.285 Figure 17. Overview of epigenetic mechanisms regulating transcription Brief description of epigenetic mechanisms underlying transcriptional regulation. Adapted from National Institutes of Health 277. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 39 Histone post-translational modifications (hPTMs) Functions regulated Histone residues modified Acetylation Transcription, Repair, Replication, Condensation K -ac Lysine methylation Transcription, Repair K -me1 K -me2 K -me3 Arginine methylation Transcription R -me1 R -me2a R - me2s Phosphorylation Transcription, Repair, Condensation S -ph T-ph Ubiquitylation Transcription, Repair K -ub Sumoylation Transcription K -su ADP ribosylation Transcription E -ar Deimination Transcription R > Cit Proline isomerization Transcription P-cis > P-trans Arising from the combination of the features above, chromatin can be classified into general conformations with different functional implications. The genomic regions that are more accessible and non-condensed are called euchromatin or open chromatin, being depleted of nucleosomes and linked to hPTMs associated with accessibility, active CREs, and transcription (FIG. 18). In contrast, the chromatin that is more condensed and compact is referred to as heterochromatin or closed chromatin. This is nucleosome-rich, usually located at the nuclear periphery, and associated with non-accessible CREs and transcriptional repression 258. An alternative intermediate state termed permissive chromatin is also known to be sufficient for pioneer TFs to start the chromatin remodelling process leading to accessibility 286 (FIG. 18). In eukaryotes, a larger genomic fraction is persistently associated with constitutive heterochromatin at repetitive regions, such as Table 1. Overview of the different classes of hPTMs An example of the repertoire of histone post-translational modifications (hPTMs) and their associated functions in eukaryotes. Adapted from Kouzarides T. 2007 285. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 40 telomeres or centromeres, whereas euchromatin is more restricted to particular regions or CREs. Figure 18. The dynamic chromatin states across the genome Alternative chromatin states across the genome: 1) inactive loci with closed chromatin (heterochromatin), 2) permissive chromatin as an intermediate state that allows the access of TFs and the initiation of chromatin remodeling towards transcription, and 3) an active locus displaying chromatin accessibility (euchromatin) and allowing the binding of TFs. Adapted from Klemm et al. 2019 259. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 47 ApiAP2 TF Phenotype (-| Block) Binding motif AP2-SP2 oocyst -|, sporoblast - PF3D7_0420300 NA CACACAC, GTGTTACAC AP2-O2 zygote -|, ookinete TGACATCA SIP2 NA GGTGCAC AP2-EXP2 / AP2-06A slow growth - AP2-06B NA ATAAGCCCA, CTCTAGAG AP2Tel sporozoites -|, liver stage - AP2-L sporozoites -|, liver stage AATTTCC AP2-LT / AP2-08 NA CACACACA PF3D7_0934400 NA - AP2-I NA GGGTCGACCC, TCTTGCC, GTGCACTA AP2-11A NA AGCATAC AP2-11B NA - AP2-G5 NA TAGAACAA AP2-O ookinete -|, oocyst TAGCTA PF3D7_12224000 NA TATATATA AP2-G asexual -|, gametocyte GxGTACxC AP2-12 NA TCTA(C/T)AA PF3D7_1305200 NA TGCACACAC AP2-FG / AP2-G3 asexual -|, gametocyte TAGCTCA(G/A)A AP2-HS NA GCGGGGC AP2-O4 ookinete -|, oocyst ATTCTAGAA AP2-G2 asexual -|, gametocyte GTTG(T/C) AP2-O3 zygote -|, ookinete - AP2-O5 zygote -|, ookinete - AP2-HC dispensable CACACACAC, AP2-EXP oocyst -|, sporoblast TGCATGCA Table 2. Description of ApiAP2 TFs. Brief description of the ApiAP2 TF family, the phenotypes they have been described to influence or to block in rodent malaria models, and the motifs at their TF binding sites. Adapted from Jeninga et al. 2019 327. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 48 4.2.2 Nucleosome landscape and chromatin accessibility In Plasmodium, well-positioned nucleosomes can be found in the promoters downstream of TSSs (‘+1 nucleosomes’), while the regions directly upstream of TSSs tend to be depleted of nucleosomes (accessible nucleosome-depleted regions, NDRs) until a '-1 nucleosome' upstream is detected (FIG. 23). This nucleosome positioning conforms to the typical eukaryote pattern: NDRs upstream of genes that must be highly expressed enable the access of the transcriptional machinery to the promoters and TSSs, flanked by strongly positioned nucleosomes, and vice versa for genes repressed or less expressed. However, nucleosomes display distinctive properties. These include lower stability, weaker binding of the H2A/H2B dimer, short spacing (DNA linkers of only 8 bp in length vs. mean linker length in eukaryotes of 20-75 bp), loss of sequence-dependent positioning capacity (but presence of flanking motifs), or a wrapping DNA length of 155 bp instead of the canonical 147 bp 290,296,341 (FIG. 24). Due to these biochemical properties that lead to attenuated histone-DNA interactions, the nucleosomes tend to be more mobile and, because of the close nucleosome spacing, they do not form higher order chromatin structures. The four canonical histone proteins in eukaryotes (H2A, H2B, H3·and H4) also present divergent sequences in P. falciparum, with 64%, 68%, 93% and 92% identity between humans and Plasmodium H2A, H2B, H3, and H4, respectively (e.g. only 2 amino acids differentiating H3 between humans and Arabidopsis thaliana) 290. The typical 10 bp periodicity of AT and GC in the nucleosomes is also less pronounced in Plasmodium 342,343. It is possible that the AT richness inhibits the formation of higher-order chromatin structures, so the chromatin is generally maintained in a more accessible state 341,344. It has also been suggested that due to the absence of the histone linker H1 in Apicomplexa, the AT stretches between nucleosomes could be a novel mechanism to achieve nucleosome positioning and control of accessibility 290. In addition to the canonical histones within the nucleosome core, lineage-specific variants include H2A.Z, H2B.Z, H3.3 and CenH3. Regarding functions, H2A.Z and H2B.Z are likely involved in transcriptional regulation 345,346, whereas H3.3, which is located at GC-rich subtelomeric regions, has been reported to be a potential memory mark for virulence gene expression 347. Finally, CenH3 appears to mark the AT-rich centromeres and is involved in chromosome segregation 295. Regarding the relationship between nucleosome positioning/occupancy and gene expression regulation, evidence to date tends to be contradictory and inconclusive. Previous studies profiling chromatin accessibility by MNase-seq and FAIRE-seq, have pointed to a positive correlation between the extent of NDRs at CREs and active Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 49 transcription 342,343,348. Nucleosome positioning has also been reported to be more relaxed in Plasmodium compared to other eukaryotes 341 (FIG. 25). This could be due to the presence of less stable nucleosomes, but it has also been suggested that the techniques applied for assaying chromatin structure and accessibility may not have been precise, and that bias in enzymatic digestion because of the AT richness or repetitive regions may have led to inconclusive results 297,349,350. The short developmental times of these parasites and their natural lack of synchrony could have also confounded reports of nucleosome stability, because of the parasite cells captured being in different developmental stages 290. All this considered, the number of studies that have appropriately and comprehensively studied nucleosome landscapes and chromatin accessibility in Plasmodium prior to this thesis was very limited 343.351 Figure 23. The nucleosome landscape around Plasmodium genes Schematic representation of the prototypical landscape and nucleosome positioning by MNaseseq around a P. falciparum. Transcriptional landmarks such as TSS, TTS, UTRs or well positioned nucleosomes are included. Extracted from Watzlowik et al. 2021 290. Figure 24. Distinctive characteristics of Plasmodium nucleosomes Schematic representation of the particularities of nucleosomes in P. falciparum when compared to human. Extracted from Watzlowik et al. 2021 290. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 50 Comparative studies between different Plasmodium stages have shown that the nucleosome landscape is mostly unaltered through development at the global level (i.e. static nucleosomes). However, the local nucleosome occupancy at the promoter of developmental loci showed significant and dynamic changes between stages, which correlated with gene expression. During the IDC it was observed that in rings and schizonts, the genome was more compact and heterochromatic (i.e. higher nucleosome occupancy), while in the trophozoite, which requires massive transcription, the nucleosomes were depleted, so the genome was more open and the larger NDRs in the promoters correlated with the higher level of transcription 352. Such extensive and drastic changes in nucleosome occupancy and chromatin accessibility have not been reported in any other eukaryote 290,343. Chromatin accessibility profiling can be used as a proxy to predict and investigate active CREs and GRNs underlying transcription 353,354. However, chromatin structure involved in transcriptional regulation has traditionally remained understudied in Plasmodium. Some CREs have been predicted largely by in silico approaches and characterized to a variable extent, but the majority do not share sequence similarity with other eukaryotes and remain pending of experimental validation 355–357. 4.2.3 Post-translational modifications of histones As mentioned above, a basic epigenetic mechanism of transcriptional regulation is based on the N-terminal tails of the histones. These harbour the post translational chemical modifications that are determining the chromatin properties and functions, such as Figure 25. Comparison between the Plasmodium and Saccharomyces nucleosome landscapes Representation of the differences in the nucleosome landscape around a transcriptionally active gene in S. cerevisae and P. falciparum. Extracted from Abel & Le Roch 2019 351. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 51 phosphorylation, methylation, or acetylation. In P. falciparum, the proportion of constitutive acetylation (linked to euchromatin) and the number of identified hPTMs (~500) are also unusually large, including several that appear to be specific to Plasmodium and Apicomplexa, or required only at certain developmental stages or conditions 275,276,358. For example, gametocyte-specific hPTMs include H3K36me2, H3K36me3, and H3K79me3, or H3K9me2 and H3K18me1 appear to be IDC-specific 358. Such a dynamic pattern points to a dominant role in the just-in-time model of transcription regulation. From a functional point of view, P. falciparum is considered to show both a general abundance of hPTMs associated with transcriptional activation and less regions with repressor hPTMs associated with repression (i.e. most of the genome displays euchromatin, with exceptions including heterochromatic subtelomeric regions and intrachromosomal domains) 275,358–360. As in the rest of eukaryotes, acetylation is commonly associated with chromatin accessibility and activation of transcription 359,361,362, while methylation can be associated with both activation or repression, depending on the affected residue, the genomic position of the modification, and the proteins recruited. An example of a protein recruited to a methylated hPTM is the heterochromatin protein 1 (HP1). It binds to H3K9me3, which is a canonical repressor mark, and interacts with several factors to form a multi-protein complex linked to heterochromatic regions and transcriptional repression 363,364. Another recently described hPTM is H3K64me3, which appears to be repressor for the set of Plasmodium proteins exported to the host cell surface 365. On the contrary, H3K4 methylation appears to be associated with euchromatin and transcriptional activity. Furthermore, the functions of many other hPTMs seem to have diverged in Plasmodium, such as H3K36me3, which typically marks active transcription in eukaryotes and, in parasites, it is associated with heterochromatin and repressed promoters 366. In accordance with the large repertoire of hPTMs, Plasmodium parasites display an extensive panel of chromatin remodelers. These include 'writers' and 'erasers' in charge of depositing and removing hPTMs (e.g. histone acetyl transferases, deacetylases, lysine methyltransferases and lysine demethylases; HATs, HDACs, HKMTs and KDMs, respectively), and 'readers' that recognize the modifications, such as proteins containing chromodomains and bromodomains (FIG. 26). Extensive functional characterization is still required, but it is already known these are crucial for a number of physiological processes, pathogenesis, virulence, and disease transmission 338,367–370. Examples of these are: the H3K9 methyltransferase KMT1 or the H3K9 deacetylase Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 52 HDAC1, which interact with the transcriptional repressor HP1, the histone deacetylases HDAC1 and HDA1, which epigenetically modulate AP2-G expression to regulate gametocytogenesis and sexual development, the histone acetyl transferase GCN5, which regulates stress- and artemisinin resistance-related genes, the HKMTs that are regulating activation of dormant stages in the liver 371, or the bromodomain proteins that bind acetylated histones in the promoters of invasion-related genes, whose expression is required for invading RBCs and undergoing the IDC 372,373. In accordance with these functions, HATs, HDACs, and HKMTs are targets of interest for therapeutic interventions in Plasmodium 374–377, for example disrupting the life cycle 378. Figure 26. Overview of the chromatin modifications of histone H3 Schematic representation of the remodeling processes that the histone H3 may undergo at specific residues, through acetylation or deacetylation by HATs or HDACs, or through methylation changes by KDMs or HKMTs in the case of methylation. Extracted from Abel & Le Roch 2019 351. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 53 4.2.4 Other mechanisms of transcriptional regulation In Plasmodium, as in other eukaryotes, the spatial organization of the genome within the nucleus is an important layer of transcriptional regulation 379–381. Pioneering studies have begun to describe the 3D nuclear organization in Plasmodium using state-of-the-art techniques for capturing chromosome conformation 382,383. They added evidence that P. falciparum lacks well-defined chromosome territories (i.e. topologically associated domains), as well as most of the conserved architectural proteins (including CTCF) 384. It has also been reported that condensation is minimal apart from the telomeres, with few central regions tethered to the heterochromatic nuclear periphery (being not comparable to other eukaryotes and conforming to a predominantly euchromatic genome). On the other hand, the nuclear pores and the nuclear structure are dynamic through the developmental life cycle, with changes in 3D architecture that correlate with the level of transcription 351,385,386 (FIG. 27). For example, nuclear pores show an increase at the more transcriptionally active trophozoites. There is also a variable colocalization of genes with euchromatin or heterochromatin depending on the transcriptional state, as well as an unusual loss of centromere interactions in P. falciparum and P. vivax sporozoites, which was suggested to be the result of global transcriptional repression and the increase of long-range contacts associated with the expression of liver stage genes 382,383,387. Finally, there are also complex features associated with virulence-related genes (i.e. coding antigens or exported proteins involved in pathogenesis, invasionrelated genes, and some TFs), including their colocalization within clusters of heterochromatin in the nuclear periphery and the formation of chromatin loops 363,388. Regarding direct methylation of DNA, its role remains unclear. In other Apicomplexa parasites such as Toxoplasma and Cryptosporidium, DNA methylation is reported to be absent, and only few studies in P. falciparum and in P. knowlesi have successfully identified this type of epigenetic mechanism 389–393. In P. falciparum, cytosine methylation has been depicted to be plant-like, with some features different from those of model eukaryotes: only one DNA strand was methylated, and methylation was detected mainly within exons and in CHH motifs. Besides, in P. falciparum DNMT2- mediated tRNA cytosine methylation has recently been found to regulate the translation Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 54 of proteins that mediate responses to nutritional stress and several metabolic pathways, including sexual commitment and homeostasis 394,395. Finally, lncRNAs (long noncoding RNAs), GC-rich ncRNAs or antisense ncRNAs, are gaining prominence as critical regulators of gene expression in Plasmodium. They have been shown to be transcribed following a stage-specific pattern through the development of human malaria parasites, contributing to parasite virulence and pathogenesis 396,397. Examples include lncRNAs associated with the GDV1 protein during gametocytogenesis, or to the multicopy var gene family. Evidence of the role of ncRNAs on Plasmodium development and pathogenesis are accumulating, but the precise mechanisms that Plasmodium ncRNAs use to influence epigenetic features and transcriptional regulation remain rather unknown 398. 4.3 Transcriptional variation Host-parasite interactions are very dynamic and the environments that the parasites may encounter within humans and mosquitoes are diverse. In this context, transcriptional regulation via epigenetic mechanisms provides means for the parasites to achieve fast and reversible phenotypic variation, both during development and to adapt to different environmental constraints (e.g. temperature, parasitaemia levels, drug Figure 27. Dynamics of 3D chromatin organization in Plasmodium development Dynamic changes in the 3D chromatin architecture through blood stages of P. falciparum, which correlate with the levels of transcription, for example of invasion-related and virulence genes, or the TF AP2-G. Adapted from Abel & Le Roch 2019 351. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 55 exposure, transmission intensity, or host nutritional status). These environmental cues may induce facultative phenotypic changes in some parasite cells, but not in others, and result in a pattern of transcriptional heterogeneity in the parasite population. This ability is crucial for parasite survival and is related to life-history traits such as virulence, pathogenesis, or transmission. The phenotypic variation can be achieved beforehand following a strategy called bet-hedging, so some parasites would be better suited to survive and overcome future selective processes or interventions. Alternatively, it could happen that transcriptional heterogeneity is generated as a direct and adaptive response after certain environmental constraints 399–401 (FIG. 28). Cell-to-cell transcriptional heterogeneity can be achieved a priori through a bethedging process which is known as clonally variant gene expression (CVGE). This refers to the capacity of parasite clones (i.e. individual cells with identical genome) to switch at different times between expression or silencing of genes termed clonally variant genes (CVGs). Thanks to CVGE, alternative phenotypes can be generated from a single genome. One type of CVGs are the variant surface antigens (VSAs). The strategy known Figure 28. Overview of the bet-hedging process in Plasmodium Representation of the generation of transcriptionally divergent cell populations through transcriptional regulation based on epigenetic marks of clonally variant genes (CVGs). These new populations would overcome environmental constraints and ensure the survival of the parasites. Adapted from Seco-Hidalgo et al. 2015 399. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 56 as antigenic variation is used by many pathogens to avoid mechanical clearance and immune recognition by the hosts 402–407. In Plasmodium, genes coding for VSAs are typically part of multicopy gene families that are located in subtelomeric clusters, enriched in heterochromatin and subjected to more recombination. Antigenic variation plays a direct role in the pathogenesis and virulence of Plasmodium parasites 289, and unlike other organisms, epigenetic regulation is known to have a dominant role in the regulation of these and other CVGs. In P. falciparum, the most well-known VSA is the erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family. It is essential for cytoadherence and pathogenesis in humans, including immune evasion and cerebral malaria 408,409. Each parasite contains around 60 different copies of var genes. The repertoire is very polymorphic between parasite strains and isolates due to recombination 410–412, and some variants are more pathogenic than others 413. In addition to CVGE, this gene family is subject to mutually exclusive expression, which means that amongst the whole family, only one copy is predominantly expressed by an individual parasite at a particular time. This copy can switch to another one periodically, and the profile can persist through generations (i.e. epigenetic memory) 413–416 (FIG. 29). This is the basis of malaria pathogenesis through cytoadherence or immune evasion as mentioned above, because the PfEMP1 copy presented onto the surface of infected RBCs periodically changes, to avoid recognition (FIG. 29). Other examples of var switching include the response to antibiotic blasticidin, with the responsible epigenetic marks persisting for generations after the antimalarial is withdrawn 415, or the exposure to heat shock (i.e. fever cycles), which appears to be mediated by a sirtuin gene 417. This phenomenon at the var genes seems to be related to ncRNAs expressed from a promoter in the introns or from adjacent genes 418–420, together with other layers of epigenetic regulation 254,421,422. For instance, these include the histone variants H3.3, H2A.Z and H2B.Z, which seem to be distinctively marking the promoter of the active var gene copy 347,423, or H3K36me3, which has been shown to be essential for repression of var genes 424. Beyond var genes, other multicopy gene families in Plasmodium are rifin (repetitive interspersed), pir (Plasmodium interspersed), or stevor (subtelomeric variable open reading frame) 425,426. These families also appear to follow to some extent the principles of antigenic variation, to mediate rosetting, and to show some degree of mutually exclusive expression 427–429. For example, rifin antigens have also been found to be targeted by human antibodies conferring protection 430,431, so the variation may also be a selective advantage for parasites. 432 Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 63 regulation in mosquitoes, there is also experimental evidence on the potential relevance of DNA methylation, but it remains controversial 251,466. Finally, the field of mosquito ncRNAs has also attracted attention, but in Anopheles only a handful of specific miRNAs have been functionally characterized 251,467. Apart from the chromatin structure, the identification of CREs have also received long-awaited attention. Despite the fact that a vast number of regulatory elements have been characterized in Drosophila over the last few years 468,469, the progress in Anopheles mosquitoes has been scarce. Several studies have investigated CREs of particular genes involved in specific functions, such as development or insecticide resistance 470. At the genome-wide level, most of the low number of CREs reported arise from in silico predictions and lack experimental validation 251. In this context, a pioneering study profiled the genome-wide chromatin accessibility of A. gambiae hemocytes to identify active CREs and predict the cognate TFs 471. However, caveats included the limited resolution and accuracy of the technique or the lack of integration of the chromatin accessibility data with gene expression profiles. There are now improved methods available for chromatin accessibility profiling (see below). All the above, together with the global burden of malaria and increasing need for insights on the transcriptional regulation occurring in the mosquito host, call for further research. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 64 6. Next-generation sequencing techniques The term Next Generation Sequencing (NGS) refers to the use of parallel highthroughput techniques to generate large datasets based on sequencing billions of nucleic acid fragments, at or beyond the genome-wide scale, during a single instrument run, and at a moderate cost 472–474. In the last two decades, the technological advances have been immense, leading to the foundation of whole new disciplines of increasing importance. Bioinformatics is the field of study concerned with the analysis of what is known as 'omic' data (FIG. 34). Inspired by the terms 'genome / genomic', many techniques include the suffix -omic to depict the molecules or features that are collectively targeted and measured. These include genomics, transcriptomics, epigenomics, metagenomics, proteomics, or metabolomics, among others. The approaches tend to be complementary, and their resolution and power are increasing dramatically over time. For example, with the advent of NGS techniques, -omic approaches have greatly improved the understanding of transcription, allowing for the modular interrogation of each one of its aspects (e.g. addressing levels such as transcription or translation independently). Integrative studies combining different omics techniques now hold the key to understanding biological or pathogenic processes and to implement precision medicine approaches 475–478.479 Figure 34. Number of multiomic-related publications Number of publications containing the terms ‘multiomics’ or ‘multi-omics’ in PubMed. Extracted from WikiMedia 479. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 65 The fields of infectious diseases and malaria have not been unaware of the socalled ‘omics revolution’ 480–484, and genome-wide studies on both Plasmodium and Anopheles spp. are now abundant and capable of addressing countless questions 291,485,486. Up to this day, new -omic and high-throughput techniques are continuously being developed and applied to malaria research. Driven by the tremendous drop in the cost of generating genomic data (FIG. 35), thousands of genomes of different species of Plasmodium parasites and Anopheles vectors can now be sequenced, enabling population and comparative genomics studies to support and regularly inform the malaria eradication agenda. In this context, the following sections provide a methodological introduction of the NGS and the multiple -omic techniques applied in this thesis.487 6.1. Transcriptomics Transcriptomics is one of the most popular categories of -omic techniques. It can be defined as the study of the transcriptome (i.e. the set of the expressed RNA molecules) in a given sample, which can be originated from cells, tissues, or whole organisms 488. RNA sequencing (RNA-seq) is currently the prototypical high-throughput method to analyze the expression of multiple mature transcripts under a given condition. It allows obtaining information on the RNA molecules corresponding to the complete genome, generating transcriptional profiles at a base resolution 489 (FIG. 36). Transcriptomic approaches can now be routinely used to assess the transcriptional status of both Plasmodium and Anopheles genes in a variety of tissues, experimental conditions, or disease statuses 490,491.492 Figure 35. The evolution of sequencing costs Dynamics of the sequencing cost of a human genome over the last decades. Adapted from the National Institutes of Health 487. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 66 6.2. Epigenomics 6.2. Epigenomics The field of epigenomics refers to the study based on high-throughput sequencing of the epigenome of a sample, encompassing the chromatin structure impacting transcriptional regulation, and the distribution of epigenetic modifications across the genetic material 493. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is a technique that, thanks to the use of complementary antibodies, allows the co-purification of the DNA fragments associated with chromatin-binding proteins of interest (e.g. TFs, Figure 36. Step-by-step walkthrough of a RNA-seq assay Schematic representation of the steps in a typical RNA-seq assay, including library preparation, high-throughput sequencing, alignment to reference sequences and obtaining base-resolution expression profiles. Adapted from Wang et al. 2009 492. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 67 chromatin remodelers, or epigenetic modifications such as hPTMs) 494,495. ChIP-seq analyses provide genome-wide maps of the distribution of these proteins or epigenetic marks at single-base resolution (FIG. 37). This approach targeting hPTMs at the nucleosomes has been widely used to perform genome-wide predictions of CREs (e.g, active promoters enriched in H3K4me3 and H3K27ac, or active enhancers enriched in H3K27ac and H3K4me1, without H3K4me3) in many organisms, including the species studied here, P. falciparum and A. gambiae. Figure 37. Step-by-step walkthrough of a ChIP-seq assay Overview of the steps in a typical ChIP-seq assay, including library preparation, sequencing, and alignment to reference sequences to obtain genome-wide epigenomic profiles. Adapted from Park 2009 495. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 68 Several protocols have also been developed to precisely characterize the accessible DNA regions that would correspond to active CREs, inferring the activity of the so-called ‘regulatory genome’ 496. These approaches include the techniques Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE-seq), the micrococcal nuclease coupled with sequencing (MNase-seq), the DNase-I hypersensitive sites coupled with sequencing (DNAse-seq), and more recently the Assay for Transposase- Accessible Chromatin coupled with sequencing (ATAC-seq) 259,497. ATAC-seq is based on a hyperactive DNA transposase that simultaneously performs cleavage and tagmentation (i.e. insertion of sequencing adapters) of the chromatin at nucleosomedepleted DNA regions (i.e. NDRs). The target DNA fragments can then be purified, amplified by PCR, and sequenced. The enrichment of sequencing reads at a particular genomic region ultimately indicates that it is an accessible NDR (e.g. containing active CREs with TF binding sites 498,499 (FIG. 38). On the contrary, regions without coverage of sequencing reads below a certain length (termed ‘nucleosome-free reads’) may be used to infer nucleosome positioning 500,501. The main advantages of the ATAC-seq approach are the higher sensitivity and resolution, the more easy-to-use and faster protocol, and the relaxed requirements for accepting low-input samples 498,499,502. 6.3. De novo-genome assemblies The majority of bioinformatic analysis for the methods described above are rooted in an initial alignment of the sequencing datasets against genomes or sequences used as reference 503. However, reference genomes may not fully represent the genomic diversity of the species or populations 504,505. The assayed samples may contain novel genomic features that would be missed due to the use of a non-representative enough reference. In the case of malaria parasites, there are now genomes available for all human-infecting species, but they are frequently fragmented and far from reference-grade quality. Even if belonging to the same species, different Plasmodium strains or populations are also likely to display genetic diversity (FIG. 39). In addition, the culturing of parasites is costly and implies drastic adaptations, such as the appearance of genomic aberrations or the inability to undergo sexual conversion 506–508. For example, the differences become clear when comparing with the most used P. falciparum reference strain (3D7). Despite being curated and updated for decades 299, only a core genome is shared by different P. falciparum strains, and sequences from field isolates frequently include novel genomic features, such as genes or copy number variations 412,509,510 (FIG. 40). Overall, for the reasons above, it would be recommended to generate de novo genome assemblies Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 69 using field isolates, ensuring that the sequences used as reference in bioinformatic analyses are as close as possible to natural conditions.511 Figure 38. Step-by-step walkthrough of an ATAC-seq assay Overview of an ATAC-seq assay, from input samples to the interpretation of the results after high-throughput sequencing. Adapted from Smith et al. 2022 511. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 70 The de novo genome assembly process is based on obtaining and fragmenting copies of a genome, followed by sequencing and assembling the overlapping reads in larger units representing consensus regions, called contigs and scaffolds. These ultimately would form the telomere-to-telomere chromosomes of the novel genomes. Currently, there are different sequencing technologies, which are based on sequencing short reads (commonly by Illumina technologies), or most recently long reads (referred to as ‘third generation’) 512. Longer reads span larger genomic regions and difficult-to- Figure 39. Overview of the core genome amongst Plasmodium strains Representation of the alignment of 15 P. falciparum assemblies from isolates worldwide to the chromosome 11 of the reference strain 3D7. A core common genome is shown in black, as opposed to variable portions in gray. Extracted from Otto et al. 2018 509. Figure 40. Overview of novel genomic features amongst Plasmodium strains Schematic representation of the alignments between multiple P. falciparum strains from different regions, highlighting the annotation that points to some genomic features (e.g. copy number variants) located only at particular samples. Extracted from Otto et al. 2018 509. Host-parasite interactions in human malaria: Introduction Transcriptional regulation and epigenetics 71 assemble repeats. Continued advances in long read technologies have greatly impacted the field, in terms of recent improvements of resolution, performance, and price 513. These are typically based on two alternative principles: the light-mediated sequencing by Pacific Bioscience technologies (PacBio) or the use of functionalized nanopores by Oxford Nanopore technologies (ONT) 514,515. In the case of Plasmodium, whose assembly process is particularly challenging, the promise of a ‘perfect’ genome assembly (i.e. complete and without gaps) is closer thanks to sequencing longer reads. However, the quality of the assemblies may still be variable, usually due to differences in sample quality and library preparation, genome size, fragment length, sequencing depth, or the presence of too many repetitive regions in the sequences. In addition, there are inherent caveats in sequencing technologies. For example, unlike shorter reads by Illumina, up-to-date ONT and PacBio technologies cannot capture repeats up to a point, so de novo assemblies based on long reads are likely not totally accurate and the annotation is hindered. Gene and protein prediction may be affected due to artefactual indels representing non-sequenced bases 516,517. This is particularly relevant to organisms with skewed genomes and compositional biases due to abundant homopolymer tracks, such as the AT-rich Plasmodium genomes. Therefore, to produce genomes of a reference-quality grade, a post-assembly manual finishing process to get ‘telomere-to-telomere’ assemblies is required (known as ‘polishing’). The polishing step used to demand large manual and time efforts, but there are now automatic methods available. However, easy-to-use pipelines are scarce or obscure, and they typically require considerable bioinformatic skills that are not available for the majority of research teams 509,518,519. Host-parasite interactions in human malaria: Research objectives Transcriptional regulation and epigenetics 72 Research objectives General objective and scope of the thesis In this thesis, we applied multiple omics data analyses to both Plasmodium parasites and Anopheles mosquitoes with the aim of investigating the regulatory genome controlling parasite development and interactions with the host, and mosquito responses to infection. Specific objectives Section 1: The Plasmodium parasite 1) We aimed to investigate the changes in chromatin accessibility underlying transcriptional regulation in P. falciparum during blood development and associated with clonally variant gene expression. Based on chromatin accessibility profiles, our goal was the mapping of active cis-regulatory elements and the prediction of binding sites for regulatory proteins (Chapter I). 2) We aimed to review our knowledge of Plasmodium spp. transcriptional regulation mechanisms during development in the Anopheles mosquito, particularly epigenetic mechanisms. We also discuss phenotypic variation of the malaria parasites as strategy for survival across its heterogeneous life cycle, such as bethedging strategies and transcriptional responses directly triggered by the environment (Chapter II). 3) We aimed to develop an easy-to-use pipeline for obtaining, correcting, and annotating de novo genome assemblies of reference-grade quality from Plasmodium spp. field isolates (Chapter III). Section 2: The A. gambiae mosquito 4) We aimed to describe the epigenomic changes at the level of histone marks and the accompanying transcriptional responses that take place in the midguts of A. gambiae infected with P. falciparum (Chapter IV). 5) We aimed to characterize the chromatin accessibility and the nucleosome landscape underlying transcriptional changes in P. falciparum-infected A. gambiae tissues (i.e. midguts and salivary glands) (Chapter V). Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 1 79 As explained above, mechanisms of transcriptional regulation rely on accessible CREs that harbour binding sites for regulatory proteins (i.e. TFs). Thus, we next performed DNA-binding motif enrichment analyses on the set of stage-specific CREs with the aim of predicting developmental regulators (i.e. enriched motifs resembling TF binding sites in stage-specific THS), and of computing GRNs that connect the TFs to potential target genes. This way, we predicted binding sites for several ApiAP2 TFs (FIG. 45) that are also expressed at the same stages, and are known to have stage-specific functions 327. We found that our THS overlapped with the binding sites of AP2-I and BDP1, involved in erythrocyte invasion associated genes, and previously reported by ChIP-seq analyses 373. Indeed, ~90% of the AP2-I sites previously reported were accessible in our data set (i.e. coincided with THS), particularly at the late stages when transcription of invasion-related genes is required (FIG. 49). For instance, in schizonts we predicted a motif similar to the binding site for AP2-I at the promoters of almost 200 genes, including several known to be related with the invasion of RBCs (FIG. 50), such as the merozoite surface proteins MSP1, MSP2, MSP6 or MSP7. Our next objective was to characterize the epigenetic mechanisms that could be associated with transcriptional regulation of CVGs. For this purpose, we obtained regions with significant differential accessibility between the P. falciparum subclones 10G and 1.2B, which display different transcriptional states at several CVGs. In general, we observed that the differential expression of CVGs was associated with differential accessibility in their promoters, so increased accessibility was positively correlated with increased transcription in each clone (FIG. 51). Examples of these include the genes clag3.1 and clag3.2, whose previously described mutually exclusive expression is now supported by differential accessibility upstream overlapping the binding sites for the TF AP2-I and the reader BDP1 (FIG. 52). Figure 49. ATAC-seq profiles at the binding sites for AP2-I Profile plots of normalized ATAC-seq signal (controlcorrected) for each stage centered at the binding sites for the TF AP2-I by Santos et al. 2017 373. Adapted from Ruiz et al. 2018 523. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 1 80 Figure 50. Network analyses in P. falciparum schizonts Network depicting the regulatory interactions between the predicted motifs corresponding to TF binding sites at T40 and their target genes (i.e. genes with the enriched motif in the promoter). White nodes represent the motifs identified, with T40.1 corresponding to AP2-I. Adapted from Ruiz et al. 2018 523. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 1 81 Figure 51. Chromatin accessibility at promoters of P. falciparum CVGs Heatmap showing the gene expression levels extracted from Rovira-Graells et al. 2012 433 and the chromatin accessibility by ATAC-seq at promoters for the set of top-30 variable CVGs previously described by Rovira-Graells et al. 2012 433. Values correspond to the P. falciparum subclones 10G and 1.2B and the developmental stages where accessibility and expression are the maximum. Adapted from Ruiz et al. 2018 523. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 1 82 However, there were also cases of CVGs with invariant accessibility at promoters despite differential expression between the subclones (FIG. 51). These included members of relevant multicopy gene families, such as rifin, Pfmc-2TM or var. In relation to var genes, the subclone 1.2B is known to predominantly express only one copy, as opposed to 10G, which expresses multiple copies at low level 433. Here, we observed several THS through the introns and gene bodies of var genes, but strikingly the only distinctive feature regarding the overexpressed copy in the 1.2B subclone was the accessibility at two adjacent genes (FIG. 53). These are part of the ncRNA gene family ‘RNA with Unknown Function 6’ (RUF6), which has been previously involved in regulation of the var gene family. We found that the only pair of accessible RUF6 genes in the 1.2 subclone were the ones surrounding the overexpressed var copy. This finding is suggestive that the underlying regulatory mechanism might involve RUF ncRNA mediated regulation and chromatin looping. Collectively, our ATAC-seq data add new insights into the precise mechanisms of transcriptional regulation underlying mutually exclusive expression of clag3 and var genes. Figure 52. Chromatin accessibility profiles at the P. falciparum gene clag3.1 Genome browser snapshot including chromatin accessibility profiles by ATAC-seq at the region of chromosome 3 containing the gene clag3.1 (PF3D7_0302500). Binding sites for AP2-I 373 and the bromodomain protein BDP1 extracted from Josling et al. 2015 372 are included. Gene expression levels from a previous microarray study by Rovira-Graells et al. 2012 433 are included. Adapted from Ruiz et al. 2018 523. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 1 83 Figure 53. Chromatin accessibility profiles at the region containing the most expressed var gene copy in P. falciparum 1.2B Genome browser snapshot including chromatin accessibility profiles by ATAC-seq at the region of chromosome 7 containing the var gene copy PF3D7_0711700, which is predominantly expressed by the 1.2B subclone, and the adjacent ncRNA-coding genes ruf6-1 and ruf6-2. Gene expression levels from a previous microarray study by Rovira-Graells et al. 2012 433 are included. Adapted from Ruiz et al. 2018 523. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 2 84 Chapter 2: The second life of Plasmodium in the mosquito host: gene regulation on the move * Original article II: Ruiz, J. L., & Gómez-Díaz, E. (2019). The second life of Plasmodium in the mosquito host: gene regulation on the move. Briefings in functional genomics, 18(5), 313-357. doi: 10.1093/bfgp/elz007 In this article, we discussed the gaps of knowledge related to the life cycle of Plasmodium parasites in the mosquito host. The aim was to review data for mosquito stages and several Plasmodium spp., regarding transcriptional regulation both in the context of developmental progression (i.e. stage-specific sets of genes with distinct functions) (FIG. 54), and related to the generation of transcriptional heterogeneity that may be involved in adaptation to environmental constraints (i.e. CVGE). Dissemination by the vectors is indispensable for malaria transmission and mosquitoes are the definitive hosts of the parasites where sexual reproduction and recombination occur. Moreover, the mosquito midguts represent the largest bottleneck through parasite development (i.e. only 0.1-0.5% of the gametocytes survive and develop into oocysts), and the parasites can develop into many mosquito species. This is a scenario that would favour the evolution of plasticity 14,223,528. In this review, we first discussed some of the reasons for the scarcity of functional genomics studies in mosquito stages, primarily linked to the low-input samples that can be obtained from mosquito tissues. In addition, there has also been an anthropocentric and diseasefocused mindset that has to some extent biased studies towards P. falciparum blood stages (i.e. when most malaria symptoms occur). As a result of these, the life cycle in the mosquitoes remains rather obscure 124,163,164, and the study of genomic and transcriptional variation in this part of the life cycle is in its infancy 154,291,529,530. Studies involving non-falciparum species and non-reference parasite strains have also been rare 508,531,532. Fortunately, the paradigms above have changed over the last decade. There is now an increasing interest in malaria research to expose the hidden life of Plasmodium parasites in the mosquito hosts (i.e. vectors of the disease), also resembling as much as possible the natural conditions in the field. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 2 85 Figure 54. Transcriptional dynamics during Plasmodium development in the mosquito Top: Heatmap showing transcriptional dynamics during Plasmodium sporogonic development. Ten stage-specific genes are shown based on multiple previously published datasets for several developmental stages and Plasmodium spp. Bottom: Barplots showing overrepresented Gene Ontology terms for each cluster of stage-specific genes. Adapted from Ruiz JL & Gómez-Díaz et al. 2019 221. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 2 86 Regarding developmental progression of the parasite in the mosquitoes, in this review, we highlighted the underlying mechanisms of transcriptional regulation. These include regulators such as ApiAP2 TFs, which have been shown to also be essential for parasite development in the mosquito (e.g. AP2-O or AP2-EXP) targeting mosquito stages-specific sets of genes (FIG. 55). Pioneering studies in P. falciparum have already described the link between epigenetic histone marks and transcription 332,335,447. The association between hPTMs and transcription is similar to that better-known in blood stages, so the functional role of hPTMs is likely to be conserved throughout the life cycle and across Plasmodium spp. Figure 55. Gene regulatory networks in Plasmodium sporogony Left: Schematic representation of some ApiAP2 TFs that are known to be overexpressed during Plasmodium development in the mosquito and examples of their targets. Right: Transcriptional dynamics of the TF-encoding genes (blue) and the top-5 more expressed known target genes. Adapted from Ruiz JL & Gómez-Díaz et al. 2019 221. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 2 87 Another aspect discussed in the review is transcriptional variation in Plasmodium, which seems to be crucial for fast adaptation to within host environmental constraints. Different phenotypes may be randomly generated via CVGE, so upon environmental change the divergent clonal parasite populations are sorted (i.e. a bet-hedging process). Alternatively, transcriptional heterogeneity may occur as an ad-hoc response to the environmental selective constraint (i.e. a direct adaptative response). In this review, we discussed the available data for transcriptional changes through bet-hedging or adaptive responses. These are known for several CVG families in blood stages related to processes such as antigenic variation and sexual commitment but remain almost completely unstudied in mosquito stages. Regarding antigenic variation, pioneering studies by our group and others 332,335 shown that in P. falciparum, several CVGs are dynamically regulated and expressed at high levels in the mosquito. Remarkably, a single member of the var gene family becomes activated. This is suggestive that var genes could play unknown roles in parasite-mosquito interactions additionally to their known immune evasion function in the blood cycle. An alternative hypothesis is that there is an epigenetic reset of the CVGs repertoire in mosquito stages, and these genes become primed (i.e. ‘preactivated’) prior transmission to the next host. The recent observation that var expression changed upon mosquito passage in P. falciparum controlled human infections, support our results 533,534. The studies above describing a single overexpressed var gene in P. falciparum also evidenced that the exclusive expression started early during the sporogonic cycle, in the oocyst stage. Correlated with the patterns of gene expression during sporogony, dynamic changes in the hPTMs occupancy were described. These conformed to the previous relationship observed for blood stages (i.e. H3K9ac and H3K4me3 linked to transcription vs. H3K9me3 linked to repression). The ApiAP2 TF AP2-EXP was described to have binding sites at the promoter of the active var gene, as well as at several other CVGs and sporozoite-specific genes 332. This suggests that TF could be a master regulator of P. falciparum sporogony. Overall, CVGE and the underlying regulation during the mosquito life cycle remain undescribed. Even if these regulatory mechanisms are unknown, in our review we also discussed the environmental cues that are known to influence and trigger phenotypic differences in the parasites during sporogony (e.g. regarding the EIP), which may be reflecting transcriptional heterogeneity. For example, these include factors such as the temperature, the pH, or the presence of host lipids in the midgut (FIG. 56). Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 2 88 Finally, in the review we advocated that the mosquito is a novel biological model for future functional genomic studies. As mentioned above, it is the definite host for Plasmodium parasites, where new genetic variation arises (i.e. sexual reproduction and recombination), and where the parasites face the strongest selective pressures. No apparent disease is suffered by the mosquitoes, which appear to limit the fitness costs to tolerate infections 124. However, the insects could be used to perform in vivo studies addressing the transcriptional regulation of the parasites during development and adaptation. This would be important because of the abundant differences that have been described between in vivo Plasmodium infections and in vitro approaches. These include transcriptomic changes in CVGs such as var or clag, loss-of-function phenotypes, or the inability to develop into sexual stages or generate antigenic diversity, amongst a plethora of drastic changes reflecting adaptation to culture 535–538. Thus, conclusions from laboratory-only studies may be far from the reality in natural conditions. To sum up, we advocate that studies focusing on Plasmodium mosquito stages, using nonreference strains or non-falciparum species, and resembling natural conditions as much as possible, would greatly benefit the field. These would inform current malaria eradication efforts, while also contributing to the development of more effective control approaches, for example the discovery of novel molecular markers or new types of drugs 124. Figure 56. Phenotypic variability in Plasmodium mosquito stages The developmental transitions during sporogony and the environmental factors within mosquitoes that could affect Plasmodium, generating alternative phenotypes at different points, for example associated with pH, temperature, immune response, host factors, or the host nutritional status. Adapted from Ruiz JL & Gómez-Díaz et al. 2019 221. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 4 95 genes. These include immunity-related genes such as CASPL2 and PGRPLC. Finally, we identified the motifs significantly enriched in the regions with differential hPTMs (i.e. potential CREs containing TFs binding sites). We searched for similarity of the motifs with known Drosophila TF binding sites and identified sequences that resemble the binding sites for fly TFs related to immunity, development, or reproduction, such as z, Dref, and Deaf1. Overall, our results point to a complex relationship between the epigenomic and transcriptional states in mosquito transcriptional responses to a P. falciparum infection. 541 Figure 58. Histone modifications-derived chromatin states identified in A. gambiae Heatmap showing four chromatin states predicted based on the combination of the enrichment profiles of four hPTMs, namely H3K9ac, H3K27ac, H3K4me3, H3K9me3. A darker blue indicates a higher level of enrichment. Adapted from Ruiz et al. 2019 541. Figure 59. Genomic locations of ChIP-seq peaks for histone modifications in A. gambiae. Density profiles showing the distance from the ATG start codon to the ChIP-seq peaks for each hPTM assayed. The dashed line corresponds to the 2Kb upstream region, representing the putative promoter region in Anopheles mosquitoes. Adapted from Ruiz et al. 2019 541. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 4 96 Figure 60. Annotation of A. gambiae genomic features to chromatin states Heatmap showing the overlap with the defined chromatin states of several genomic features, including promoters, gene bodies, intergenic regions or ChIP-seq peaks. Darker blue in the first column represents a larger fraction of the genome occupied by each chromatin state, and for the rest it represents the likelihood of finding each state. Adapted from Ruiz et al. 2019 541. Figure 61. Correlation between transcription and histone modifications at promoters Heatmaps showing the gene expression levels and the enrichment of hPTMs by ChIP-seq at the promoters. The rho coefficient for the Spearman correlation is included for each histone mark. Adapted from Ruiz et al. 2019 541. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 4 97 Figure 62. ChIP-seq signal at A. gambiae genes with different intron content Profile plots showing the input-corrected normalized ChIP-seq signal for each hPTM centered at A. gambiae gene bodies (left), and at the bodies of genes grouped by their average intronic composition (right). Adapted from Ruiz et al. 2019 541. Figure 63. Changes in histone modifications and gene expression levels between the Infection and Control conditions Violin plots showing the ratio between the Infection and Control conditions for the gene expression levels by RNA-seq and each one of the hPTMs assayed by ChIP-seq. The plots correspond to gene clusters that are more expressed in the Infection condition (left) or the Control condition (right). The rho coefficient for the Spearman correlation is shown when significant. Adapted from Ruiz et al. 2019 541. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 5 98 Chapter 5: The regulatory genome of the malaria vector Anopheles gambiae: integrating chromatin accessibility and gene expression * Original article V: Ruiz, J. L., Ranford-Cartwright, L. C., & Gómez-Díaz, E. (2021). The regulatory genome of the malaria vector Anopheles gambiae: integrating chromatin accessibility and gene expression. NAR genomics and bioinformatics, 3(1), lqaa113. doi: 10.1093/nargab/lqaa113 In this article, we focused on the study of the epigenetic mechanisms underlying transcriptional regulation in mosquitoes infected with the human malaria parasite P. falciparum. To this aim, we characterized the chromatin accessibility and the nucleosome landscapes using ATAC-seq, and the transcriptomic profiles by RNA-seq. Samples corresponded to P. falciparum-infected A. gambiae midguts and salivary glands at 7 days and 14 days post blood meal, respectively. Independent experimental infections with the P. falciparum 3D7 strain were used as biological replicates. We also integrated publicly available epigenomics datasets for the same tissues that we obtained in a previous study (i.e. ChIP-seq of H3K9ac, H3K27ac, H3K4me3, and H3K9me3 in the previous chapter). First, the ATAC-seq technique had never been assayed in A. gambiae, so we assessed its performance. We observed the canonical distribution of chromatin accessibility peaks immediately preceding the annotated TSS. The adjacent nucleosomes also conformed to the canonical +1/-1 distribution (FIG. 64), and they were also more abundant at exons and introns (FIG. 65). Additionally, we confirmed the positive correlation between chromatin accessibility at promoters and transcription of associated genes (FIG. 66). The integration of ChIP-seq data also conformed to previous observations, with H3K9ac and H3K27ac occupancy positively correlating with accessibility, H3K4me3 showing no correlation, and H3K9me3 showing a negative correlation (FIG. 66). We also found that the significant accessible regions (i.e. THS) located predominantly upstream of genes, at the 5' UTRs and promoters (FIG. 67), but we also found unexpected large numbers of THS located in intergenic regions and particularly in intronic regions (~30% of all THS). We then investigated the epigenetic states of the THSs that located in introns, and we observed that ~50% of intronic THS coincided with significant enrichment of H3K4me4 (i.e. a hPTM typical of promoters), suggesting that these could correspond to alternative TSS. In support of the regulatory function of intronic CREs, we found that intron-rich genes display more THS at the introns than at the upstream promoters (and the opposite for intron-poor genes) (FIG. 68). Intronic accessibility also correlated positively with accessibility at the promoters, as well as with the transcription of the Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 5 99 associated genes. Among the genes with accessible regulatory sites located at introns, we found examples of genes with known immunology-related functions or with roles in Plasmodium infection, such as tep or def1. 542 Figure 64. ATAC-seq profiles at A. gambiae TSSs Profile plots showing the ATAC-seq signal centered at A. gambiae TSSs. The nucleosome-free signal corresponds to chromatin accessibility, and the mononucleosome signal marks the adjacent nucleosomes located at closed chromatin. Grey profiles represent random signal. Adapted from Ruiz et al. 2021 542. Figure 65. Location of the ATAC-seq mononucleosomal reads Barplots showing the fraction of ATAC-seq reads that were aligned to different genomics features. Adapted from Ruiz et al. 2021 542. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 5 100 Figure 66. Correlation between transcription and histone modifications and chromatin accessibility at promoters Heatmaps showing the ATAC-seq nucleosome-free signal at promoters, the gene expression levels by RNA-seq of the associated genes, and the enrichment of the hPTMs assayed in Ruiz et al. 2019 541. Data is for midguts and the significant Spearman correlation is shown for each case. Adapted from Ruiz et al. 2021 542. Figure 67. Genomic location of A. gambiae ATAC-seq peaks Barplots showing the annotation to genomic features for the THSs from each assayed tissue and nearby genes with or without 5’ UTRs annotated. Adapted from Ruiz et al. 2021 542. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 5 101 Next, to characterise tissue-specific GRNs, we compared chromatin accessibility and transcriptional profiles in mosquito midguts and salivary glands. We found that 85% (~21,250) of all differentially accessible regions between tissues were more accessible in salivary glands, and described a positive correlation between gene expression and changes in accessibility, both at THS in 5’ UTRs/promoters and at introns (FIG. 69). Overall, we found 1,920 significant differentially expressed genes (DEGs) containing differentially accessible regions between tissues located at 5’ UTRs/promoters and introns. Overrepresented GO terms for these tissue-specific DEGs, conformed to the functional activities expected for each tissue, such as genes coding for trypsin involved in digestion and proteases involved in catabolism in the midguts (FIG. 70). Among DEGs with tissue-specific THS, we also found examples of relevant immunity and infectionrelated proteins, such as the TF rel2 or the antimicrobial gam1 (FIG. 71). Figure 68. ATAC-seq peaks annotated to A. gambiae genes with different intron content Barplots showing the number of THSs annotated to different genomic features for subsets of genes grouped by their intronic composition, namely intron-rich, intron-medium and intron-poor. Adapted from Ruiz et al. 2021 542. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 5 102 Figure 69. Changes in chromatin accessibility and gene expression levels between the A. gambiae tissues Heatmaps showing the changes of chromatin accessibility at promoters and expression for the subset of genes that are significantly differentially expressed and that display differentially accessible regions in their promoters. Adapted from Ruiz et al. 2021 542. Figure 70. Overrepresented GO terms at each A. gambiae tissue Barplots showing the overrepresented Gene Ontology terms for each set of differentially expressed genes with differentially accessible regions at each assayed tissue. Adapted from Ruiz et al. 2021 542. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 5 103 Next, we seek in our dataset of tissue-specific CREs binding motifs for TFs involved in activating or silencing transcription. We successfully identified several motifs associated with increased or decreased transcription at each tissue, which resembled Drosophila TFs with matching activating or repressor roles (e.g. serpent and hunchback, respectively). Finally, we used chromatin accessibility data to characterize novel regulatory sites in A. gambiae. First, we added evidence of the activity of ~700 enhancers, which had previously been annotated in the mosquito mainly based on computational predictions, and in our tissues coincided with THS. Next, using a homology-based approach and accurate enhancer maps of D. melanogaster, we predicted more than 1,000 novel enhancers, ~90 of which seemed to be active in our tissues (i.e. overlapped THS). Approximately 80% of the enhancer-annotated genes in the mosquito tissues matched the enhancer target reported by others in Drosophila, which served as validation of our approach. We also found a positive correlation between accessibility of the enhancer-like THS and the transcription of the annotated genes, and between the accessibility and gene expression changes linked to tissue-specificity. This added evidence to the regulatory role of the enhancer candidates. An example of functional relationship is the lrim1 gene, which is related to responses to infection, and whose enhancer was previously described by others 543. Here, the lrim1 enhancer Figure 71. Chromatin accessibility and transcriptional profiles at the region containing the A. gambiae gene gam1 Genome browser snapshot including chromatin accessibility profiles by ATAC-seq and gene expression profiles by RNA-seq at the region of the chromosome 3R containing the gene gam1 (AGAP008645). 5’ UTRs, THSs and the differentially accessible regions are included. Adapted from Ruiz et al. 2021 542. Host-parasite interactions in human malaria: Summary of results Transcriptional regulation and epigenetics Chapter 5 104 overlaps a differentially accessible THS between tissues, and accordingly the gene is a DEG more expressed in the tissue where the enhancer is more accessible (FIG. 72). Figure 72. Chromatin accessibility and transcriptional profiles at the region containing the A. gambiae gene lrim1 Genome browser snapshot including chromatin accessibility profiles by ATAC-seq and gene expression profiles by RNA-seq at the region of the chromosome 2L containing the gene lrim1 (AGAP006348). 5’ UTRs, THSs, differentially accessible regions, and a previously described enhancer by Nardini et al. 2019 543 are included. Adapted from Ruiz et al. 2021 542. Host-parasite interactions in human malaria: Discussion Transcriptional regulation and epigenetics 111 characterized the genome-wide occupancy of the histone marks H3K27ac, H3K9ac, H3K9me3 and H3K4me3 in infected and non-infected A. gambiae midguts, reporting the relationship between chromatin states at regulatory regions and gene expression levels. Our findings agreed with observations in other eukaryotes: H3K9ac and H3K27ac at mosquito promoters were associated with active transcription, H3K4me3 did not show correlation, and H3K9me3 was associated with repression 270,571,572. We also observed simultaneous enrichment of H3K4me3 and H3K9me3, which could point to a poised transcriptional state. This bivalent state of some promoters is typically related to lowly expressed genes that need to be activated quickly at a certain point in time, such as genes encoding TFs, involved in development or gene imprinting 573. Similar to what has been described for Drosophila, Anopheles gene architecture is linked to differences in the enrichment of histone marks in the gene body (i.e. genes with longer introns display different chromatin states, with broader enrichment of H3K27ac and H3K9ac). Another observation is that the majority of the histone marks appeared to be associated with promoters and gene bodies. However, we observed a large fraction of enrichment at distal elements (i.e. ~40% of the sites located at distances greater than 2 Kb from the nearest downstream gene). Enhancers remain rather unknown in mosquitoes, so these could be novel regulatory sites. In agreement with this hypothesis, this set of distal regions appeared to be enriched in H3K27ac, which is a histone mark typical of active enhancers 278,574, and H3K9ac/H3K4me3 depleted, both promoter-specific histone marks278. That analysis allowed us to propose 4,204 novel enhancer candidates in the midguts of A. gambiae based on chromatin states. Our study laid the foundations for future studies on mosquito enhancers as promising targets for novel vector control strategies. Our experimental design also included mosquito samples infected with P. falciparum and non-infected (i.e. control condition). Therefore, in Article IV we were also able to characterize the chromatin and transcriptional changes induced by P. falciparum and a set of differentially regulated infection-responsive genes. We identified ~2,500 regions with differential enrichment of histone marks between infected and control samples. Importantly, a fraction of these were located at promoters or intragenic regions (~100 sites), or appeared located at distal upstream regions (~130 sites). We annotated these sites to 278 Plasmodium-responsive genes. For some of these, the enrichment in activating or repressor histone marks was correlated with the transcriptional status of the nearest gene. However, this correlation was not generalizable and there were also regions with differential enrichment of histone marks that were not associated with Host-parasite interactions in human malaria: Discussion Transcriptional regulation and epigenetics 112 transcriptional differences between the control and infected conditions. A possible explanation is that there may be a certain threshold of enrichment of histone marks necessary to effectively influence transcription 575. In addition, contrary to our expectations, we only found a few Plasmodium-responsive genes known to be related with the immune response against malaria infection. A possible explanation is that mosquito midguts were sampled at 7 days post-blood meal, which corresponds to the time point of mature P. falciparum oocysts, whereas the better-known peak of early immune responses against Plasmodium it is known to occur between 2 and 24 hours (i.e. when parasites face the largest bottleneck during development in the mosquito). Finally, our approach also allowed the prediction of binding sites for transcription factors, some of which might be involved in mosquito responses against infection. To this aim, we leveraged the Drosophila TF databases included in the HOMER software 576. Looking at regions showing differential enrichment of histone marks, we found motifs matching the consensus sequences of known Drosophila TFs involved in immunity and development. Examples are Deaf1, known to be involved in the immune response of Drosophila against microbes 577, or dorsal, which beyond its role in development is known to be a downstream regulator of Toll signalling 578. In article IV, we first attempted to identify mosquito enhancers based on prototypical chromatin states at distal regions enriched at particular histone marks. Chromatin accessibility is a much better proxy to functionally characterize regulatory elements and TF binding events in vivo 458,511. However, in A. gambiae, previous studies involving chromatin accessibility profiling were limited to one applying FAIRE-seq in mosquito hemocytes 471. Several Anopheles enhancers have been predicted in silico, but the vast majority of them lack experimental validation. This is striking given the importance of human malaria vectors for global health, and when compared with the situation in close model organisms such as Drosophila, with tens of thousands of well characterized enhancers 579. To address this gap and complementing our previous work, in Article V we aimed to profile chromatin accessibility in mosquitoes by applying for the first time the ATAC-seq technique 542. To study tissue-specific gene regulatory networks in A. gambiae, we integrated transcriptomic and ATAC-seq data for midguts and salivary glands of P. falciparum-infected mosquitoes. As a proof of concept, our results met our expectations based on what we know from other insects, mainly Drosophila spp. 580,581. In both midguts and salivary glands, chromatin accessibility was predominantly located in introns and promoters. We also integrated the ATAC-seq data with our datasets of histone marks in A. gambiae discussed above, in article IV 541. We observed a positive Host-parasite interactions in human malaria: Discussion Transcriptional regulation and epigenetics 113 correlation between accessibility and activating chromatin states (i.e. enrichment of H3K9ac and H3K27ac), and negative correlation for the repressor H3K9me3, which is associated with chromatin condensation (i.e. heterochromatin). The accessibility upstream of genes, and at introns, was also positively correlated with gene expression. Such a pattern for introns agrees with observations from the model D. melanogaster, being well-known that introns have relevant roles in transcriptional regulation 582,583. In article IV, we observed that A. gambiae genes with larger intron content displayed more enriched gene bodies in H3K27ac 541. In article V we confirmed the positive correlation between transcription, H3K27ac enrichment and chromatin accessibility in introns. We also reported that the gene structure had an impact on chromatin accessibility, so genes with larger intron content display more intronic accessibility compared to the promoters, and the opposite for more exonic genes. This points to the promoters being of variable importance depending on the intronic compositions of the genes. This regulatory mechanism is known as intron-mediated enhancement, and while poorly understood, it is suggested to be conserved in all of the eukaryotes, including Drosophila 584. Our results point to conservation also in Anopheles mosquitoes. Another aspect addressed in this article is the chromatin changes underlying tissue-specific regulation in Plasmodium-infected mosquitoes. We integrated and compared RNA-seq and ATAC-seq data between midguts and salivary glands to report thousands of regions that are differentially accessible between the tissues and annotated to differentially expressed genes. The correlation between the magnitude of change in accessibility and gene expression between tissues (i.e. the fold changes) was positive. Regarding tissue-specific gene regulatory networks, the set of genes differentially regulated in midguts and salivary glands displayed gene ontology terms that agreed with the biological functions, processes or metabolic pathways expected for each tissue For example, these include digestion in midguts 585 or metabolism of amino acids in salivary glands 586. We also predicted the TFs that may be binding to the differentially accessible cis-regulatory elements. We reported coincidence with binding sites of Drosophila TFs which in some cases are also known to be specific for the same tissues in the model organism. These include examples of immune-related TFs in the midgut and salivary glands, such as relish or serpent, which appear to be functionally conserved between Drosophila and Aedes and Anopheles mosquitoes. It is important to note that the gene regulatory networks that we have described for the first time in A. gambiae are likely relevant not only to the physiology and tissue-specific functions of these insects, but also for responses against pathogens, including Plasmodium. For example, we provide the Host-parasite interactions in human malaria: Discussion Transcriptional regulation and epigenetics 114 location of active regulatory sites for several immune-related genes, such as srpn6, gam1 or def1, which were also shown to be Plasmodium-responsive in Article IV 541. The integration of transcriptomic and chromatin accessibility datasets in A. gambiae allowed us to predict active regulatory elements, but we also aimed to further classify them into promoters and enhancers. When comparing our set of accessible regulatory sites with previous A. gambiae enhancers predicted in silico (~700 enhancers), we found ~40% of coincidence. In addition to these, we reported ~1,100 mosquito sequences that showed homology to publicly available D. melanogaster enhancers. Of these, ~100 coincided with accessible regulatory sites in our study, and thus may be active enhancers in the mosquito tissues, conditions, and time points assayed here. Considering these two approaches, we ultimately defined ~800 accessible enhancer candidates in mosquitoes. These were mainly located at introns or exons and for ~80% of the candidates, our annotation based on the nearest gene coincided with the ortholog gene target previously defined by others. These observations also support the reports in other organisms, including Drosophila, that despite being capable of regulating over long distances, the majority of enhancers are proximal or intragenic 468,587. Regarding the potential function, for this set of mosquito enhancers we reported positive correlation between chromatin accessibility and transcription of the target genes, and for a fraction, the accessibility coincided with H3K27ac enrichment. Finally, concerning mosquito responses against malaria, we observed that ~140 of the enhancer candidates above annotated to the set of malaria infection-responsive genes defined in Article IV 541. An example is the lrim1 locus, which is an immune related gene whose function has been associated with mosquito midgut responses 588,589. Our transcriptomic data showed that LRIM1 is more expressed in the salivary glands, and in agreement we could report an upstream enhancer, experimentally validated by others 543, with increased accessibility in the salivary glands. Overall, with this set of hundreds of novel mosquito enhancers at the disposal of the community, future studies would now be needed to comprehensively validate and characterize their function. These could require techniques such as STARR-seq, which has already been applied to Anopheles mosquitoes 590, transgenesis, or Hi-C, which would also account for the 3D genome organization and the potential regulation of malaria responsive genes via chromatin loops. Host-parasite interactions in human malaria: Conclusions Transcriptional regulation and epigenetics 115 Conclusions 1. Chromatin accessibility profiling allows for the in vivo study of transcriptional regulation in P. falciparum, identifying cis-regulatory elements likely involved in the dynamics of open chromatin and predicting their cognate transcription factors. 2. The dynamics of chromatin accessibility in regulatory regions, together with chromatin states based on the enrichment of histone marks, correlate with lifestage specific transcriptional profiles of malaria parasites. 3. The integration of epigenomic and transcriptomic datasets in transcriptionally heterogeneous P. falciparum subclones provides new insights into the regulation of mutually exclusive and clonally variant gene expression profiles, which are critical for malaria pathogenesis and virulence. 4. During Plasmodium development in the Anopheles mosquitoes, stage-specific transcriptional programs are under the spatiotemporal control of transcription factors, which belong to the ApiAP2 family and are specific to this part of the life cycle. 5. In the mosquito vector, environmental cues such as temperature variations or the host nutritional status can modulate phenotypic differences in the parasites, including traits such as growth and multiplication rates, progression through the life-cycle or infectivity. However, we lack knowledge about mechanisms underlying parasite phenotypic variation and families of genes showing clonally variant gene expression in mosquito stages. 6. Beyond the use of culture-adapted reference genomes, the sequencing, assembly, and correction of de novo genomes from field isolates generally enable more accurate genomic studies, including of parasites such as Plasmodium and Trypanosoma species. The ILRA pipeline is a new tool to streamline the automatic polishing of long reads de novo genomes. It has been tested on several novel P. falciparum genome assemblies, and it could be widely implemented in numerous sequencing projects addressing malaria parasites, amongst other organisms. 7. 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