A metabolism-chromatin axis promotes ribosome heterogeneity in the human malaria parasite

A metabolism-chromatin axis promotes ribosome heterogeneity in the human malaria parasite | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A metabolism-chromatin axis promotes ribosome heterogeneity in the human malaria parasite Justine E. Couble , Tiziano Vignolini , Gregory Dore , Bérangère Lombard , Michael Richard , Damarys Loew , Michael Büttner , Rafael Dueñas-Sánchez , Gernot Poschet , Jessica M. Bryant , View ORCID Profile Sebastian Baumgarten doi: https://doi.org/10.1101/2025.03.22.644534 Justine E. Couble 1 Institut Pasteur, Université Paris Cité, INSERM U1347, G5 Parasite RNA Biology Group , F- 75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tiziano Vignolini 1 Institut Pasteur, Université Paris Cité, INSERM U1347, G5 Parasite RNA Biology Group , F- 75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gregory Dore 1 Institut Pasteur, Université Paris Cité, INSERM U1347, G5 Parasite RNA Biology Group , F- 75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bérangère Lombard 2 Institut Curie, PSL Research University, CurieCoreTech Mass Spectrometry Proteomics , Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michael Richard 2 Institut Curie, PSL Research University, CurieCoreTech Mass Spectrometry Proteomics , Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Damarys Loew 2 Institut Curie, PSL Research University, CurieCoreTech Mass Spectrometry Proteomics , Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michael Büttner 3 Metabolomics Core Technology Platform, Centre for Organismal Studies, Heidelberg University , 69120 Heidelberg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rafael Dueñas-Sánchez 3 Metabolomics Core Technology Platform, Centre for Organismal Studies, Heidelberg University , 69120 Heidelberg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gernot Poschet 3 Metabolomics Core Technology Platform, Centre for Organismal Studies, Heidelberg University , 69120 Heidelberg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jessica M. Bryant 4 Institut Pasteur, Université Paris Cité, INSERM U1201, CNRS EMR9195, Biology of Host-Parasite Interactions Unit , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sebastian Baumgarten 1 Institut Pasteur, Université Paris Cité, INSERM U1347, G5 Parasite RNA Biology Group , F- 75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sebastian Baumgarten For correspondence: sebastian.baumgarten{at}pasteur.fr Abstract Full Text Info/History Metrics Preview PDF ABSTRACT The transmission of the most virulent human malaria parasite, Plasmodium falciparum , relies on its survival in the contrasting environments of the human host and mosquito vector. One of the most fascinating adaptations to this lifestyle is the specific silencing of individual rDNA genes in the human host that are de-repressed following host-to-vector transmission. In this study, we defined the epigenetic signatures of rRNA transcription and found that rDNA silencing relies on aerobic glycolysis, the sole energy-generating pathway in the human host. We show that disruption of NAD + regeneration during lactate fermentation promotes rDNA de-repression and identify the sirtuin histone deacetylase Sir2a as the mediator between fluctuating NAD + levels and a functional transcriptional outcome. Hence, rDNA activation appears to be coupled to the metabolic state of the parasite as it transitions from aerobic glycolysis to mitochondrial respiration during host-to-vector transmission. INTRODUCTION Ribosomes are the macromolecular complexes of all living organisms that translate messenger RNA transcripts into proteins. Instead of comprising a homogeneous population that passively synthesize proteins, it is becoming increasingly clear that even within the same organism, ribosomes can feature remarkable compositional and structural heterogeneity, possibly giving rise to specialized functions 1 . Different ribosome populations can exist in distinct cell types of multicellular organisms 1 , and modifications of ribosomes in single-celled organisms can arise during changes of growth conditions 2 or in response to certain stressors 3 . A uniquely extreme example of such ribosome heterogeneity is found in Plasmodium falciparum, the unicellular parasite that causes the most severe form of human malaria. The parasite’s life cycle between the human host and mosquito vector is driven by a tightly regulated gene expression program 4 – 7 that relies on epigenetic and post-transcriptional mechanisms 8 . One of the most striking phenomena in gene transcription throughout its life cycle is the switch in expression amongst different rDNA loci 9 . In contrast to most described eukaryotic genomes, P. falciparum does not encode repeats of rDNA, but only five rDNA genes, each of which is located on a different chromosome 10 , 11 . These distinct rDNA genes can be broadly classified into A-types (A1 and A2), expressed as the sole rRNA type in the human host and S-types (S1, S2, and S3), which are additionally activated during development in the mosquito vector. This expression pattern makes Plasmodium one of the only known examples wherein distinct rRNA genes are assembled into possibly divergent ribosomes within the same cell. rRNA comprises up to 95% percent of total RNA in a cell, and the continuous biogenesis of ribosomes can take up to 80% of a cell’s energy budget 12 , 13 . Thus, rRNA transcription is one of the most tightly regulated processes of any living cell, and many organisms have evolved regulatory mechanisms to precisely silence and activate rDNA genes to adjust ribosome amounts in response to nutrient availability and environmental condition 14 . In P. falciparum, multiple and seemingly unrelated conditions have been observed to activate silent rDNA genes in vitro, including exposure to suboptimal growth temperature (< 37°C) 15 – 17 , glucose starvation 15 , and disruption of histone deacetylation 17 . Yet how P. falciparum is able to silence mosquito-stage rDNA during development in the human host and precisely de-repress them during developmental progression through the lifecycle remains unknown. In this study, we characterize the epigenetic signature of rRNA transcription and combine assays on different parasite growth conditions with functional forward genetics to show that rDNA silencing in the human host is sensitive to NAD + /NAM ratios that are maintained through the activity of lactate dehydrogenase. In combination with quantitative chromatin immunoprecipitation and protein-DNA proximity labelling, we reveal that the NAD + -dependent histone deacetylase Sir2a promotes rDNA repression, acting as a mediator of the cell’s metabolic state and local rDNA chromatin environment. With the metabolic dependence of rDNA silencing, these data provide a model of how rDNA de-repression tightly follows the natural transition from aerobic glycolysis to oxidative phosphorylation as the parasite progresses from the human host to the mosquito vector. RESULTS rDNA transcription across the P. falciparum life cycle To accurately measure the abundance of each rRNA type, we first performed total RNA sequencing across the parasite life cycle. A-type rRNA is the exclusive rRNA type throughout the development in human blood, including the asexual replicative cycle and gametocytes ( Figure 1A ). Notably, the parasite expresses a heterogeneous mixture of A- and S-type rRNAs in the mosquito stages, suggesting that silenced S-type rRNA are de-repressed in the vector. Additionally, we identified two novel rDNA loci constituting a third rRNA class that is expressed specifically during the oocyst stage, termed “O-type”. This type represents a minor fraction of expressed rRNA ( Figure 1B ), are highly divergent from each other (Table S1), and both surprisingly lack the 18S rDNA at their genomic loci. They are transcribed from two rDNA loci – O1 and O2 – located on chromosome 8, which brings the total number of rDNA loci in the genome to seven, all of which are located in subtelomeric regions ( Figure 1C , S1A , Table S1). Download figure Open in new tab Figure S1 A) Schematic representation of the localization of the rDNA loci in the P. falciparum genome. Only chromosomes with rDNA loci are represented. B) Fold-change of the relative abundance of two A1 28S SNVs relative to the asexual stage in gametocytes (G), oocysts (O) and sporozoites (S). C) ChIP/input ratio track (log 2 ) of H3K9ac at silent rDNA loci. D) HP1 ChIP/input ratio track 20 (i.e. compacted, transcriptionally silenced chromatin) and ATAC-seq (assay for transposase accessible chromatin, i.e. open, transcriptionally active chromatin) on the different rDNA loci 21 . The rDNA loci are colored in the genome track. E) Micro-C derived intra- and interchromosomal interaction matrices of chromosomes containing a rDNA locus. All matrices are shown at 5 kb resolution, except intrachromosomal interactions of chromosome 1 (Pf3D7_01_v3, 2 kb). Interchromosomal and top triangle of the intrachromosomal matrices show interaction frequencies (see color bars). Bottom triangle of the intrachromosomal matrices show log 2 -scaled observed/expected interaction frequency ratios. Download figure Open in new tab Figure 1 A) Proportion of A1/2, S1 and S2/3 28S rRNA in asexual stages (A), gametocytes (G), oocysts (O) and sporozoites (S) based on FPKM values of each rRNA type. B) Expression levels (FPKM: fragments per kilobase of exon per one million mapped reads) of O1 (left) and O2 (right) 28S rRNA. Black dot: mean; vertical lines: Standard error of the mean (SEM). Life-cycle stages abbreviations as in A). C) Schematic illustration of the P. falciparum life cycle indicating rRNA types that are expressed in addition to the A-type rRNA at each developmental stage. D) Synteny graph of the seven P. falciparum rDNA loci. Grey lines indicate sequence similarity ≥70% over a region of ≥100 bp. Edges of the shown regions are the start/end positions of the upstream and downstream genes. E) H3K9ac ChIP/input ratio track at the A2 rDNA locus F) HP1 ChIP/input ratio track 20 at the S1, S3 and O2 rDNA loci. G) Interchromosomal contact map of chromosome 5 and 7 at 5 kb resolution generated by Micro-C 22 . Location of A1 and A2 rDNA loci are indicated by the dotted line. Insert: Magnification of the A1/A2 interaction bin (5 kb resolution). H) Top: Intrachromosomal contact map of chromosome 7. Middle and bottom: ChIP/input ratio track of HMGB1 and HMGB2. I) Detailed view of ChIP/input ratio tracks of HMGB1 (top) and HMGB2 (bottom) at the A2 rDNA locus. The putatively upstream regulatory regions of the seven rDNA loci are highly diverse in length (1.8 kb – 12 kb) and overall sequence identity ( Figure 1D , Table S1). The upstream intergenic regions of the S2 and S3 rDNA loci are amongst the longest found in the P. falciparum genome and encode long non-coding RNAs 16 that share partial sequence similarity with the upstream region of the newly described O2 locus ( Figure 1D ). The divergence of the rDNA upstream regions suggests that different and/or multiple regulatory mechanisms exist to regulate transcription of each rDNA locus. This is surprising given that the 18S and 28S sequences are nearly identical between A1 and A2, as well as between S2 and S3 (Table S1). Indeed, when we compared the relative levels of rRNA transcribed from each A-type locus by counting the abundance of their defining single nucleotide variants (SNV), we found that the contribution of each A-type rDNA locus to the total pool of A-type 28S rRNA is similar between the human blood stages and oocysts, but that A1 28S rRNA levels double in the sporozoite stage ( Figure S1B ). This suggest that even near identical rDNA are subject to differential transcriptional control or post-transcriptional processing. Epigenetic state and chromatin organization of rDNA loci in asexual stages A hallmark of active transcription in P. falciparum is histone 3, lysine 9 acetylation (H3K9ac) upstream of a transcribed gene 18 . Chromatin immunoprecipitation followed by sequencing (ChIP-seq) of H3K9ac showed a significant peak at the A2 rDNA locus ( Figure 1E ), but not at any of the silent rDNA loci in asexually replicating cells ( Figure S1C ) identifying upstream histone acetylation as an epigenetic signature of active rRNA transcription. On the other hand, a conserved feature of rDNA silencing in many eukaryotes is the spread of heterochromatin along adjacent rDNA loci in repeat arrays 19 . To gain further insight into the epigenetic state of rDNA loci, we used previously published heterochromatin protein 1 (HP1 20 ) ChIP-seq and ATAC-seq 21 datasets. Despite the lack of rDNA repeats, we find that the individual silent rDNA loci in asexual stage parasites in human blood are occupied by HP1 ( Figure 1F ), while the transcriptionally active A-type rDNA locus is encompassed in open, accessible chromatin ( Figure S1D ) that mirrors the presence of upstream acetylation ( Figure 1E ). The substantially lower HP1 levels at silent rDNA loci compared to adjacent (sub-)telomeric regions that are constitutively heterochromatinized ( Figure 1F , Figure S1D ) might indicate a “poised”, facultative heterochromatic state that still enables rapid transcriptional activation. Of note, HP1 occupies the S1 and S2/3 rDNA genic region, whereas HP1 at the O2 locus is only enriched in the upstream, ncRNA-encoding region ( Figure 1F ). Among all known eukaryotes, rRNA is typically transcribed by polymerase I within the nucleolus. In P. falciparum , rDNA loci were similar shown to cluster in the nucleolus with DNA FISH 17 . To resolve these interactions at high resolution, we analyzed Micro-C data in blood stage asexual parasites 22 . Despite their locations on different chromosomes, we found that the two actively transcribed A-type rDNA loci in asexual stage parasites strongly interact with each other ( Figure 1G ). Conversely, no direct interaction was found among any of the silent rDNA loci ( Figure S1D ). In addition, the A-type rDNA locus represents a boundary in local DNA-DNA interactions ( Figure 1H , top), indicating high levels of rRNA transcription in a compartment within the parasite’s nucleus. To identify proteins potentially involved in the structuring of the rDNA chromatin environment and/or recruitment of PolI to active rDNA loci, we annotated the PolI machinery in P. falciparum. This search identified high-mobility group proteins 1 (HMGB1, PF3D7_1202900) and 2 (HMGB2, PF3D7_0817900) as putative homologs of human nucleolar transcription factor 1 (Table S2). This protein is required for PoII recruitment and subsequent remodeling of active rDNA chromatin and rRNA transcription 23 . Epitope-tagging of the two proteins with 3x hemagglutinin (3xHA) ( Figure S2A-E ) followed by ChIP-seq revealed that they specifically associate with the active, but not the silent rDNA loci ( Figure 1H,I Figure S2F,G , Table S3), providing evidence for an orthogonal role of these proteins similar to other eukaryotic nucleolar transcription factors. Download figure Open in new tab Figure S2 A) Representation of the pSLI strategy to integrate a 3xHA-tag at the 3’ end of the HMGB1 and HMGB2 loci. GGG and GSG: linkers; 2A: skip peptide; Neo: neomycin-resistance gene; yDHODH: yeast dihydroorotate dehydrogenase. B) Nanopore tracks displaying reads (in purple) encompassing the pSLI plasmid, the HMGB1 locus and the surrounding genomic region, indicating correct integration of the plasmid downstream of the HMGB1 locus. C) PCR of wild-type NF 54 gDNA and HMGB2-HA gDNA using primer pair p1/p2 ( Figure S2A , Table S10) showing integration of the plasmid. D) Western blot of a co-immunoprecipitation of HMGB1-HA performed with protein lysates extracted from wild-type parasites (negative control, left) and HMGB1-HA (right). Input: Input protein lysate; SN: Supernatant; IP: Immunoprecipitate. Aldolase is used as a loading control. Numbers on the left indicate molecular weight (MW) in kilodaltons (kDA). E) As in D), but for HMGB2-HA. F) Giemsa images of HMGB1-HA and HMGB2-HA parasites cultured at two different growth temperatures (37°C and 32°C) at the time of sampling for ChIP-seq (37°C: 36 h.p.i; 32°C: 56 h.p.i), showing a similar developmental stage. G) ChIP/input ratio tracks of HMGB1 and HMGB2 at the S1, S3, O1 and O2 locus. rDNA silencing is sensitive to changes of NAD + /NAM levels Decreased temperatures have been shown to promote the de-repression of silent rDNA loci in asexually replicating blood stage parasites, providing clues as to how these genes are differentially regulated between the human host and mosquito vector 15 – 17 . By culturing parasites at 32°C (a temperature at which the parasites can still complete the asexual replicative cycle ( Figure S3A, S3B ) rather than 37°C, we confirmed that S2/S3-type rRNAs show the most pronounced upregulation ( Figure 2A , Table S4). The S1 and O-type rRNA feature a similar pattern, although to a lesser degree, and A-type rRNA do not show any change in rRNA transcription ( Figure 2A ), and there was no change in preferential transcription of the A1 versus A2 type rDNA locus ( Figure S3C ). The large increase in transcription of S2/S3 rRNA is further accompanied by the recruitment of HMGB1 to these loci at 32°C ( Figure 2B , S2F, Table S5), indicating that this upregulation is not a result of promiscuous transcription, but of targeted differential regulation. Download figure Open in new tab Figure S3 A) Growth curve showing parasitemia over 100 hours at 37°C and 32°C. Uninfected red blood cells (uRBC) served as background reference. Error bars: standard deviation of the mean (sdm) of nine replicates (three independent blood donors * three technical replicates). B) Giemsa images of wild-type NF54 parasites cultured at two different growth temperatures (37°C and 32°C) at the time of sampling for total RNA-seq (37°C: 36 h.p.i; 32°C: 56 h.p.i), showing a similar developmental stage. C) Fold-change of the relative abundance of two A1 28S SNVs at 32°C relative to 37°C. Black dot: mean; vertical line: SEM. D) Developmental age of parasites grown at 37°C and 32°C derived from mRNA-seq in comparison to a bulk reference transcriptome. 61 E) Developmental age of parasites grown at 37°C and 32°C derived from mRNA-seq in comparison to scRNA sequencing. Reference developmental stages of the scRNA data are shown on the left 6 . F) Schematic of NAD + consumption and regeneration during glycolysis ending either with Acetyl-CoA that enters the TCA cycle (left) or lactate fermentation (right). NAD + can be used as a co-substrate and be regenerated in the Preiss-Handler Pathway. Numbers for NAD + , NADH, ADP and ATP are per molecule of glucose. ADP: adenosine diphosphate; ATP: adenosine triphosphate; NAD + /NADH: nicotinamide adenine dinucleotide, oxidized and reduced form; NAM: Nicotinamide; NA: nicotinic acid; NaMN: nicotinate mononucleotide; NaAD: nicotinate adenine dinucleotide; BCKDH: branched chain ketoacid dehydrogenase; LDH1: lactate dehydrogenase; PARP: poly (ADP-ribose) polymerases. G) Giemsa images of P. falciparum wild-type NF54 parasites grown at 37°C and 32°C at the time of sampling for the metabolomics experiments. H) Representation of the pSLI strategy to integrate a 3xHA-tag and a glmS ribozyme at the 3’end of the nicotinamidase locus. GGG and GSG: linkers; 2A: skip peptide; Neo: neomycin-resistance gene; yDHODH: yeast dihydroorotate dehydrogenase. I) PCR of wild-type NF54 and Nico-glmS gDNA using primer pair p3/p2 ( Figure S3H , Table S10) showing integration of the plasmid. J) Growth curve showing parasitemia of the Nico-glmS cell line with (+Gln) and without (-Gln) glucosamine over 4 days. Uninfected red blood cells (uRBC) served as background reference. Error bars: sdm of nine replicates (three independent blood donors x three technical replicates. K) Western blot analysis of Nico-glmS parasites after 24 and 48h of glucosamine treatment. Aldolase and H3 are used as loading controls for the cytoplasmic (C) and nuclear (N) fractions. Numbers on the left indicate molecular weight (MW) in kilodaltons (kDA). Download figure Open in new tab Figure 2 A) Fold-change difference of rRNA expression at 32°C compared to 37°C for the five different rDNA loci. Black dot: mean; vertical lines: SEM. B) ChIP/input ratio track of HMGB1 at 32°C (light blue) and 37°C (dark blue) at the S3 rDNA locus. C) Fold-change (log 2 , y-axis) between parasites grown at 32°C compared to 37°C plotted over the mean abundance of each gene (x-axis, log 10 scale). Transcripts that are significantly up or downregulated ( p -adj < 0.01) are displayed in pink. D) NAD + /NAM ratios in wild-type NF 54 parasites grown at 37°C and 32°C. Black dot: mean; vertical lines: SEM. n = 9. E) NAD + /NAM ratios in Nico-glmS parasites grown with (+Gln) or without (-Gln) glucosamine. Black dot: mean; vertical lines: SEM. n = 6. F) Fold-change difference of 28S rRNA expression in Nico-glmS parasites grown with (+Gln) or without (-Gln) glucosamine. n = 3. G) NAD + /NAM ratios in LDH-glmS parasites grown with (+Gln) or without (-Gln) glucosamine. Black dot: mean; vertical lines: SEM. n = 5. H) Fold-change difference of 28S rRNA expression in LDH-glmS parasites grown with (+Gln) or without (-Gln) glucosamine. Black dot: mean; vertical lines: SEM. n = 3. I) Fold-change difference of 28S rRNA expression between wild-type 3D7 and Sir2a-KO P. falciparum parasites. n = 3. J) and K) Histone PTMs of H4 (J) and H3 (K) in wild-type 3D7 and Sir2a-KO parasites obtained by Mass Spectrometry. Aggregated Z-score of mean intensities of six biological replicates are represented. Only significantly up- or down-regulated peptides are shown (see Table S8 for complete list of PTMs). me: methylation; ac: acetylation; la: lactylation. Note: carboxyethylation and lactylation PTMs have an identical mass and cannot unequivocally be distinguished To explore how a decrease in growth temperature could lead to a de-repression of silenced rDNA, we performed messenger RNA sequencing of parasites grown at 32°C and 37°C ( Figure 2C , Figure S3D,E , Table S6). Among the most significantly downregulated genes was nicotinamidase (Nico, PF3D7_0320500), which encodes a metabolic enzyme that catabolizes nicotinamide (NAM) to nicotinic acid in the Preiss-Handler pathway 24 ( Figure 2C , S3F). NAM itself can result from the activity of enzymes consuming NAD + as a co-substrate 25 ( Figure S3F ). We therefore compared the ratio of NAD + to NAM by targeted, quantitative metabolomics and found significantly lower NAD + /NAM ratios in parasites grown at 32°C ( Figure 2D , Figure S3G , Table S7). To confirm that this is the result of decreased nico transcript levels, we directly knocked-down nico 26 ( Figure S3H,I ). Depletion of Nico did not affect parasite growth ( Figure S3J,K ), and targeted metabolomics confirmed a significant drop in the NAD + /NAM ratio between control and knock-down cells ( Figure 2E , S4A , Table S7). Importantly, this decrease of the NAD + /NAM ratio in the Nico knockdown is accompanied by a de-repression of previously silent S-type rRNA, similar to that which takes place upon growth at 32°C ( Figure 2F , S4B, Table S4). Download figure Open in new tab Figure S4 A) Giemsa images of indicated parasite cell lines and treatments at the time of sampling for metabolomics experiments. Gln: glucosamine. B) Giemsa images of indicated parasite cell lines and treatments at the time of sampling for total RNA-seq experiments. Gln: glucosamine. While it has been known that S-type rDNA can be de-repressed by decreasing growth temperatures, our data suggest that low temperature does not directly control rRNA transcription, but leads to a metabolic shift featuring decreased NAD + /NAM ratios. Indeed, glucose starvation has similarly been found to de-repress silent rDNA 15 , providing further evidence that it is the metabolic state that influences rRNA transcription rather than a temperature-mediated signal. On the other hand, nico transcription decreases by only half in mosquito midgut stages ( Figure S5A ), suggesting that additional factors influencing NAD + metabolism are involved in the de-repression of rRNA during the human-to-mosquito transition. Download figure Open in new tab Figure S5 A) Expression of the nicotinamidase (Nico, PF3D7_0320500, left), branched chain ketoacid dehydrogenase (BCKDHA, PF3D7_1312600, middle) and lactate dehydrogenase (LDH1, PF3D7_1324900, right) along the parasite lifecyle. FPKM: fragments per kilobase of exon per one million mapped reads. A: asexual stages; G: gametocytes; O: oocysts; S: sporozoites. Data retrieved from 62 , 63 . B) Representation of the pSLI strategy to integrate a 3xHA-tag and a glmS ribozyme at the 3’end of the LDH1 locus. GGG and GSG: linkers; 2A: skip peptide; Neo: neomycin-resistance gene; yDHODH: yeast dihydroorotate dehydrogenase. C) PCR of wild-type NF54 and LDH-glmS gDNA using primer pair p4/p2 ( Figure S5B , Table S10) showing integration of the plasmid. D) Western blot analysis of the LDH-glmS cell line after 6, 24 and 48h of glucosamine (Gln) treatment. Aldolase is used as a loading control. Numbers on the left indicate molecular weight (MW) in kilodaltons (kDA). E) Giemsa images of LDH-glmS cells grown without (-Gln, top) or with (+Gln) glucosamine. Time represents hours after glucosamine additions. F) Giemsa images of wild-type 3D7 and Sir2a-KO parasites at the time of sampling for histone-PTM LC-MS/MS experiments. Disruption of aerobic glycolysis de-represses silent rDNA genes To provide further evidence for the role of metabolites – i.e. NAD + /NAM ratios - in rDNA transcriptional regulation, we investigated enzymes involved in the dramatically changing metabolism of the parasite between human blood stages and mosquito stages. During the asexual replicative cycle in the human blood, P. falciparum generates all ATP via aerobic glycolysis, or glycolysis that ends with the fermentation of glucose-derived pyruvate to lactate despite available oxygen 27 ( Figure S3F ). It follows that the single mitochondrion of the parasite is dramatically reduced, and neither the TCA cycle nor oxidative phosphorylation are essential for asexual replication in the human blood 28 – 32 . However, for development in the mosquito vector, the parasite extensively restructures and builds up its mitochondria 31 , 32 . This switch of metabolic pathways from only aerobic glycolysis to predominantly oxidative phosphorylation is accompanied by decreased expression of lactate dehydrogenase (LDH1, PF3D7_1324900), responsible for the fermentation of pyruvate to lactate, and increased expression of branched chain ketoacid dehydrogenase (BCKDHA, PF3D7_1312600), which catabolizes pyruvate to acetyl-CoA that enters the TCA cycle ( Figure S3F , S5A ). Importantly, aerobic glycolysis regenerates NAD + used in glycolysis via the activity of LDH1, maintaining elevated NAD + levels, whereas increased mitochondrial activity promotes NAD + consumption via the TCA cycle, leading to a global decrease in NAD + levels 33 ( Figure S3F ). To test whether LDH1 is essential for maintaining cellular NAD + /NAM levels, we performed inducible knock-down of ldh1 in blood stage parasites ( Figure S5B,C ), which resulted in rapid depletion of LDH1 protein ( Figure S5D ) and an arrest in the growth cycle ( Figure S5E ). In addition, LDH1 knockdown led to a significant decrease in the NAD + /NAM ratio ( Figure 2G ), possibly due to the decreased rates of NAD + regeneration ( Figure S4A , Table S7). Moreover, we found the same pattern of silent S-type rDNA de-repression upon LDH1 knockdown ( Figure 2H , S4B, Table S4). Interestingly, LDH1 expression is also significantly downregulated in parasites grown at 32°C (Table S6), possibly leading to an additive effect together with the downregulation of Nico. Altogether, this data provides further evidence that silencing of rDNA transcription in the human host is sensitive to NAD + /NAM ratios and depends on the maintenance of aerobic glycolysis. The NAD + -dependent HDAC Sir2a is required for rDNA silencing One possible way that fluctuating NAD + levels influence transcriptional regulation of rDNA is via chromatin-modifying enzymes. NAD + is an essential co-factor in many cellular processes 34 , including the activity of sirtuins, or class III histone deacetylases (HDACs) 35 . The P. falciparum genome encodes two divergent sirtuin homologs, Sir2a and Sir2b. Sir2a activity depends on NAD + and at the same time is inhibited by its own product - NAM – making it sensitive to changes in cellular NAD + /NAM ratios 25 ( Figure S3F ). Both sirtuins were found to be involved in the regulation of heterochromatinized subtelomeric virulence genes 36 – 38 . In addition, Sir2a (but not Sir2b) has also been shown to repress rDNA transcription 17 . Total RNA-seq comparing wild-type 3D7 and Sir2a knock-out (‘Sir2a-KO’) parasites 39 ( Figure S4B ) corroborate this finding, with S-type 28S rRNA showing the strongest upregulation when Sir2a is deleted ( Figure 2I , Table S4). To confirm whether Sir2a could act as a ‘translator’ of cellular NAD + levels on a transcriptional level, we first aimed to validate whether Sir2a can deacetylate histones in vivo. Among the histone post-translational modifications (PTMs) that were quantified using mass-spectrometry, we found that histone peptides containing H3K9ac, H3K14ac and H4K12ac were significantly and consistently more abundant in Sir2a-KO parasites compared to wild-type 3D7 parasites ( Figure 2J,K , S5F, Table S8). These data provide in vitro evidence that Sir2a is a histone deacetylase. Since no genome-wide binding data are currently available for Sir2a, we attempted ChIP-seq of an epitope-tagged Sir2a to determine whether it directly binds to rDNA loci ( Figure S6A,B ). However, recovery of Sir2a from the nuclear fraction was inefficient ( Figure S6C ). We therefore adapted a protein-DNA proximity labeling approach 40 , fusing the bacterial M.EcoGII 6mA DNA methyltransferase (‘madID’) to Sir2a ( Figure S6D-F ). Thus, the interaction of Sir2a with a specific genomic locus allows the madID fusion protein to add 6mA modifications onto surrounding adenines ( Figure 3A ). 6mA sites can then be detected by direct DNA sequencing using Oxford Nanopore technology and compared to non-modified, wildtype genomic DNA. A cell line that inducibly expresses the madID protein with an N-terminal nuclear localization signal (NLS-madID) served as a negative control ( Figure S6G-I ). 6mA could readily be detected on DNA collected from both Sir2a-madID and NLS-madID using 6mA antibodies ( Figure S6J ). In addition, the majority of putatively modified bases identified by direct DNA sequencing in Sir2a-madID cells compared to wild-type cells were adenines ( Figure 3B ). In contrast, no nucleotide preference was detected for NLS-madID, and the overall number of putatively modified sites was substantially lower, providing first evidence for increased recruitment of the madID protein in the Sir2a-madID cell line and its activity ( Figure 3B ). Download figure Open in new tab Figure S6 A) Representation of the pSLI strategy to integrate a 3xHA-tag at the 3’end of the Sir2a locus. GGG and GSG: linkers; 2A: skip peptide; Neo: neomycin-resistance gene; yDHODH: yeast dihydroorotate dehydrogenase. B) PCR of wild-type 3D7 and Sir2a-KO gDNA using primer pair p5/p6 ( Figure S6A , Table S10) showing integration of the plasmid. C) Western blot of a co-immunoprecipitation of Sir2a-HA performed with separated cytoplasmic (left) and nuclear protein extracts (right). Input: Input protein lysate; SN: Supernatant; IP: Immunoprecipitate. The higher band likely corresponds to a Sir2a-HA-neomycin fusion protein due to incomplete skipping at the T2A peptide site (calculated molecular weight of Sir2a-HA + neomycin: 64.4 kDA). Numbers on the left indicate molecular weight (MW) in kilodaltons (kDA). D) Representation of the pSLI strategy to integrate a madID-3xHA-tag at the 3’end of the Sir2a locus. GGG and GSG: linkers; 2A: skip peptide; Neo: neomycin-resistance gene; yDHODH: yeast dihydroorotate dehydrogenase. E) PCR of wild-type 3D7 and Sir2a-madID gDNA using primer pair p7/p8 ( Figure S6D , Table S10) showing integration of the plasmid. F) Nanopore tracks displaying reads (in purple) encompassing both the pSLI plasmid, the Sir2a locus and the surrounding genomic region, indicating correct integration of the plasmid downstream of the Sir2a locus. G) Representation of the pFIO_hsp86_NLS_madID plasmid used to inducibly overexpress the nuclear-localized madID protein under a hsp86 promoter. H) PCR of NLS-madID gDNA without (-Rap, left) and with (+Rap, right) 200nM rapamycin treatment using primer pair p9/p10. yDHODH: yeast dihydroorotate dehydrogenase; NLS: nuclear localization signal; loxP: Cre recombinase binding sites. I) Western blot analysis of the NLS-madID cell line without (-Rap, left) and with (+Rap, right) 200nM rapamycin treatment. Aldolase and H3 are used as loading controls for the cytoplasmic (C) and nuclear (N) fractions. Numbers on the left indicate molecular weight (MW) in kilodaltons (kDA). J) Dot blot assay using anti-6mA antibodies against gDNA from Sir2a-madID cells (left) and NLS-madID cells without (-Rap, middle) and with (+Rap, right) 200 nM rapamycin treatment. Numbers on the left indicate total amount of gDNA used. Download figure Open in new tab Figure 3 A) Schematic illustration of the Sir2a-madID approach followed by ONT sequencing. B) Number of modified sites in the Sir2a-madID (red) and NLS-madID (grey) cell line categorized by nucleotide identity. A: adenine; C: cytosine; G: guanine; T: thymine. C) Global view of 6mA densities for Sir2-madID (‘Sir2a’) and NLS-madID (‘madID’) together with ChIP/input ratio tracks of HP1, H3K9ac, H3K14ac, H4K12ac and H4K16ac. Heterochromatic regions are highlighted in grey. Dotted insert indicates the location of the A2 rDNA locus. madID data are shown as number of modified sites per 1 kb normalized to the AT content of each bin. D) Genome-wide comparison of modified sites identified in the Sir2-madID (‘Sir2a’) and NLS-madID (‘madID)’ cell line between euchromatic regions (EC, n = 76) and HP1-occupied regions (HC, n = 89). E) ChIP/input ratio tracks of H3K9ac, H3K14ac, H4K12ac and H4K16ac at the A2 rDNA locus. F) Number of putatively modified sites located in the top 100 most (‘T’) and least (‘B’) differentially enriched peaks of each PTM that were identified by ChIP-seq in Sir2a-KO parasites. G) Detailed view of 6mA densities in Sir2a-madID (‘Sir2a’) and NLS-madID (‘madID’) together with calibrated ChIP data for four histone PTMs at the A2 rDNA locus. Blue: Sir2a-KO; brown: wild-type 3D7 . madID data are shown as number of modified sites per 10 bp normalized to the AT content of each bin. H) Same as g) for the S2 rDNA locus. Interestingly, 6mA in Sir2a-madID is not equally distributed, but is preferentially located in euchromatic regions of the genome ( Figure 3C,D ). Whereas sirtuins in other eukaryotes are often implicated in the maintenance of heterochromatin 41 , these data identify Sir2a as a possible modulator of histone acetylation across transcriptionally permissive chromatin regions. A notable exception is the specific and high Sir2a enrichment within the intron of var genes, a region known to be important for var gene biology ( Figure S7A ). This finding suggests that Sir2a could regulate var gene transcription via histone deacetylation and subsequent transcriptional repression of var intron-derived transcripts 42 – 44 . Download figure Open in new tab Figure S7 A) Detailed view of calibrated ChIP-seq data for four histone PTMs at two subtelomeric var genes. Blue: Sir2a-KO; brown: Wild-type 3D7 . Sir2a-madID (‘Sir2a’) and NLS-madID (‘madID’) are shown as number of modified sites per 10 bp bin normalized to the AT content of each bin. B) ChIP/input track of the second replicates for H3K9ac, H3K14ac, H4K12ac and H4K16ac on chromosome 7. C) ChIP/input ratio tracks of H3K9ac, H3K14ac, H4K12ac and H4K16ac at the S1 rDNA locus. D) ChIP/input ratio tracks of H3K9ac, H3K14ac, H4K12ac and H4K16ac at the S2 rDNA locus. E) ChIP/input ratio tracks of H3K9ac, H3K14ac, H4K12ac and H4K16ac at the S3 rDNA locus. F) Illustration representing the principle of calibrated ChIP-seq using spike-in chromatin. Yeast and P. falciparum chromatin are crosslinked and sonicated separately. A known quantity of yeast chromatin is added to each P. falciparum sample and the common ChIP-seq protocol is followed. The reads are aligned to both genomes and the spike-in is used to normalize the P. falciparum signal (see Material and Methods). G) Detailed view of 6mA densities in Sir2a-madID (‘Sir2a’), NLS-madID (‘madID’) and calibrated ChIP data for four histone PTMs at the S1 (left) and S3 (right) rDNA locus. Blue: Sir2a-KO; brown: wild-type 3D7 . madID data are shown as number of modified sites per 10 bp normalized to the AT content of each bin. H) Proportion of A1/2, S1,S2/3, O1 and O2 28S rRNA during midgut development of P. falciparum . Time indicates hours post bloodmeal. Data were re-analyzed from 55 (see Material and Methods) Download figure Open in new tab Figure S8 Uncropped images of DNA gels and Western Blot membranes used in this study. To elucidate whether Sir2a can indeed act as a modulator of histone acetylation levels at silent rDNA loci, we first performed ChIP-seq of H3K9ac, H3K14ac, H4K13ac and H4K16ac (a modification targeted by Sir2 homologs in other eukaryotes 45 – 47 in wild-type 3D7 cells. All four histone PTMs are predominantly located within transcriptionally permissive euchromatin 18 , in a pattern similar to that seen for Sir2a-madID ( Figure 3C , S7B, Table S9). More importantly, we find that peaks of H3K14ac, H4K12ac and H4K16ac located upstream of the A2 rDNA locus are among the highest and most significant enrichments across the genome ( Figure 3E , Table S9), similar to that seen for H3K9ac ( Figure 1E ) and that they are absent from silent rDNA loci ( Figure S7C,D,E ) We next adapted a spike-in ChIP-seq approach that allows accurate quantification of changes in PTM enrichment between two samples, i.e. Sir2a-KO and wild-type 3D7 parasites 48 ( Figure S7F ). Integrating the quantitative ChIP enrichment signals with 6mA densities showed that histone PTM peaks that feature the greatest increase of enrichment in Sir2a-KO cells compared to wild-type 3D7 cells also feature significantly higher 6mA densities (i.e. Sir2a occupancy) than the histone PTM peaks with the lowest increase of enrichment ( Figure 3F ), thus directly linking Sir2a binding with histone (de-)acetylation levels. Most importantly, upon Sir2a deletion, we find substantial increases for all four modifications upstream of the active A2 rDNA locus, a region that also features increased 6mA densities (i.e. Sir2a occupancy, Figure 3G , Table S9). In direct comparison to the active rDNA locus however, Sir2a occupancies are 2- to 20-fold higher upstream of all major silent rDNA loci ( Figure 3H , S7G), and accordingly we find that all histone PTM levels also substantially increase in their upstream regions when Sir2a is disrupted ( Figure 3H , S7G). Altogether, these data indicate that high Sir2a occupancy in the upstream regions of silent rDNA loci leads to continuous histone deacetylation and maintenance of a transcriptionally repressed state. DISCUSSION The P. falciparum lifecycle features a highly regulated transcriptional program in which it silences specific rDNA types in the human host and de-represses them during mosquito development. The data in this study suggest that the silencing of mosquito-stage rDNA loci in the human host relies on high NAD + /NAM ratios that are maintained through aerobic glycolysis and translated on a molecular level by the HDAC Sir2a. Even though the five canonical and two divergent (O-type) rDNA loci are not organized in a ‘classical’ rDNA repeat array and have distinct upstream regions, we identified several conserved epigenetic features of active and silent rDNA chromatin. Active rDNA loci are characterized by open chromatin, histone acetylation at regulatory upstream regions, direct interaction in 3D space, and enrichment of nucleolar transcription factors (i.e. HMGB1/2). Silent S-type rDNA loci are heterochromatinized via HP1 enrichment over their genic region and high Sir2a occupancy at their upstream regions. Our combination of quantitative ChIP-seq of histone acetylation and Sir2a protein-DNA labelling revealed that Sir2a binds to and de-acetylates histones genome-wide, especially in euchromatic regions. Thus, histone acetylation could prove to be a key modulatory link between changing environmental conditions and the transcriptional switches between rDNA loci that take place in the transition from human blood to mosquito. Sir2a might thereby not be specific for the modification of rDNA loci; however, knockout of Sir2a and subsequent increase in histone acetylation could render rDNA chromatin more permissive to binding of HMGB1 and 2, which are always highly abundant. Accordingly, other Sir2a-regulated genes might rely on the additional presence of stage-specific transcription factors (e.g. AP2s 49 ) to be fully activated. Although highly de-repressed upon our in vitro perturbations, S-type rRNA do not reach the same absolute expression levels as they normally do in mosquito stages (Table S4). One possible explanation is that in addition to the increase in enrichment of activating histone modifications and HMGB proteins, silencing histone modifications or chromatin-associated proteins (i.e. H3K9me3 and HP1) might have to be removed. Interestingly, the recently identified histone demethylases Jumonji1 and 2 50 , which can remove H3K9me3, are themselves dependent on a-ketoglutarate 51 . As an intermediate of the TCA cycle, a-ketoglutarate levels might be higher in mosquito stages, allowing Jumonji demethylases to help activate H3K9me3-silenced rDNA loci. This pathway could potentially represent another link between rDNA transcription and metabolic state of the parasite. Thus, the metabolically-linked chromatin composition – i.e. histone acetylation versus H3K9me3 – determines accessibility of rDNA loci to transcriptional activators. Importantly, we consistently found a specific expression pattern of the main rRNA types upon perturbation of temperature, Nico, or LDH1 – an upregulation of S1 and S2/3 rRNA and no significant change in A1/A2 rRNA transcription - which suggests a specific and common regulation mechanism in all conditions and genetic backgrounds tested. We propose that the common denominator is the change in NAD + /NAM levels. In this study, we combined distinct growth conditions with forward genetic approaches to independently modulate the NAD + /NAM ratios. Together, these data link two of the most substantial changes in the transition from the human host to the mosquito vector, i.e. the restructuring of the premier energy-generating metabolic pathway and the de-repression of silent rDNA. Key to this model is the finding that in organisms that maintain an active mitochondria but engage in aerobic glycolysis, the suppression of the latter leads to a decrease in NAD + levels due to a lack of NAD + regeneration by LDH 33 . A key question for Plasmodium therefore is: when does the parasite naturally switches from aerobic glycolysis to oxidative phosphorylation that could lead to a decrease in NAD + regeneration? During gametocytogenesis in human blood, the parasite extensively expands its mitochondria, down-regulates LDH1, and up-regulates BCKDH. Yet inhibition of the TCA cycle does not significantly alter NAD + levels at this stage, suggesting that NAD + homeostasis is not primarily influenced by it 29 , 52 , 53 . Moreover, inhibition of oxidative phosphorylation does not prohibit gametocyte development 30 , supporting the hypothesis that parasites enter a metabolically semi-quiescent stage during which rRNA transcription profiles do not change ( Figure 1A ). Following transmission and during gamete to ookinete development (∼24 hours), NAD + levels were found to stay stable, with S-type rRNA remaining repressed 7 , 54 , 55 ( Figure S7F ). However, genes involved in aerobic respiration and mitochondrial activity are among the most upregulated as the parasites develops into oocysts, which coincides with the de-repression of S-type rRNA 7 . These data suggest that the parasite switches to oxidative phosphorylation at the onset of oocyst development, at the same time silent rDNA are de-repressed 7 . In most eukaryotes, not all rDNA genes within an rDNA repeat are transcribed simultaneously. Because rDNA transcription, ribosome biogenesis, and protein synthesis require large amounts of energy 12 , 13 , they need to be balanced with the resources available to the cell. As the first and rate-limiting step in this cascade, many organisms evolved mechanisms that specifically ensure the repression of rDNA transcription under unfavorable conditions (i.e. starvation, nutrient stress. This can be achieved either by targeting the PolI initiation complex 56 – 58 or by directly changing the chromatin environment of rDNA repeats in the nucleolus 14 , 59 . In the latter situation, increased NAD + /NADH levels during nutrient stress in human cells lead to elevated deacetylation activity of the sirtuin HDAC SirT1 (part of the eNoSC complex), resulting in heterochromatinization and silencing of rDNA 14 . Although NAD + levels and sirtuin activity are central to this mechanism, a major difference with P. falciparum rDNA regulation is that instead of controlling the total amount of rRNA during times of nutrient stress, the parasite silences specific rDNA types as it uses distinct metabolic pathways during its progression through the lifecycle. During asexual development in the human blood, P. falciparum develops in a glucose-rich environment that allows rapid replication without oxidative phosphorylation, which generates more ATP per molecule of glucose than lactate fermentation during aerobic glycolysis. Yet the preference of lactate fermentation over oxidative phosphorylation is found in many cell types and organisms during periods of rapid proliferation 60 and is thought to be driven by the higher need for NAD + than ATP, for example as a co-factor for various enzymes 33 . Given that the parasite already tightly controls the substantial restructuring from aerobic glycolysis to oxidative phosphorylation during host-vector transmission and that lactate fermentation maintains high NAD + /NAM ratios, linking this switch to the (de-) repression of rDNA might therefore allow the parasite to precisely control ribosome heterogeneity without the necessity of an additional, independent regulatory mechanism. Altogether, our results provide a model of how the metabolic state of P. falciparum is translated into a functional transcriptional outcome to developmentally control the expression of distinct rRNA transcripts. MATERIALS AND METHODS Parasite culture Asexual blood-stage P. falciparum parasites were cultured in human RBCs (obtained from the Etablissement Francais du Sang with approval number HS 2021-24819) in RPMI-1640 medium (Thermo Fisher # 53400-025) supplemented with 10% v/v Albumax I (Thermo Fisher no. 11020039), hypoxanthine (0.1 mM final concentration, CC-Pro # Z-41-M) and 10 mg gentamicin (Sigma # G1397-10ML) at 4% haematocrit and under 5% O2, 3% CO2 at 37°C. Parasite development was monitored by Giemsa staining. For synchronization, late-stage parasites were enriched by plasmion flotation followed by ring-stage enrichment via sorbitol (5%) lysis 6 hours later. For sampling of highly synchronous parasites during the IDC, the synchronous schizonts were enriched by plasmion flotation shortly before reinvasion, followed by sorbitol treatment 6 hours later. The 0 h time point was considered to be 3 h after plasmion flotation. Gametocytes were obtained following to the protocol by Fivelman et al. 64 Briefly, synchronous asexual, late-stage parasites (30-35 h.p.i.) were concentrated at ∼2.5% parasitaemia and 2.5% hematocrit using plasmion flotation. The next day, 75% of the spent culture medium was replaced with fresh medium, and the ring-stage parasites (∼10-15% parasitemia) were left to develop into trophozoites at high parasitaemia for an additional 24h. The culture was then diluted in fresh media to 3% parasitaemia and kept for an additional 24 hours. The growth medium of the resulting high-parasitaemia, ring-stage culture was then replaced with RPMI supplemented with 5% human serum, 5% Albumax, 0.1 mM hypoxanthin, 10 mg Gentamicin and 50 mM N-acetylglucosamine (NAG, Sigma # A3286). Growth media was then changed daily for 5 days with the addition of NAG, and then without NAG for an additional six days. Stage V gametocytes were harvested at day 11. Generation of cell lines All cell lines for genomic integration were generated with the selection-linked integration method 65 starting with the modified pSLI-sandwich plasmid as described below. The pSLI-sandwich plasmid was digested with SalI and AvrII to remove the 2xFKBP and GFP tags. A 3xHA tag was PCR-amplified from plasmid pUF-dCas9 66 using primers HA_F/HA_R and cloned in the digested pSLI-sandwich plasmid. To enhance the efficiency of the separation between Neomycin and the protein of interest, a GSG linker was added in between the two coding sequences. Annealed forward and reverse oligonucleotides (GSG_F/GSG_R, Table S10) were cloned at the SalI restriction site, yielding the pSLI_3xHA_GSG_T2A_NeoR_hDHFR plasmid. The GlmS ribozyme was amplified using primers GlmS_F/GlmS_R from the plasmid pUF-dCas9 66 and cloned into the XhoI restriction site of the plasmid pSLI_3xHA_GSG_T2A_NeoR_hDHFR, yielding the pSLI_3xHA_GSG_T2A_NeoR_GlmS _hDHFR plasmid. For both plasmids, the hDHFR resistance cassette was changed to a yeast dihydroorotate dehydrogenase (yDHODH) resistance cassette, conferring resistance to DSM1. To do so, the original plasmids were digested with BamHI and HindIII to excise the hDHFR cassette and the yDHODH sequence was PCR-amplified with yDHODH_F and yDHODH_R primers and cloned in the digested plasmids, yielding pSLI_3xHA_GSG_T2A_NeoR_yDHODH and pSLI_3xHA _GSG_T2A_NeoR_ GlmS _yDHODH plasmids. The madID coding sequence 40 was codon-optimized for translation in P. falciparum and synthesized by GenScript. To add the fusion gene to a protein of interest, the synthesized madID gene was amplified using primers madID_F and madID_R and cloned into the AvrII restriction site of plasmid pSLI_3xHA_GSG_T2A _NeoR_yDHODH, yielding pSLI_madID-3xHA_GSG_T2A _NeoR_yDHODH. Homology regions serving as template for the integration of the plasmid at the 3’ end of the genes of interest were PCR-amplified from P. falciparum genomic DNA with the primer pairs HMGB1_F/HMGB1_R, HMGB2_F/HMGB2_R, Sir2a_F/Sir2a_R Nico_F/Nico_R, LDH_F/LDH_R, and Sir2a_F/Sir2a-madID_R (Table S10). The PCR products were cloned in the NotI/AvrII digested plasmids pSLI_3xHA_GSG_T2A _NeoR_yDHODH (for HMGB1, HMGB2, Sir2a-HA, see Figure S2A , S6A ), pSLI_3xHA _GSG_T2A_NeoR_ GlmS_yDHODH (for Nico and LDH1, see Figure S3F , S5D ) and pSLI_madID-3xHA_GSG_T2A_NeoR_yDHODH (for Sir2a-madID, see Figure S6D ) For the NLS-madID control construct, the pFIO-hsp86_no-mCherry plasmid 67 , was digested with NsiI and EcoRV. A yDHODH resistance cassette was amplified from plasmid pUF-dCas9 with primers pFIO_yDHODH_F/pFIO_yDHODH_R and cloned into the digested pFIO-hsp86_no-mCherry plasmid. The resulting plasmid was further digested with HindIII and XhoI. Annealed loxP oligos (Lox_HindIII_F/ Lox_HindIII_R) and the madID-HA tag (amplified from and pSLI_madID-3xHA_GSG_T2A_NeoR_yDHODH with primers pFIO_madID_F/pFIO_madID_R) were cloned into the digested plasmid in a single reaction. A NLS sequence was obtained by annealing oligos pFIO_NLS_F/pFIO_NLS_R and cloning into the HindIII restriction site of the plasmid, yielding pFIO_hsp86_NLS_madID ( Figure S6G ). The Sir2a-KO cell line used in this study was first described in Duraisingh et al 36 . All primers can be found in Table S10. All PCR reactions were performed using the KAPA HiFi DNA Polymerase (Roche # 07958846001) following the manufacturer’s protocol, but with lower elongation temperature (62°C or 68°C). Cloning and plasmid amplification were performed using the In-Fusion HD cloning kit (Clontech # 639649) and XL10-Gold ultracompetent E. coli (Agilent Technologies # 200315) following the manufacturer’s protocol. All plasmids were sequenced either by Sanger or Nanopore full-plasmid sequencing to verify that no mutation appeared. All pSLI plasmids were transfected into ring-stage P. falciparum (strain NF54) parasites by electroporation following standard protocols 66 and using 100 μg plasmid. The pFIO_hsp86_NLS_madID was transfected into the P. falciparum NF54 DiCre cell line 68 . To select for plasmid uptake, transfected parasites were cultured with 1.5 μM DSM1 (MR4/BEI Resources) and drug-resistant parasites emerged after three to four weeks. Positive selection for integration of the pSLI plasmids was performed via the addition of 400 μg/mL G418 (Sigma # G8168). For all cell lines, parasites were collected by saponin lysis (0.075% in DPBS) and gDNA was extracted using the Qiagen DNeasy Blood & Tissue Kit (Thermo Fisher # 69504). Plasmid integration was verified by PCR with corresponding primers listed in Table S10 Since no specific primers for the verification of successful integration of the HA-tag at the HMGB1 locus were found, integration was verified by DNA sequencing using Oxford Nanopore sequencing (see below and Figure S2B ) Parasite growth assay To measure parasite growth kinetics the cell lines were tightly synchronized by plasmion/sorbitol to a 6h window as described above. The ring-stage parasites were diluted separately to 0.2% parasitemia (5% hematocrit) in the blood of three different donors. For the Nico-glmS cell line, the culture was split and glucosamine (Sigma # G1514) was added to one half of the culture (2.5 mM final concentration). The growth curve was performed in a 96-well plate (200 μl culture per well) with three technical replicates per condition and per blood. Parasitemia was measured every 24 h by staining parasite nuclei using SYBR Green I (Sigma # S9430) and quantifying infected RBCs using a Cytoflex flow cytometer. For the growth curve at 32°C and 37°C, the culture was split after synchronization, and additional timepoints were collected at the time of schizont rupture to precisely measure the duration of the life cycle at the different temperatures. All data were post-processed and analysed using FlowJo. Since knockdown of LDH-glmS following addition of glucosamine at the ring-stage did not allow for the completion of one full cell cycle, growth dynamics were compared by Giemsa-staining. The parasites were tightly synchronized by plasmion/sorbitol to a 6h window and diluted to 1% parasitemia (5% hematocrit). The culture was then split, and glucosamine was added (2.5 mM final concentration) to one half after the sorbitol. Parasite growth was monitored by Giemsa staining every 12h for 74h ( Figure S5E ). Western Blot For HMGB1-HA and HMGB2-HA cell lines ( Figure S2D,E ), synchronized late-stage parasites were collected by saponin lysis (0.075% saponin [Sigma # S790] in Dulbecco’s phosphate-buffered saline [DPBS, Thermo Fisher # 14190-144]) at 37°C. Parasites were washed twice with ice-cold DPBS, and lysed by resuspending the cell pellet in TLB (Total Lysis Buffer: 20mM Tris-HCl pH 7.5, 50mM NaCl, 1mM DTT, 0.1% SDS and protease inhibitor [PI, Roche # 11836170001]) and sonicated with a Bioruptor Pico (5 cycles 30sec ON / 30sec OFF) at 4°C. Cell debris was removed by centrifugation (13,500g, 10min, 4°C) and the supernatant was transferred to a new tube. 25 µl of protein G Dynabeads were washed twice with TLB, resuspended in TLB with 1 µl of anti-HA antibody (Abcam # ab9110) and rotated for 2h. The beads were washed twice with TLB, and added to the HMGB1/2 protein lysates. The HA-tagged proteins were bound by overnight rotation, the beads were washed twice with TLB and immunoprecipitated proteins were eluted by resuspending the beads in TLB supplemented with NuPage Sample Buffer (Thermo Fisher # NP0008) and NuPage Reducing Agent (Thermo Fisher # NP0004) and incubation at 70°C for 10 min. For the Nico-glmS cell line ( Figure S3K ), glucosamine was added to half of the culture immediately before the synchronization by plasmion/sorbitol. Cells were collected after 24h and 48h of glucosamine treatment and lysed with 0.075% saponin in DPBS at 37°C. Cells were washed twice with DPBS at 4°C. For separation of the cytoplasmic and nuclear protein fractions, the cell pellet was first resuspended in 1 ml CLB (Cytoplasmic Lysis Buffer :25 mM Tris-HCl pH 7.5, 10 mM NaCl, 1% IGEPAL CA-630, 1 mM DTT, 1.5 mM MgCl2, 1xPI) and incubated on ice for 30 min with regular flicking. The cytoplasmic lysate was cleared by centrifugation (13,500g, 10 min, 4°C). The nuclei pellet was gently washed twice with CLB, resuspended in 100 μl NLB (Nuclear Lysis Buffer: 25 mM Tris-HCl pH 7.5, 1 mM DTT, 1.5 mM MgCl2, 600 mM NaCl, 1% IGEPAL CA-630, PI) and sonicated with a Bioruptor Pico (5 cycles 30sec ON / 30sec OFF). This nuclear lysate was cleared by centrifugation (13,500g, 10 min, 4°C). Protein samples were supplemented with NuPage Sample Buffer and Reducing Agent and denatured for 10 min at 70°C. The LDH-glmS cell line ( Figure S5D ), parasites were synchronized by plasmion/sorbitol, and glucosamine (2.5 mM final concentration) was added immediately after the sorbitol to half of the culture. Cells were collected after 6h, 24h and 48h of glucosamine treatment, lysed and proteins were extracted with TLB as described above. Protein samples were supplemented with NuPage Sample Buffer and Reducing Agent before denaturation for 10 min at 70°C. For the Sir2a-HA cell line ( Figure S6C ), late-stage parasites were collected and lysed with 0.075% saponin in DPBS at 37°C. Cells were washed twice with DPBS at 4°C. The separation of the cytoplasmic and nuclear protein fractions was performed as described above, using CLB and NLB. Protein immunoprecipitation was performed using anti-HA antibody (Abcam # ab9110) with overnight rotation in CLB for cytoplasmic fractions and NLB for nuclear fractions. Immunoprecipitated proteins were eluted by resuspending the beads in NLB or CLB supplemented with NuPage Sample Buffer and NuPage Reducing Agent and incubation at 70°C for 10 min. For NLS-madID ( Figure S6I ), the cell line was synchronized by plasmion-sorbitol to a 6h window. Rapamacin was added (200nM final concentration) to half of the culture after the sorbitol, and cells were collected at 40 h.p.i. The separation between nuclear and cytoplasmic fractions was performed as described above. For all Western Blots, the samples were separated on a NuPage 4–12% Bis-Tris gel (Thermo Fisher #NP0321) using MOPS running buffer (Thermo Fisher # NP0001) at 100 V for 1.5 h and transferred to a PVDF membrane using Trans-Blot Turbo transfer system (Bio-Rad) using the mixed molecular weight program. The membrane was blocked for 1 hour in 1% milk in 0.1% Tween20 in PBS (PBST). Histone H3 was detected with anti-H3 (Abcam no. ab1791: 1:1,000 in 1% milk-PBST) primary antibody, followed by donkey anti-rabbit (GE # NA934-1ML) secondary antibody conjugated to HRP (1:5,000). HA-tagged proteins and PfAldolase were detected using HRP conjugated anti-HA (Ozyme 14031S, 1:1,000 in 1% milk PBST), and anti-PfAldolase (Abcam # ab38905, 1:5,000 in 1% milk PBST) antibodies, respectively. The HRP signal was developed using the SuperSignal West Pico chemiluminescent substrate (Thermo Fisher # 34580) and imaged with a ChemiDoc XRS+ (Bio-Rad). Histone mass-spectrometry Histone extraction P. falciparum (strain 3D7) and Sir2a-KO cell lines were tightly synchronized using plasmion/sorbitol and 6 replicates, each corresponding to ∼2x10 ^8 parasites, were collected at 40 h.p.i. ( Figure S5F ) and lysed with 0.075% saponin in DPBS. The pellet was transferred to 1.5 ml Protein LowBind Eppendorf tube, washed twice with 1 ml cold DPBS (4,000g, 5 min, 4°C), snap-frozen and stored at -80°C until ready to be processed. The pellet was resuspended in 500 μL of cold buffer A (15 mM Tris–HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl 2 , 1 mM CaCl 2 , 250 mM sucrose, 0.3% NP40, PI) and incubated on ice for 10-20min, flicking the tube regularly. The pellet was then washed twice with buffer A without NP40 to remove detergent (1,000g, 5 min, 4°C) and 5X pellet volume of 0.25 M HCl was added. The tube was rotated for 4h at 4°C, centrifuged (3,400g, 5 min, 4°C) and the supernatant containing histones was transferred to a new tube. A second spin was performed (3,400g, 5 min, 4°C) to remove pellet traces and the supernatant was again transferred to a new tube. Tricholoroacetic acid was added to a final concentration of 20% and the sample incubated on ice for 1h. After spinning (3,400g, 5 min, 4°C), the supernatant was carefully removed and washed with 500 μl acetone + 0.1% HCl. The histones were then washed twice using 100% acetone. The pellet was dried for 30 min under a chemical hood, and resuspended in 40 μl of loading buffer (NuPage Sample Buffer and NuPage Reducing Agent in ddH 2 O). The samples were then loaded on a 15% home-made acrylamide gel and ran at 30 mA in SDS-PAGE running buffer. The gel was stained with LabSafe GEL Blue (VWR 786-35) for 1 h and washed with distilled water until clear. Gel slice corresponding to histones were excised and in-gel digested by using Tryps/LysC (Promega). Peptides extracted from each band were loaded onto homemade C18 StageTips (packed with AttractSPE Disk Bio C18, Affinisep) for desalting. Peptides were eluted using 40 / 60 acetonitrile / H2O + 0.1% formic acid and vacuum concentrated to dryness. Peptides were resuspended in loading buffer (0.3% TFA in miliQ water) before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS Analysis Online LC was performed with an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific). Peptides were first trapped onto a C18 column (75 μm inner diameter × 2 cm; nanoViper Acclaim PepMap TM 100, Thermo Scientific) with buffer A (2 / 98 acetonitrile / H 2 O + 0.1% formic acid) at a flow rate of 2.5 µL/min over 4 min to concentrate the samples. Separation was performed on a 50 cm nanoviper column (i.d.75 µm, C18, Acclaim PepMap TM RSLC, 2 μm, 100Å, Thermo Scientific) regulated to a temperature of 50°C with a linear gradient of 2% to 30% buffer B (100% acetonitrile, 0.1% formic acid) at a flow rate of 300 nL/min over 91 min. MS full scans were performed in the ultrahigh-field Orbitrap mass analyzer in ranges m/z 375–1500 (resolution of 120L000 at m/z 200; maximum injection time 25 ms; AGC 300%). The top 20 most intense ions were subjected to Orbitrap for further fragmentation via high energy collision dissociation (HCD) activation and a resolution of 15L000 with the auto gain control (AGC) target set to 100%. We selected ions with charge state from 2+ to 6+ for screening. Normalized collision energy (NCE) was set at 30 and the dynamic exclusion of 40s. Histone PTM analysis For PTM identification, the data were searched against the Plasmodium falciparum histone sequences using Mascot. Only peptides that could be specifically assigned to a single histone (i.e. are proteotypic) were included in the analysis. Enzyme specificity was set to trypsin and a maximum of five-missed cleavage sites were allowed. Oxidized methionine, carbamidomethyled cysteine, N-terminal acetylation, acetylation, methylation (mono, di and tri), ubiquitination, propionylation, butyrylation, succinylation, malonylation, hydroxybutyrylation, glutarylation and crotonylation, palmitoylation, carboxyethylation of lysine, methylation (mono and di) of arginine, monomethylation of glutamic acid and aspartic acid were set as variable modifications and with a maximum of nine modifications for all Mascot searches. Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.02 Da for MS/MS peaks. Importantly, lysine carboxyethylation and lactylation PTMs have an identical mass and cannot unequivocally be distinguished. For post-translational modifications (PTMs) quantification, Skyline was used for processing the data (version 23.1.0.380) MacCoss Lab Software, Seattle, WA; https://skyline.ms/project/home/software/Skyline/begin.view ) and extracted Ion chromatograms from each peptide ion and peak area were integrated. Peptides were grouped according to their specific protein sequences and then PTMs quantifications were run independently for each peptide group (Table S8). The resulting files were further processed using myProMS v3.10 [PMID: 17610305] ( https://github.com/bioinfo-pf-curie/myproms ). For each site, the peak areas of corresponding ions were log2-transformed, and the distribution was normalized by the equivalent (log 2 -transformed) distribution of non-modified ions using the R package preprocessCore 69 . To evaluate the statistical significance of the change in protein abundance, a linear model (adjusted on peptides and biological replicates) was performed, and a T-test was applied on the fold change estimate. The p-values were then corrected for multiple testing using the Benjamini-Hochberg procedure. To represent the data with a heatmap, the intensities of the peptides were first transformed using the Z-score. Indeed, since for a given modification, the intensities are not comparable according to the peptide or the charge state, we choose to transform them. Finally, we aggregated the different values (using the mean) by replicates for each modification. This permits to obtain only one value by replicate, for each modification. RNA sequencing and analysis Total RNA sample preparation and sequencing For the comparison of 37°C and 32°C ( Figure 2A ), P. falciparum parasites (strain NF54) were tightly synchronized by plasmion-sorbitol in a 6h window, diluted to 2% parasitemia and half of the culture was put at 32°C. Parasites grown at 37°C were collected at 36 h.p.i., while parasites grown at 32°C were collected at 56 h.p.i., which corresponded to the equivalent developmental stage (Figures S3A, S3B). For the LDH-glmS cell line, parasites were synchronized by plasmion/sorbitol to a 6h window and diluted to 2% parasitemia. At 12 h.p.i., the culture was split and glucosamine (2.5 mM final concentration) was added to half of the culture. Parasites were collected at 36 h.p.i. ( Figure S4B ). Late stage P. falciparum Nico-glmS parasites were split and glucosamine (2.5 mM final concentration) was added to half of the culture. After 6h, the parasites were synchronized by a plasmion/sorbitol in a 6h window and diluted to 2% parasitemia. Parasites were collected at 36 h.p.i. ( Figure S4B ). For the comparison of P. falciparum wild-type (strain 3D7) and Sir2a-KO parasites, cells were synchronized by plasmion/sorbitol to a 6h window and diluted to 2% parasitemia. Parasites were collected at 36 h.p.i. ( Figure S4B ). For all asexual stages and the stage V gametocytes ( P. falciparum strain NF54, Figure 1A ), parasites were lysed with 0.075% saponin in DPBS at 37°C (or 32°C for the cells cultivated at this temperature). The parasite cell pellet was washed once with DPBS and then resuspended in 700 μl QIAzol reagent (Qiagen # 79306). Total RNA was extracted using the Direct-zol RNA Microprep (Zymo # R2060), including an on-column DNase I digestion according to the manufacturer’s protocol. Midgut oocysts (8 days post bloodmeal) and salivary gland (21 days post bloodmeal) sporozoites were obtained by dissecting Anopheles stephensi mosquitoes infected with P. falciparum (strain NF54, Figure 1A ). Dissected midguts and salivary glands were placed in 250 mL of PBS (between 50 and 300 mosquitoes per replicate) and lysed by adding 750 mL of TRIzol-LS (Thermo Fisher # 10296010) followed by vigorous pipetting. Total RNA was obtained by chloroform extraction followed by isopropanol precipitation. The precipitated RNA was washed twice with 75 % ethanol and resuspended in RNase-free water. All sequencing Libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit (NEB # E7760S) and sequenced (150bp paired-end) on the Illumina NextSeq 500 platform. mRNA sample preparation and sequencing P. falciparum (strain NF54) parasites were synchronized by plasmion/sorbitol to a 6h window. The culture was split after synchronization and one half incubated at 32°C. The cells were collected at 15 h.p.i. ( Figure S3D,E ). Cells were lysed and RNA was extracted as described above. mRNA was enriched using the Dynabeads mRNA Purification Kit (Thermo Fisher # 61006). Library preparation and sequencing was performed as for the total RNA sequencing samples (see above). RNA-seq data processing and analysis Stringent read alignment filtering requires the removal of reads that align to multiple regions in the reference genome, generally discarding reads mapping equally well to the near identical A1/A2 and S2/S3 loci (Table S1). To be able to maintain stringent alignment filtering and at the same time accurately measure rRNA abundance, the A1 and S2 rDNA loci of the P. falciparum reference genome (PlasmoDB, version 64 70 ) were masked from the beginning of the 18S to the end of the 28S sequence using bedtools ‘maskfasta’ 71 , leaving only one mappable copy of each rDNA type (i.e. A2 and S3). A rDNA locus of An. stephensi was added as separate chromosome to the masked genome of P. falciparum and used as mapping reference for all samples originating from infected mosquitoes (i.e. oocysts and sporozoites). For all RNA-seq experiments, raw sequencing data were basecalled and demultiplexed with bcl2fastq and sequencing adapters were trimmed using trimmomatic 72 . Trimmed reads were aligned to the masked P. falciparum genome (see above) using STAR 73 with default options and option ‘--outFilterMultimapNmax 1’. Optical duplicates were removed using samtools ‘fixmate’ and ‘markdup’ and only alignments with both mates were retained (samtools view -f 0x2). For the mRNA-seq experiment ( Figure 2B , Table S6), gene counts were calculated from the filtered alignments using htseq-count 74 with options ‘-t exon -s reverse -r pos’. Fragments per kilobase of exon per one million mapped reads (FPKM) values were calculated and differential gene expression analysis was performed in R using DESeq2 75 . The mRNA transcriptome-based calculation of developmental age for parasites grown 37°C and 32°C ( Figure 2B , S3D) was calculated in R using the method developed in Lemieux et al. 61 and using the transcriptome of Bozdech et al. 4 as baseline reference. Approximation of the developmental age by comparison to scRNA 6 ( Figure S3E ) was calculated as follows: For each gene, the average FPKM across all three replicates was calculated and the expression of all genes was correlated to each individual transcriptome of Dogga et al. 6 in R using cor(method = “pearson”). Resulting Pearson R 2 values were plotted over the UMAP1 and UMAP3 coordinates of each individual cell in R using ggplot2 76 . To stringently align reads to the different rDNA loci, reads were aligned using STAR with default options except for ‘--alignEndsType EndToEnd’, ‘ --scoreDelOpen -100’ and ‘-- scoreInsOpen -100’ and subsequently filtered as above. To further remove misalignments among non-masked rDNA loci, only alignments with one or less mismatches per read pair (samtools view -e ‘[nM] <=1’) and an alignment length ≥120bp were retained. FPKM values and fold-changes of rRNA transcripts were calculated in R 77 (Table S1). To quantify the change of contribution of transcription from A1 and A2 loci to the total pool of A-type 28S rRNA transcripts, filtered alignments were visualized using the Integrative Genomics Viewer (50) and the frequency of the single nucleotide variants (SNVs) at the A2 locus (i.e. reads originating from the A1 locus mapping to the A2 locus ( Figure S1B ) was calculated. Fold-changes of SNV abundance were calculated relative to the asexual stage sample. For rRNA expression during midgut development ( Figure S7H ), the raw fastq files from Mohammed et al. 55 were mapped to the masked reference genome that includes a rDNA locus of An. stephensi . Alignments were filtered as described above for the total RNA sequencing samples. FKPM values for the calculation of relative contributions of each rDNA type were calculated in R using DESeq2 75 . Chromatin Immunoprecipitation In general, we performed two independent biological replicates for all ChIP-seq experiments presented in this study. P. falciparum chromatin preparation HMGB1-HA and HMGB2-HA cell lines were synchronized by plasmion/sorbitol to a 6h window. After the synchronization, half of the culture was incubated at 32°C. Parasites cultivated at 37°C were collected at 36 h.p.i., while parasites incubated at 32°C were collected at 56 h.p.i., which corresponded to an equivalent developmental stage (Figures S2F, S3A). For histone PTM ChIP-seq, wild-type P. falciparum (strain 3D7) and Sir2a-KO cell lines 36 were synchronized by plasmion/sorbitol to a 6h window and collected at 16 h.p.i. Chromatin Immunoprecipitation was performed as described previously 66 . Briefly, synchronized parasite cultures were lysed with saponin (0.075% in DPBS), washed with DPBS at 37°C (or 32°C for parasites cultivated at this temperature) and resuspended in DPBS at 25°C. For HMGB1-HA and HMGB2-HA samples, parasites were first cross-linked for 20 min by adding glycolbis(succinimidylsuccinate) (EGS) to a final concentration of 1.5 mM. All samples (pre-cross-linked or not) were cross-linked for 10 min by adding methanol-free formaldehyde (Thermo Fisher # 28908) to 1% final concentration with gentle agitation. The cross-linking reaction was quenched by adding 2.5 M glycine to a final concentration of 0.125 M and incubated at RT for another 5 min. Parasites were centrifuged (3,250g, 5 min, 4°C), washed with DPBS, snap-frozen, and stored at −80°C until further use. Yeast chromatin preparation A culture of Saccharomyces cerevisiae was grown overnight in yeast peptide dextrose media (Bacteriological peptone 20 g/L, Glucose 20 g/L, Yeast extract 10 g/L) at 30°C with agitation (300 rpm) to reach the stationary phase. The optical density (OD) was measured and the culture was diluted to an OD of 0.4. Cells were collected during the exponential phase at OD = 0.8 by centrifugation (5 min, 30°C, 2,500 g ) and crosslinking was performed by resuspending the cells in 2% formaldehyde and incubating at 25°C for 5 min with rotation. 3 M Tris was added to a final concentration of 1.5 M to quench the crosslinking reaction, and the mixture was rotated at 25°C for 1 min. After centrifugation (3 min, 4°C, 2,500 g ), the pellets were washed once with ice-cold PBS, and once with ddH 2 O. After centrifugation (20 sec, 4°C, 3,300 g ), the supernatant was removed and the pellets were snap-frozen and stored at -80 °C. Cell pellets were thawed on ice and resuspended in 1X yeast ChIP lysis buffer (50 mM Hepes-KOH pH 8, 140 mM NaCl, 1mM EDTA, 1% Triton X100, 0.1% sodium deoxycholate, 1mM PMSF and 1X protease inhibitors). The cell walls were disrupted using a Beads Beater with zirconium beads and samples were recovered by centrifugation. The chromatin was sheared by sonication (8 cycles ‘30sec ON / 30 sec OFF’ in a Diagenode Pico BioRuptor) and centrifuged (20 min, 21,130 g , 4°C). The supernatant was transferred to a new tube, aliquoted and stored at -80°C. Yeast chromatin was mixed with P. falciparum chromatin as described below. Immunoprecipitation Each sample was resuspended in 2 ml nuclear extraction buffer (10 mM HEPES pH 8, 10 mM KCl, 0.1 mM EDTA pH 8, and complete protease inhibitor (PI, Roche no. 11836170001). IGEPAL CA-630 was added to a final concentration of 0.25% and the cells were lysed with a prechilled douncer homogenizer (200 strokes). The nuclei were pelleted by centrifugation (13,500g, 10 min, 4°C), and the supernatant (cytoplasmic fraction) was removed while the pellet was resuspended in 1.8 mL of cold SDS Lysis buffer (50 mM Tris– HCl pH 8, 10 mM EDTA pH 8, 1% SDS, PI). Chromatin was sheared to ∼250 bp fragments by 10 sonication cycles (once cycle: 30s ON / 30s OFF) using a Diagenode Pico Bioruptor. Insoluble materials were removed by centrifugation (10 min at 13,500g at 4°C) and DNA concentration was measured by Qubit. For the wild-type and Sir2a-KO histone-PTM ChIP-seq experiments, P. falciparum and yeast chromatin was mixed at the following ratios ( P. falciparum:yeast ): H4K16ac: 2.5:1; H3K9ac, H3K14ac, H4K12ac: 1:1. 100ng of DNA was stored as input sample at -20°C for later processing (see below). For each immunoprecipitation, 25 μl of Protein G Dynabeads (Invitrogen 10004D) were pre-washed twice with 1 ml ChIP dilution buffer (16.7 mM Tris–HCl pH 8, 150 mM NaCl, 1.2 mM EDTA pH 8, 1% Triton X-100, 0.01% SDS) and resuspended in 1 ml ChIP dilution buffer. For HMGB1-HA and HMGB2-HA samples, 1 μg of anti-HA (Abcam # ab9110) was added to the beads and incubated at 4°C for 2h with rotation. For the wild-type and Sir2a-KO samples, 1 μg of either anti-H3K9ac (Merck # 07-532), anti-H3K14ac (Abcam # ab52946), anti-H4K12ac (Active Motif # 39066) or anti-H4K16ac (Merck # 07-329) was added and incubated at 4°C for 2h with rotation. The beads were washed twice with 1 ml ChIP dilution buffer and 1μg of chromatin lysate was diluted 10-fold in ChIP dilution buffer, added to the bead-antibody complexes and incubated at 4°C overnight with constant rotation. Following immunoprecipitation, the beads were washed sequentially in 1 ml of low salt wash buffer (20 mM Tris–HCl pH 8, 150 mM NaCl, 2 mM EDTA pH 8, 1% Triton X-100, 0.1% SDS), High salt wash buffer (20 mM Tris–HCl pH 8, 500 mM NaCl, 2 mM EDTA pH 8, 1% Triton X-100, 0.1% SDS), LiCl wash buffer (10 mM Tris–HCl pH 8, 250 mM LiCl, 1 mM EDTA pH 8, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA pH 8). For each wash, the beads were rotated for 5 min at 4°C except for the last TE wash, which was performed at room temperature (RT). The beads were then resuspended in 200 μl of elution buffer (50 mM Tris–HCl pH 8 10 mM EDTA pH 8.0, 1% SDS) and protein-DNA complexes were eluted by 30 min incubation at 65°C. The beads were collected on a magnetic rack and the supernatant transferred to a new tube. DNA purification and library preparation The immunoprecipitated DNA and the input DNA were reverse-crosslinked by incubating the samples at 65°C for 10h. 200 μl of TE buffer were added and the samples were treated with RNase A (0.2 mg/mL final concentration) for 2h at 37°C, followed by a 2 h incubation at 55°C with proteinase K (0.2 mg/mL final concentration). DNA was purified by adding 400 μl phenol:chloroform:isoamyl alcohol, and phases were separated by centrifugation (13,500g, 10 min, 4°C) after vigorous vortexing. 16 μl of 5 M NaCl (200 mM final concentration) and 30 μg glycogen were added to the aqueous phase, and DNA was precipitated by adding 800 μl 100% EtOH 4°C and incubating for 30 min at −20°C. DNA was pelleted by centrifugation (20,000g, 10 min, 4°C) and washed with 500 μl 80% EtOH at 4°C. After centrifugation, the DNA pellet was air-dried and resuspended in 30 μl of 10 mM Tris-HCl pH 8.0. Libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit (NEB E7645S) and sequenced (150 bp paired end) on the NextSeq 500 platform (Illumina). ChIP-seq data processing and analysis Raw read pre-processing and adapter trimming were performed using bcl2fastq and trimmomatic 72 as described above for RNA-seq samples. Trimmed reads were aligned to the masked genome (see above) using bowtie2 78 with settings ‘--no-mixed --no-discordant -- end-to-end --sensitive’. Alignments were subsequently filtered for PCR duplicates using samtools 79 ‘fixmate’ and ‘markdup’ and only alignments with a mapping quality ≥ 30 were retained (samtools view -q 30). To calculate differences in HMGB1 occupancy at rDNA loci at 32°C and 37°C, IP/input enrichments were first calculated for both conditions and replicates independently using macs2 80 ‘callpeak’ with options ‘--nomodel --extsize 120’. The resulting tag numbers, pileup and lambda files for the IP and input samples of each replicate and condition were then used to calculate significant enrichment differences using macs2 ‘bdgdiff ‘. For all ChIP-seq experiments, coverage tracks representing the ratio of IP over Input were generated using deeptool’s 81 ‘bamCompare’ with options ‘-bs 10 --scaleFactorsMethod None -- operation ratio --normalizeUsing CPM’. IP/input coverage tracks were visualized using the Integrative Genomics Viewer 82 . For the analysis of histone PTMs in the wild-type 3D7 and Sir2a-KO parasites, raw sequencing reads were pre-processed as described above. The reads were then mapped to the masked P. falciparum genome (PlasmoDB v64 70 ) using tinymapper ( https://github.com/js2264/tinyMapper ), with option ‘--mode ChIP’ and using the S. cerevisiae S288C genome (version R64) as calibration reference. Only correctly paired reads with a mapping quality >= 30 were retained (bowtie2 option ‘-f 0x001 -f 0x002’ -q 30) for downstream analysis within tinymapper. Significant peaks for each replicate and histone PTM were identified within tinymapper using macs2 and by using the input sample as background. To calculate quantitative enrichment values for each PTM, for each sample and replicate, the number of ChIP reads mapping to the P. falciparum genome (in counts per million [CPM]) was normalized to the total number of ChIP reads mapping to the S. cerevisiae calibration genome (Table S9). Overlapping consensus peaks between replicates were identified using bedtool’s ‘intersect’ 71 (Table S9). For the comparison of 6mA densities ( Figure 3F ), first the absolute fold-change at the peak summit was calculated using the calibrated enrichment values for all significant peaks identified in the Sir2a-KO samples (i.e. calibrated enrichment Sir2a-KO/ calibrated enrichment wild-type 3D7 ). For the 100 peaks showing the highest and lowest change in peak enrichment, 6mA densities identified by Sir2a-madID (see below) were calculated within the peak region. Of note, even peaks with the lowest change of absolute peak enrichment feature a fold-change of Sir2a-KO/wild-type 3D7 > 1 (Table S9). The raw ChIP and input fastq files for HP1 20 were retrieved from NCBI SRA (ChIP: SRR12281320; Input: SRR12281322) and processed as described above. Bedgraph files for ATAC (assay for transposable accessible chromatin) sequencing data corresponding to 35 h.p.i. were retrieved from NCBI GEO (accession: GSM2789027) and visualized using the Integrative Genomics Viewer 82 . Micro-C analysis Post-processed, normalized matrices for the 36 h.p.i. timepoint were retrieved from Singh et al. 22 . Importantly, only alignments with a mapping quality (MAPQ) ≥ 30 were retained during the generation of interaction matrices. This ensures that reads that map equally well to either A-type or S2/3-type locus are removed and otherwise would inflate the interaction frequencies between these loci. Intra- and interchromosomal interactions ( Figure 1G , S1E) were plotted using hicExplorer’s ‘hicPlotmatrix’ 83 at 5 kb resolution, except intrachromosomal interactions of Pf3D7_01_v3 which were plotted at 2 kb resolution. Intrachromosomal, log 2 -scaled observed/expected interaction frequency ratios ( Figure S1E ) were visualized using juicebox 84 . Annotation of PolI factors For the annotation of Polymerase I-related factors in P. falciparum , proteins in human, Arabidopsis and yeast with GO term annotations related to PolI activity were retrieved from QuickGO 85 (Table S2). Corresponding protein sequences were searched using BLASTp against the P. falciparum proteome (PlasmoDB, v64 70 ) with options ‘-evalue 1e-3 - max_target_seqs 2’ and only hits with a SwissProt annotation was retained. The identified P. falciparum proteins were then used in a reciprocal BLASTp 86 search against the model organism protein sequences and only the best hit was kept (option ‘-max_target_seqs 1’) (Table S2). Metabolomics Sample collection Asexual blood-stage P. falciparum parasites (wild-type NF 54 , LDH-glmS and Nico-glmS cell lines) were tightly synchronized by plasmion-sorbitol in a 6h window, iRBCs were concentrated by plasmagel flotation at 33 h.p.i., resuspended at 2.5x10 7 cells/mL in cell culture media and incubated in 12-well plates before collection at 40hpi. For LDH_GlmS, glucosamine (2.5 mM final concentration) was added at 16 h.p.i. to half of the culture, to obtain a total of 24h of glucosamine treatment. For Nico-glmS, glucosamine (2.5 mM final concentration) was added at 6h before synchronization to half of the culture, to obtain a total of 48h of glucosamine treatment. For the 32°C vs 37°C temperature experiment, half of the culture was put at 32°C after the plasmion at 33 h.p.i. Due to slower growth, the cells incubated at 32°C were collected 2h later, at 42 h.p.i. ( Figure S3G ). For each condition, 6-10 replicates of 5x10 ^7 cells were collected: the cells were transferred to a 2 ml tube, centrifuged at 14,000 g for 30 sec at 37°C, washed with 1 ml of ice-cold DPBS, centrifuged at 14,000 g for 30 sec at 4°C and the pellet was snap-frozen in liquid nitrogen and stored at -80°C. LC-MS determination of energy carrier metabolites Frozen cell pellets were processed following an adjusted protocol targeting energy carriers such as NAD/NADH and NADP/NADPH. This method was extended by including polarity switching and additional metabolites of interest. Briefly, cell pellets were extracted on ice using 250□µl cooled extraction buffer (Acetonitrile: MeOH : 15 mM ammonium acetate in H 2 O (3:1:1), pH 10). Subsequently, samples were sonicated to ensure complete disruption of all cells using a sonication bath (Transsonic 460, Elma) for 5Lmin at the highest frequency on ice. Afterwards, samples were centrifuged for 15Lmin at 4°C and 13,000g, and the resulting supernatant was transferred to a new LC-MS grade autosampler vial and immediately frozen at -80°C if instrument was not directly available for measuring. For metabolite separation and detection, an ACQUITY I-class PLUS UPLC system (Waters) coupled to a QTRAP 6500+ (AB SCIEX) mass spectrometer with electrospray ionization (ESI) source was used. In detail, metabolites were separated on an ACQUITY Premier BEH Amide Vanguard Fit column (100LmmL×L2.1Lmm, 1.7□µm, Waters) with constant column temperature of 35°C. Separation of NAD/NADH, NADP/NADPH and additional energy carriers was achieved using mobile phase A (50/50 ACN/water with 5 mM ammonium acetate and 0.04% ammonium hydroxide; pH 10) and mobile phase B (90/10 ACN/water with 5 mM ammonium acetate and 0.04% ammonium hydroxide), following a gradient of the A/B phase ratio (0.5 min 5%/95% at 0.4 ml/min, 4.5 min 90%/10% at 0.35 ml/min, 5 min 100%/0% at 0.3 ml/min and 5min 5%/95% at 0.4 ml/min). Data acquisition was performed using Analyst 1.7.2 (AB SCIEX) and processed using the OS software suite 2.0.0 (AB SCIEX). Areas under the curve (auc) for each metabolite were normalized to 1x10 6 cells and NAD/NAM ratios were calculated for each replicate. To estimate relative changes of NAD/NAM, all replicates were normalized to the average of the wild-type/untreated sample. Normal distribution of the data were estimated in R using functions shapiro.test() and p-values were computed using a unpaired Welch Two Sample t-test using function t.test(paired = F). ONT sampling and library preparation for HMGB1 integration check Late stage HMGB1-HA parasites were collected by saponin lysis (0.075% saponin in DPBS) at 37°C. The parasite cell pellet was washed twice with ice-cold DPBS, snap-frozen and stored at -80°C. gDNA was extracted using the Qiagen DNeasy Blood & Tissue Kit (Qiagen # 69504). The gDNA was further purified by phenol/chloroform extraction and ethanol precipitation. Briefly, 200 µl of Phenol/Chloroform/Isoamyl Alcohol (25:24:1) was added to 200 µl of gDNA in elution buffer. The samples were vortexed and centrifuged (4°C, 21,000 g , 5 min). 180 µl of the upper phase was transferred to a new tube and an equal volume of Phenol/Chloroform/Isoamyl Alcohol was added to repeat the extraction. After agitation and centrifugation, the upper phase was transferred to a new tube and 1/10 volume of 3 M sodium acetate was added, together with 1 µl of 10 mg/ml glycogen and two volumes of 100% ethanol. The mixture was centrifuged (4°C, 21,000 g , 20 min) and the resulting DNA pellet was washed with 80% ethanol, air-dried and resuspended in ddH2O. Libraries were prepared using the SQK-RAD004 kit and sequencing was performed using a FLO-MIN106 flow cell on a MinION sequencer. Real-time basecalling was performed using the fast model in Guppy (v 6.5.7) implemented in MinKNOW (v23.04.5). The expected pSLI plasmid integration was manually integrated at the HMGB1 locus of the reference P. falciparum (PlasmoDB v64 70 ) genome and annotation. Raw fastq reads were mapped to the modified genome sequence using minimap2 87 with option ‘-x map-ont’ and the resulting alignments visualized using the Integrative Genomics Viewer 82 . ONT sampling and library preparation for madID For NLS-madID, cells were synchronized by plasmion/sorbitol to a 6h window. To dimerize and activate the Cre recombinase, rapamacin was added (200 nM final concentration) after the sorbitol, and cells were collected at 40 h.p.i. Successful excision of the yDHODH locus was confirmed by PCR ( Figure S6H ). For Sir2a-madID, the cell line was synchronized by plasmion/sorbitol to a 6h window and collected at 40 h.p.i. Red blood cells were lysed with 0.075% saponin in DPBS at 37°C. The parasite cell pellet was washed twice with ice-cold DPBS, snap-frozen and stored at -80°C. gDNA was extracted using the Qiagen DNeasy Blood & Tissue Kit (Qiagen # 69504). gDNA was purified by treatment with RNase A (0.2 mg/mL final concentration) for 2h at 37°C, followed by a 2 h incubation at 55°C with proteinase K (0.2 mg/mL final concentration). The obtained gDNA was further purified by phenol/chloroform extraction and ethanol precipitation. Libraries were prepared using the native barcoding SQK-NBD114.96 kit using the recommendations for long gDNA fragments. Sequencing was performed using a MinION flow cell (R10.4.1) on a MinION sequencer. Identification of madID-modified DNA sites Raw pod5 files were merged using pod5 ‘merge’ ( https://github.com/nanoporetech/pod5-file-format ). Basecalling and mapping to the unmasked reference genome of P. falciparum was performed in a single step using dorado basecaller (v0.8.2, https://github.com/nanoporetech/dorado ) and minimap2 87 , respectively, using options ‘--emit-moves --emit-sam --kit-name SQK-NBD114-96 --reference PlasmoDB-64_Pfalciparum3D7_Genome.fasta [email protected] ’. Model ‘ [email protected] ’ was used for base calling. Individual samples were demultiplexed using dorado ‘demux’ (v0.8.2) and alignments with a MAPQ score >= 60 were filtered using samtoos ‘view’ (option -q 60). For each sample (i.e. gDNA, NLS-madID and Sir2a-madID), raw signal alignments were performed using uncalled4 88 ‘align’ with model ‘dna_r10.4.1_400bps_9mer’. For each genomic position and sample, the normalized mean read signal current was calculated using uncalled4 88 ‘refstats’ with options ‘--stats mean --layers dtw.current’. Genomic positions with a normalized mean read signal current difference >= 2 between NLS-madID vs gDNA (i.e. ‘madID’, Figure 3 ) and Sir2a-madID vs gDNA (i.e. ‘Sir2a-madID’, Figure 3 ) were retained as putatively modified sites. For genome wide visualizations, the number of modified sites was normalized to the AT content of each bin ( Figure 3C,G ,H, S7A,G). For the comparison of modified sites in heterochromatic and euchromatic regions ( Figure 3D ), the total number of modified sites was normalized to the length of each region (i.e. number of modified sites per kilobase). Location of heterochromatic and euchromatic regions was retrieved from Singh et al. 22 . P-values for the comparison of 6mA densities in euchromatic and heterochromatic regions ( Figure 3D ) and between peak regions ( Figure 3F ) were computed in R using a Wilcoxon Rank Sum Test with function wilcoxon.test(paired = F). Dot blot The gDNA of NLS-madID (with and without rapamycin treatment) and Sir2a-madID was extracted as described above and denatured for 5 minutes at 65°C. Serial dilutions were performed to blot 2.5μl of sample corresponding to 100 ng, 10 ng and 1 ng of gDNA on a nylon membrane (Biodyne # 77016), and the membrane was air-dried for 10 min. The membrane was UV-crosslinked (125 mJ/cm 2 at 254 nM for 1min) and blocked for 1h in 5% milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween20, pH 7.5). The membrane was then incubated with anti-6mA antibody (Abcam # ab151230) diluted at 1/1,000 in blocking solution for 2h at RT, washed three times with TBST for 5 min and incubated with HRP anti-rabbit (Sigma # NA934) diluted at 1/2,500 in blocking solution for 1h at RT. The membrane was washed three times with TBST for 5 min, developed using the SuperSignal West Pico chemiluminescent substrate (Thermo Fisher # 34580) and imaged with a ChemiDoc XRS+ (Bio-Rad). Acknowledgements We thank the Metabolomics Core Technology Platform of the Heidelberg University supported by the CellNetworks Core Technology Platform CCTP for metabolite analyses. We thank Christophe Chapard for providing help with yeast culturing and collection, Julien Guizetti for sharing plasmids for madID cloning and Patrick Poullet for the continuous development of myProMS used for proteomics analyses. Work in the laboratory of S.B. is supported by an ERC Starting Grant (# 947819) and baseline funding of the Institut Pasteur. J.M.B. is supported by ANR ANR-21-CE15-0002-02 ApiMORCing and ANR-21-CE15-0010-01 PlasmoVarOrg. REFERENCES 1. ↵ Genuth , N.R. , and Barna , M . ( 2018 ). The Discovery of Ribosome Heterogeneity and Its Implications for Gene Regulation and Organismal Life . Mol Cell 71 , 364 – 374 . doi: 10.1016/j.molcel.2018.07.018 . OpenUrl CrossRef PubMed 2. ↵ Sas-Chen , A. , Thomas , J.M. , Matzov , D. , Taoka , M. , Nance , K.D. , Nir , R. , Bryson , K.M. , Shachar , R. , Liman , G.L.S. , Burkhart , B.W. , et al. ( 2020 ). Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping . Nature 583 , 638 – 643 . doi: 10.1038/s41586-020-2418-2 . OpenUrl CrossRef PubMed 3. ↵ Yang , Y.-M. , and Karbstein , K . ( 2022 ). The chaperone Tsr2 regulates Rps26 release and reincorporation from mature ribosomes to enable a reversible, ribosome-mediated response to stress . Sci Adv 8 , eabl4386 . doi: 10.1126/sciadv.abl4386 . OpenUrl CrossRef PubMed 4. ↵ Bozdech , Z. , Llinas , M. , Pulliam , B.L. , Wong , E.D. , Zhu , J. , and DeRisi , J.L . ( 2003 ). The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum . PLoS Biol 1 , 85 – 100 . doi: 10.1371/journal.pbio.0000005 . OpenUrl CrossRef Web of Science 5. Howick , V.M. , Russell , A.J.C. , Andrews , T. , Heaton , H. , Reid , A.J. , Natarajan , K. , Butungi , H. , Metcalf , T. , Verzier , L.H. , Rayner , J.C. , et al. ( 2019 ). The Malaria Cell Atlas: Single parasite transcriptomes across the complete Plasmodium life cycle . Science 365 . doi: 10.1126/science.aaw2619 . OpenUrl Abstract / FREE Full Text 6. ↵ Dogga , S.K. , Rop , J.C. , Cudini , J. , Farr , E. , Dara , A. , Ouologuem , D. , Djimdé , A.A. , Talman , A.M. , and Lawniczak , M.K.N . ( 2024 ). A single cell atlas of sexual development in Plasmodium falciparum . Science (1979) 384 . doi: 10.1126/science.adj4088 . OpenUrl CrossRef PubMed 7. ↵ Yan , Y. , Cheung , E. , Verzier , L.H. , Appetecchia , F. , March , S. , Craven , A.R. , Du , E. , Probst , A.S. , Rinvee , T.A. , de Vries , L.E. , et al. ( 2024 ). Mapping Plasmodium transitions and interactions in the Anopheles female . bioRxiv . doi: 10.1101/2024.11.12.623125 . OpenUrl Abstract / FREE Full Text 8. ↵ Cortés , A. , and Deitsch , K.W . ( 2017 ). Malaria Epigenetics . Cold Spring Harb Perspect Med 7 . doi: 10.1101/cshperspect.a025528 . OpenUrl Abstract / FREE Full Text 9. ↵ Waters , A.P. , Syin , C. , and McCutchan , T.F . ( 1989 ). Developmental regulation of stage-specific ribosome populations in Plasmodium . Nature 342 , 438 – 440 . doi: 10.1038/342438a0 . OpenUrl CrossRef PubMed Web of Science 10. ↵ Gardner , M.J. , Hall , N. , Fung , E. , White , O. , Berriman , M. , Hyman , R.W. , Carlton , J.M. , Pain , A. , Nelson , K.E. , Bowman , S. , et al. ( 2002 ). Genome sequence of the human malaria parasite Plasmodium falciparum . Nature 419 , 498 – 511 . doi: 10.1038/nature01097 . OpenUrl CrossRef PubMed Web of Science 11. ↵ Mancio-Silva , L. , Zhang , Q. , Scheidig-Benatar , C. , and Scherf , A . ( 2010 ). Clustering of dispersed ribosomal DNA and its role in gene regulation and chromosome-end associations in malaria parasites . Proc Natl Acad Sci U S A 107 , 15117 – 15122 . doi: 10.1073/pnas.1001045107 . OpenUrl Abstract / FREE Full Text 12. ↵ MacInnes , A.W . ( 2016 ). The role of the ribosome in the regulation of longevity and lifespan extension . Wiley Interdiscip Rev RNA 7 , 198 – 212 . doi: 10.1002/wrna.1325 . OpenUrl CrossRef PubMed 13. ↵ Shore , D. , and Albert , B . ( 2022 ). Ribosome biogenesis and the cellular energy economy . Curr Biol 32 , R611 – R617 . doi: 10.1016/j.cub.2022.04.083 . OpenUrl CrossRef PubMed 14. ↵ Murayama , A. , Ohmori , K. , Fujimura , A. , Minami , H. , Yasuzawa-Tanaka , K. , Kuroda , T. , Oie , S. , Daitoku , H. , Okuwaki , M. , Nagata , K. , et al. ( 2008 ). Epigenetic control of rDNA loci in response to intracellular energy status . Cell 133 , 627 – 639 . doi: 10.1016/j.cell.2008.03.030 . OpenUrl CrossRef PubMed Web of Science 15. ↵ Fang , J. , Sullivan , M. , and McCutchan , T.F . ( 2004 ). The effects of glucose concentration on the reciprocal regulation of rRNA promoters in Plasmodium falciparum . J Biol Chem 279 , 720 – 725 . doi: 10.1074/jbc.M308284200 . OpenUrl Abstract / FREE Full Text 16. ↵ Sharma , I. , Fang , J. , Lewallen , E.A. , Deitsch , K.W. , and McCutchan , T.F . ( 2023 ). Identification of a long noncoding RNA required for temperature induced expression of stage-specific rRNA in malaria parasites . Gene 877 , 147516 . doi: 10.1016/j.gene.2023.147516 . OpenUrl CrossRef PubMed 17. ↵ Mancio-Silva , L. , Lopez-Rubio , J.J. , Claes , A. , and Scherf , A . ( 2013 ). Sir2a regulates rDNA transcription and multiplication rate in the human malaria parasite Plasmodium falciparum . Nat Commun 4 , 1530 . doi: 10.1038/ncomms2539 . OpenUrl CrossRef PubMed 18. ↵ Salcedo-Amaya , A.M. , van Driel , M.A. , Alako , B.T. , Trelle , M.B. , van den Elzen , A.M.G. , Cohen , A.M. , Janssen-Megens , E.M. , van de Vegte-Bolmer , M. , Selzer , R.R. , Iniguez , A.L. , et al. ( 2009 ). Dynamic histone H3 epigenome marking during the intraerythrocytic cycle of Plasmodium falciparum . Proc Natl Acad Sci U S A 106 , 9655 – 9660 . doi: 10.1073/pnas.0902515106 . OpenUrl Abstract / FREE Full Text 19. ↵ Santoro , R. , Li , J. , and Grummt , I . ( 2002 ). The nucleolar remodeling complex NoRC mediates heterochromatin formation and silencing of ribosomal gene transcription . Nat Genet 32 , 393 – 396 . doi: 10.1038/ng1010 . OpenUrl CrossRef PubMed Web of Science 20. ↵ Carrington , E. , Cooijmans , R.H.M. , Keller , D. , Toenhake , C.G. , Bártfai , R. , and Voss , T.S . ( 2021 ). The ApiAP2 factor PfAP2-HC is an integral component of heterochromatin in the malaria parasite Plasmodium falciparum . iScience 24 . doi: 10.1016/j.isci.2021.102444 . OpenUrl CrossRef PubMed 21. ↵ Toenhake , C.G. , Fraschka , S.A.-K. , Vijayabaskar , M.S. , Westhead , D.R. , van Heeringen , S.J. , and Bártfai , R. ( 2018 ). Chromatin Accessibility-Based Characterization of the Gene Regulatory Network Underlying Plasmodium falciparum Blood-Stage Development . Cell Host Microbe 23 , 557 – 569 .e9. doi: 10.1016/j.chom.2018.03.007 . OpenUrl CrossRef PubMed 22. ↵ Singh , P. , Serizay , J. , Couble , J. , Cabahug , M.D. , Rosa , C. , Chen , P. , Scherf , A. , Koszul , R. , Baumgarten , S. , and Bryant , J.M . ( 2024 ). Micro-C reveals MORC/ApiAP2-mediated links between distant, functionally related genes in the human malaria parasite . Preprint , doi: 10.1101/2024.08.28.610079 http://10.1101/2024.08.28.610079 . OpenUrl Abstract / FREE Full Text 23. ↵ Kwon , H. , and Green , M.R . ( 1994 ). The RNA polymerase I transcription factor, upstream binding factor, interacts directly with the TATA box-binding protein . J Biol Chem 269 , 30140 – 30146 . OpenUrl Abstract / FREE Full Text 24. ↵ O’Hara , J.K. , Kerwin , L.J. , Cobbold , S.A. , Tai , J. , Bedell , T.A. , Reider , P.J. , and Llinás , M . ( 2014 ). Targeting NAD+ metabolism in the human malaria parasite Plasmodium falciparum . PLoS One 9 . doi: 10.1371/journal.pone.0094061 . OpenUrl CrossRef PubMed 25. ↵ Merrick , C.J. , and Duraisingh , M.T . ( 2007 ). Plasmodium falciparum Sir2: an unusual sirtuin with dual histone deacetylase and ADP-ribosyltransferase activity . Eukaryot Cell 6 , 2081 – 2091 . doi: 10.1128/EC.00114-07 . OpenUrl Abstract / FREE Full Text 26. ↵ Prommana , P. , Uthaipibull , C. , Wongsombat , C. , Kamchonwongpaisan , S. , Yuthavong , Y. , Knuepfer , E. , Holder , A.A. , and Shaw , P.J . ( 2013 ). Inducible knockdown of Plasmodium gene expression using the glmS ribozyme . PLoS One 8 , e73783 . doi: 10.1371/journal.pone.0073783 . OpenUrl CrossRef PubMed 27. ↵ Olszewski , K.L. , and Llinás , M . ( 2011 ). Central carbon metabolism of Plasmodium parasites . Mol Biochem Parasitol 175 , 95 – 103 . doi: 10.1016/j.molbiopara.2010.09.001 . OpenUrl CrossRef PubMed 28. ↵ Painter , H.J. , Morrisey , J.M. , Mather , M.W. , and Vaidya , A.B . ( 2007 ). Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum . Nature 446 , 88 – 91 . doi: 10.1038/nature05572 . OpenUrl CrossRef PubMed Web of Science 29. ↵ Ke , H. , Lewis , I.A. , Morrisey , J.M. , McLean , K.J. , Ganesan , S.M. , Painter , H.J. , Mather , M.W. , Jacobs-Lorena , M. , Llinás , M. , and Vaidya , A.B . ( 2015 ). Genetic investigation of tricarboxylic acid metabolism during the Plasmodium falciparum life cycle . Cell Rep 11 , 164 – 174 . doi: 10.1016/j.celrep.2015.03.011 . OpenUrl CrossRef PubMed 30. ↵ Sparkes , P.C. , Famodimu , M.T. , Alves , E. , Springer , E. , Przyborski , J. , and Delves , M.J . ( 2024 ). Mitochondrial ATP synthesis is essential for efficient gametogenesis in Plasmodium falciparum . Commun Biol 7 , 1525 . doi: 10.1038/s42003-024-07240-z . OpenUrl CrossRef PubMed 31. ↵ Evers , F. , Cabrera-Orefice , A. , Elurbe , D.M. , Kea-te Lindert , M. , Boltryk , S.D. , Voss , T.S. , Huynen , M.A. , Brandt , U. , and Kooij , T.W.A . ( 2021 ). Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum . Nat Commun 12 , 3820 . doi: 10.1038/s41467-021-23919-x . OpenUrl CrossRef PubMed 32. ↵ Evers , F. , Roverts , R. , Boshoven , C. , Kea-Te Lindert , M. , Verhoef , J.M.J. , Sommerdijk , N. , Sinden , R.E. , Akiva , A. , and Kooij , T.W.A . ( 2025 ). Comparative 3D ultrastructure of Plasmodium falciparum gametocytes . Nat Commun 16 , 69 . doi: 10.1038/s41467-024-55413-5 . OpenUrl CrossRef PubMed 33. ↵ Luengo , A. , Li , Z. , Gui , D.Y. , Sullivan , L.B. , Zagorulya , M. , Do , B.T. , Ferreira , R. , Naamati , A. , Ali , A. , Lewis , C.A. , et al. ( 2021 ). Increased demand for NAD+ relative to ATP drives aerobic glycolysis . Mol Cell 81 , 691 – 707 .e6. doi: 10.1016/j.molcel.2020.12.012 . OpenUrl CrossRef PubMed 34. ↵ Covarrubias , A.J. , Perrone , R. , Grozio , A. , and Verdin , E . ( 2021 ). NAD+ metabolism and its roles in cellular processes during ageing . Nat Rev Mol Cell Biol 22 , 119 – 141 . doi: 10.1038/s41580-020-00313-x . OpenUrl CrossRef PubMed 35. ↵ Imai , S. , Armstrong , C.M. , Kaeberlein , M. , and Guarente , L . ( 2000 ). Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase . Nature 403 , 795 – 800 . doi: 10.1038/35001622 . OpenUrl CrossRef PubMed Web of Science 36. ↵ Duraisingh , M.T. , Voss , T.S. , Marty , A.J. , Duffy , M.F. , Good , R.T. , Thompson , J.K. , Freitas , L.H. , Scherf , A. , Crabb , B.S. , and Cowman , A.F . ( 2005 ). Heterochromatin silencing and locus repositioning linked to regulation of virulence genes in Plasmodium falciparum . Cell 121 , 13 – 24 . doi: 10.1016/j.cell.2005.01.036 . OpenUrl CrossRef PubMed Web of Science 37. Tonkin , C.J. , Carret , C.K. , Duraisingh , M.T. , Voss , T.S. , Ralph , S.A. , Hommel , M. , Duffy , M.F. , Silva , L.M. da , Scherf , A. , Ivens , A. , et al. ( 2009 ). Sir2 paralogues cooperate to regulate virulence genes and antigenic variation in Plasmodium falciparum . PLoS Biol 7 , e84 . doi: 10.1371/journal.pbio.1000084 . OpenUrl CrossRef PubMed 38. ↵ Freitas-Junior , L.H. , Hernandez-Rivas , R. , Ralph , S.A. , Montiel-Condado , D. , Ruvalcaba-Salazar , O.K. , Rojas-Meza , A.P. , Mâncio-Silva , L. , Leal-Silvestre , R.J. , Gontijo , A.M. , Shorte , S. , et al. ( 2005 ). Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites . Cell 121 , 25 – 36 . doi: 10.1016/j.cell.2005.01.037 . OpenUrl CrossRef PubMed Web of Science 39. ↵ Duraisingh , M.T. , Voss , T.S. , Marty , A.J. , Duffy , M.F. , Good , R.T. , Thompson , J.K. , Freitas-Junior , L.H. , Scherf , A. , Crabb , B.S. , and Cowman , A.F . ( 2005 ). Heterochromatin silencing and locus repositioning linked to regulation of virulence genes in Plasmodium falciparum . Cell 121 , 13 – 24 . doi: 10.1016/j.cell.2005.01.036 . OpenUrl CrossRef PubMed Web of Science 40. ↵ Sobecki , M. , Souaid , C. , Boulay , J. , Guerineau , V. , Noordermeer , D. , and Crabbe , L . ( 2018 ). MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells . Cell Rep 25 , 2891 – 2903 .e5. doi: 10.1016/j.celrep.2018.11.027 . OpenUrl CrossRef PubMed 41. ↵ Vaquero , A. , Scher , M. , Erdjument-Bromage , H. , Tempst , P. , Serrano , L. , and Reinberg , D . ( 2007 ). SIRT1 regulates the histone methyl-transferase SUV39H1 during heterochromatin formation . Nature 450 , 440 – 444 . doi: 10.1038/nature06268 . OpenUrl CrossRef PubMed Web of Science 42. ↵ Bryant , J.M. , Regnault , C. , Scheidig-Benatar , C. , Baumgarten , S. , Guizetti , J. , and Scherf , A . ( 2017 ). CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum . mBio 8 . doi: 10.1128/mBio.00729-17 . OpenUrl Abstract / FREE Full Text 43. Zhang , Q. , Huang , Y. , Zhang , Y. , Fang , X. , Claes , A. , Duchateau , M. , Namane , A. , Lopez-Rubio , J.-J. , Pan , W. , and Scherf , A . ( 2011 ). A critical role of perinuclear filamentous actin in spatial repositioning and mutually exclusive expression of virulence genes in malaria parasites . Cell Host Microbe 10 , 451 – 463 . doi: 10.1016/j.chom.2011.09.013 . OpenUrl CrossRef PubMed Web of Science 44. ↵ Amit-Avraham , I. , Pozner , G. , Eshar , S. , Fastman , Y. , Kolevzon , N. , Yavin , E. , and Dzikowski , R . ( 2015 ). Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum . Proceedings of the National Academy of Sciences 112 . doi: 10.1073/pnas.1420855112 . OpenUrl Abstract / FREE Full Text 45. ↵ Cavero , S. , Herruzo , E. , Ontoso , D. , and San-Segundo , P . ( 2016 ). Impact of histone H4K16 acetylation on the meiotic recombination checkpoint in Saccharomyces cerevisiae . Microbial Cell 3 , 606 – 620 . doi: 10.15698/mic2016.12.548 . OpenUrl CrossRef PubMed 46. Vaquero , A. , Scher , M.B. , Lee , D.H. , Sutton , A. , Cheng , H.-L. , Alt , F.W. , Serrano , L. , Sternglanz , R. , and Reinberg , D . ( 2006 ). SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis . Genes Dev 20 , 1256 – 1261 . doi: 10.1101/gad.1412706 . OpenUrl Abstract / FREE Full Text 47. ↵ Dang , W. , Steffen , K.K. , Perry , R. , Dorsey , J.A. , Johnson , F.B. , Shilatifard , A. , Kaeberlein , M. , Kennedy , B.K. , and Berger , S.L . ( 2009 ). Histone H4 lysine 16 acetylation regulates cellular lifespan . Nature 459 , 802 – 807 . doi: 10.1038/nature08085 . OpenUrl CrossRef PubMed Web of Science 48. ↵ Orlando , D.A. , Chen , M.W. , Brown , V.E. , Solanki , S. , Choi , Y.J. , Olson , E.R. , Fritz , C.C. , Bradner , J.E. , and Guenther , M.G . ( 2014 ). Quantitative ChIP-Seq normalization reveals global modulation of the epigenome . Cell Rep 9 , 1163 – 1170 . doi: 10.1016/j.celrep.2014.10.018 . OpenUrl CrossRef PubMed 49. ↵ Singhal , R. , Prata , I.O. , Bonnell , V.A. , and Llinás , M . ( 2024 ). Unraveling the complexities of ApiAP2 regulation in Plasmodium falciparum . Trends Parasitol 40 , 987 – 999 . doi: 10.1016/j.pt.2024.09.007 . OpenUrl CrossRef PubMed 50. ↵ Matthews , K.A. , Senagbe , K.M. , Nötzel , C. , Gonzales , C.A. , Tong , X. , Rijo-Ferreira , F. , Bhanu , N. V. , Miguel-Blanco , C. , Lafuente-Monasterio , M.J. , Garcia , B.A. , et al. ( 2020 ). Disruption of the Plasmodium falciparum Life Cycle through Transcriptional Reprogramming by Inhibitors of Jumonji Demethylases . ACS Infect Dis 6 , 1058 – 1075 . doi: 10.1021/acsinfecdis.9b00455 . OpenUrl CrossRef PubMed 51. ↵ Cascella , B. , and Mirica , L.M . ( 2012 ). Kinetic Analysis of Iron-Dependent Histone Demethylases: α-Ketoglutarate Substrate Inhibition and Potential Relevance to the Regulation of Histone Demethylation in Cancer Cells . Biochemistry 51 , 8699 – 8701 . doi: 10.1021/bi3012466 . OpenUrl CrossRef PubMed 52. ↵ Allman , E.L. , Painter , H.J. , Samra , J. , Carrasquilla , M. , and Llinás , M . ( 2016 ). Metabolomic Profiling of the Malaria Box Reveals Antimalarial Target Pathways . Antimicrob Agents Chemother 60 , 6635 – 6649 . doi: 10.1128/AAC.01224-16 . OpenUrl Abstract / FREE Full Text 53. ↵ MacRae , J.I. , Dixon , M.W. , Dearnley , M.K. , Chua , H.H. , Chambers , J.M. , Kenny , S. , Bottova , I. , Tilley , L. , and McConville , M.J . ( 2013 ). Mitochondrial metabolism of sexual and asexual blood stages of the malaria parasite Plasmodium falciparum . BMC Biol 11 , 67 . doi: 10.1186/1741-7007-11-67 . OpenUrl CrossRef PubMed 54. ↵ Shi , Y. , Wan , L. , Jiao , M. , Zhong , C. , Cui , H. , and Yuan , J . ( 2024 ). Elevated NAD + drives Sir2A-mediated GCβ deacetylation and OES localization for Plasmodium ookinete gliding and mosquito infection . Preprint , doi: 10.1101/2024.10.03.616407 http://10.1101/2024.10.03.616407 . OpenUrl Abstract / FREE Full Text 55. ↵ Mohammed , M. , Dziedziech , A. , Sekar , V. , Ernest , M. , Alves E Silva , T.L. , Balan , B. , Emami , S.N. , Biryukova , I. , Friedländer , M.R. , Jex , A. , et al. ( 2023 ). Single-Cell Transcriptomics To Define Plasmodium falciparum Stage Transition in the Mosquito Midgut . Microbiol Spectr 11 , e0367122 . doi: 10.1128/spectrum.03671-22 . OpenUrl CrossRef 56. ↵ Stefanovsky , V.Y. , Pelletier , G. , Hannan , R. , Gagnon-Kugler , T. , Rothblum , L.I. , and Moss , T . ( 2001 ). An immediate response of ribosomal transcription to growth factor stimulation in mammals is mediated by ERK phosphorylation of UBF . Mol Cell 8 , 1063 – 1073 . doi: 10.1016/s1097-2765(01)00384-7 . OpenUrl CrossRef PubMed Web of Science 57. Hannan , K.M. , Brandenburger , Y. , Jenkins , A. , Sharkey , K. , Cavanaugh , A. , Rothblum , L. , Moss , T. , Poortinga , G. , McArthur , G.A. , Pearson , R.B. , et al. ( 2003 ). mTOR-dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF . Mol Cell Biol 23 , 8862 – 8877 . doi: 10.1128/MCB.23.23.8862-8877.2003 . OpenUrl Abstract / FREE Full Text 58. ↵ Zhao , J. , Yuan , X. , Frödin , M. , and Grummt , I . ( 2003 ). ERK-dependent phosphorylation of the transcription initiation factor TIF-IA is required for RNA polymerase I transcription and cell growth . Mol Cell 11 , 405 – 413 . doi: 10.1016/s1097-2765(03)00036-4 . OpenUrl CrossRef PubMed Web of Science 59. ↵ Bierhoff , H. , Schmitz , K. , Maass , F. , Ye , J. , and Grummt , I . ( 2010 ). Noncoding transcripts in sense and antisense orientation regulate the epigenetic state of ribosomal RNA genes . Cold Spring Harb Symp Quant Biol 75 , 357 – 364 . doi: 10.1101/sqb.2010.75.060 . OpenUrl Abstract / FREE Full Text 60. ↵ Vander Heiden , M.G. , Cantley , L.C. , and Thompson , C.B. ( 2009 ). Understanding the Warburg effect: the metabolic requirements of cell proliferation . Science 324 , 1029 – 1033 . doi: 10.1126/science.1160809 . OpenUrl Abstract / FREE Full Text 61. ↵ Lemieux , J.E. , Gomez-Escobar , N. , Feller , A. , Carret , C. , Amambua-Ngwa , A. , Pinches , R. , Day , F. , Kyes , S.A. , Conway , D.J. , Holmes , C.C. , et al. ( 2009 ). Statistical estimation of cell-cycle progression and lineage commitment in Plasmodium falciparum reveals a homogeneous pattern of transcription in ex vivo culture . Proc Natl Acad Sci U S A 106 , 7559 – 7564 . doi: 10.1073/pnas.0811829106 . OpenUrl Abstract / FREE Full Text 62. ↵ Zanghì , G. , Vembar , S.S. , Baumgarten , S. , Ding , S. , Guizetti , J. , Bryant , J.M. , Mattei , D. , Jensen , A.T.R. , Rénia , L. , Goh , Y.S. , et al. ( 2018 ). A Specific PfEMP1 Is Expressed in P. falciparum Sporozoites and Plays a Role in Hepatocyte Infection . Cell Rep 22 , 2951 – 2963 . doi: 10.1016/j.celrep.2018.02.075 . OpenUrl CrossRef PubMed 63. ↵ Baumgarten , S. , Bryant , J.M. , Sinha , A. , Reyser , T. , Preiser , P.R. , Dedon , P.C. , and Scherf , A . ( 2019 ). Transcriptome-wide dynamics of extensive m6A mRNA methylation during Plasmodium falciparum blood-stage development . Nat Microbiol 4 , 2246 – 2259 . doi: 10.1038/s41564-019-0521-7 . OpenUrl CrossRef PubMed 64. ↵ Fivelman , Q.L. , McRobert , L. , Sharp , S. , Taylor , C.J. , Saeed , M. , Swales , C.A. , Sutherland , C.J. , and Baker , D.A . ( 2007 ). Improved synchronous production of Plasmodium falciparum gametocytes in vitro . Mol Biochem Parasitol 154 , 119 – 123 . doi: 10.1016/j.molbiopara.2007.04.008 . OpenUrl CrossRef PubMed Web of Science 65. ↵ Birnbaum , J. , Flemming , S. , Reichard , N. , Soares , A.B. , Mesén-Ramírez , P. , Jonscher , E. , Bergmann , B. , and Spielmann , T . ( 2017 ). A genetic system to study Plasmodium falciparum protein function . Nat Methods 14 , 450 – 456 . doi: 10.1038/nmeth.4223 . OpenUrl CrossRef PubMed 66. ↵ Bryant , J.M. , Baumgarten , S. , Dingli , F. , Loew , D. , Sinha , A. , Claës , A. , Preiser , P.R. , Dedon , P.C. , and Scherf , A . ( 2020 ). Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite . Mol Syst Biol 16 , e9569 . doi: 10.15252/msb.20209569 . OpenUrl CrossRef PubMed 67. ↵ Voß , Y. , Klaus , S. , Lichti , N.P. , Ganter , M. , and Guizetti , J . ( 2023 ). Malaria parasite centrins can assemble by Ca2+-inducible condensation . PLoS Pathog 19 , e1011899 . doi: 10.1371/journal.ppat.1011899 . OpenUrl CrossRef PubMed 68. ↵ Tibúrcio , M. , Yang , A.S.P. , Yahata , K. , Suárez-Cortés , P. , Belda , H. , Baumgarten , S. , van de Vegte-Bolmer , M. , van Gemert , G.-J. , van Waardenburg , Y. , Levashina , E.A. , et al. ( 2019 ). A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the ‘genus-species’‘Plasmodium falciparum’ Life Cycle . mBio 10 , e01170 – 19 . doi: 10.1128/mBio.01170-19 . OpenUrl CrossRef PubMed 69. ↵ Bolstad , B.M. , Irizarry , R.A. , Astrand , M. , and Speed , T.P . ( 2003 ). A comparison of normalization methods for high density oligonucleotide array data based on variance and bias . Bioinformatics 19 , 185 – 193 . doi: 10.1093/bioinformatics/19.2.185 . OpenUrl CrossRef PubMed Web of Science 70. ↵ Alvarez-Jarreta , J. , Amos , B. , Aurrecoechea , C. , Bah , S. , Barba , M. , Barreto , A. , Basenko , E.Y. , Belnap , R. , Blevins , A. , Böhme , U. , et al. ( 2024 ). VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center in 2023 . Nucleic Acids Res 52 , D808 – D816 . doi: 10.1093/nar/gkad1003 . OpenUrl CrossRef PubMed 71. ↵ Quinlan , A.R. , and Hall , I.M . ( 2010 ). BEDTools: a flexible suite of utilities for comparing genomic features . Bioinformatics 26 , 841 – 842 . doi: 10.1093/bioinformatics/btq033 . OpenUrl CrossRef PubMed Web of Science 72. ↵ Bolger , A.M. , Lohse , M. , and Usadel , B . ( 2014 ). Trimmomatic: A flexible trimmer for Illumina sequence data . Bioinformatics 30 , 2114 – 2120 . doi: 10.1093/bioinformatics/btu170 . OpenUrl CrossRef PubMed Web of Science 73. ↵ Dobin , A. , Davis , C.A. , Schlesinger , F. , Drenkow , J. , Zaleski , C. , Jha , S. , Batut , P. , Chaisson , M. , and Gingeras , T.R . ( 2013 ). STAR: ultrafast universal RNA-seq aligner . Bioinformatics 29 , 15 – 21 . doi: 10.1093/bioinformatics/bts635 . OpenUrl CrossRef PubMed Web of Science 74. ↵ Anders , S. , Pyl , P.T. , and Huber , W . ( 2015 ). HTSeq--a Python framework to work with high-throughput sequencing data . Bioinformatics 31 , 166 – 169 . doi: 10.1093/bioinformatics/btu638 . OpenUrl CrossRef PubMed Web of Science 75. ↵ Love , M.I. , Huber , W. , and Anders , S . ( 2014 ). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 . Genome Biol 15 , 550 . doi: 10.1186/s13059-014-0550-8 . OpenUrl CrossRef PubMed 76. ↵ Wickham , H. ( 2016 ). ggplot2: Elegant Graphics for Data Analysis ( Springer-Verlag New York ). 77. ↵ R Development Core Team ( 2012 ). R: A language and environment for statistical computing . Preprint at R Foundation for Statistical Computing . 78. ↵ Langmead , B. , and Salzberg , S.L . ( 2012 ). Fast gapped-read alignment with Bowtie 2 . Nat Methods 9 , 357 – 359 . doi: 10.1038/nmeth.1923 . OpenUrl CrossRef PubMed Web of Science 79. ↵ Li , H. , Handsaker , B. , Wysoker , A. , Fennell , T. , Ruan , J. , Homer , N. , Marth , G. , Abecasis , G. , Durbin , R. , and Genome Project Data Processing, S . ( 2009 ). The Sequence Alignment/Map format and SAMtools . Bioinformatics 25 , 2078 – 2079 . doi: 10.1093/bioinformatics/btp352 . OpenUrl CrossRef PubMed Web of Science 80. ↵ Zhang , Y. , Liu , T. , Meyer , C.A. , Eeckhoute , J. , Johnson , D.S. , Bernstein , B.E. , Nusbaum , C. , Myers , R.M. , Brown , M. , Li , W. , et al. ( 2008 ). Model-based analysis of ChIP-Seq (MACS) . Genome Biol 9 , R137 . doi: 10.1186/gb-2008-9-9-r137 . OpenUrl CrossRef PubMed 81. ↵ Ramírez , F. , Ryan , D.P. , Grüning , B. , Bhardwaj , V. , Kilpert , F. , Richter , A.S. , Heyne , S. , Dündar , F. , and Manke , T . ( 2016 ). deepTools2: a next generation web server for deep-sequencing data analysis . Nucleic Acids Res 44 , W160 – 5 . doi: 10.1093/nar/gkw257 . OpenUrl CrossRef PubMed 82. ↵ Robinson , J.T. , Thorvaldsdóttir , H. , Winckler , W. , Guttman , M. , Lander , E.S. , Getz , G. , and Mesirov , J.P . ( 2011 ). Integrative genomics viewer . Nat Biotechnol 29 , 24 – 26 . doi: 10.1038/nbt.1754 . OpenUrl CrossRef PubMed Web of Science 83. ↵ Wolff , J. , Rabbani , L. , Gilsbach , R. , Richard , G. , Manke , T. , Backofen , R. , and Grüning , B.A . ( 2020 ). Galaxy HiCExplorer 3: a web server for reproducible Hi-C, capture Hi-C and single-cell Hi-C data analysis, quality control and visualization . Nucleic Acids Res 48 , W177 – W184 . doi: 10.1093/nar/gkaa220 . OpenUrl CrossRef PubMed 84. ↵ Durand , N.C. , Robinson , J.T. , Shamim , M.S. , Machol , I. , Mesirov , J.P. , Lander , E.S. , and Aiden , E.L . ( 2016 ). Juicebox Provides a Visualization System for Hi-C Contact Maps with Unlimited Zoom . Cell Syst 3 , 99 – 101 . doi: 10.1016/j.cels.2015.07.012 . OpenUrl CrossRef PubMed 85. ↵ Binns , D. , Dimmer , E. , Huntley , R. , Barrell , D. , O’Donovan , C. , and Apweiler , R . ( 2009 ). QuickGO: a web-based tool for Gene Ontology searching . Bioinformatics 25 , 3045 – 3046 . doi: 10.1093/bioinformatics/btp536 . OpenUrl CrossRef PubMed Web of Science 86. ↵ Altschul , S.F. , Gish , W. , Miller , W. , Myers , E.W. , and Lipman , D.J . ( 1990 ). Basic local alignment search tool . J Mol Biol 215 , 403 – 410 . doi: 10.1016/S0022-2836(05)80360-2 . OpenUrl CrossRef PubMed Web of Science 87. ↵ Li , H . ( 2018 ). Minimap2: pairwise alignment for nucleotide sequences . Bioinformatics 34 , 3094 – 3100 . doi: 10.1093/bioinformatics/bty191 . OpenUrl CrossRef PubMed 88. ↵ Kovaka , S. , Hook , P.W. , Jenike , K.M. , Shivakumar , V. , Morina , L.B. , Razaghi , R. , Timp , W. , and Schatz , M.C . ( 2024 ). Uncalled4 improves nanopore DNA and RNA modification detection via fast and accurate signal alignment . Preprint , doi: 10.1101/2024.03.05.583511 http://10.1101/2024.03.05.583511 . OpenUrl Abstract / FREE Full Text 89. Perez-Riverol , Y. , Bai , J. , Bandla , C. , García-Seisdedos , D. , Hewapathirana , S. , Kamatchinathan , S. , Kundu , D.J. , Prakash , A. , Frericks-Zipper , A. , Eisenacher , M. , et al. ( 2022 ). The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences . Nucleic Acids Res 50 , D543 – D552 . doi: 10.1093/nar/gkab1038 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted March 22, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Couble , Tiziano Vignolini , Gregory Dore , Bérangère Lombard , Michael Richard , Damarys Loew , Michael Büttner , Rafael Dueñas-Sánchez , Gernot Poschet , Jessica M. 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