The PfAP2-G2 transcription factor is a critical regulator ...Oct 27, 2020 · PfAP2-G2 (PF3D7_1408200) plays a critical role in the maturation of Plasmodium falciparum gametocytes

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ThePfAP2-G2transcriptionfactorisacriticalregulatorofgametocytematuration

SupritaSingh1,JoanaM.Santos1,*,LindseyM.Orchard1,NaomiYamada3$,Riëttevan

Biljon1,HeatherJ.Painter1,#,ShaunMahony2,ManuelLlinás1,2,3,‡

1Department of Biochemistry and Molecular Biology, The Pennsylvania State

University,UniversityPark,PA,USA16802

Huck Center for Malaria Research, The Pennsylvania State University, University

Park,PA,USA16802

2Center for Eukaryotic GeneRegulation,Department of Biochemistry&Molecular

Biology,ThePennsylvaniaStateUniversity,UniversityPark,PA,USA16802

3DepartmentofChemistry,ThePennsylvaniaStateUniversity,UniversityPark,PA,

USA16802

*Presentaddress:UniversitéParis-Saclay,CEA,CNRS,InstituteforIntegrativeBiology

oftheCell(I2BC),91198,Gif-sur-Yvette,France.

$Present address: GSK R&D Functional Genomics, 1250 S. Collegeville Rd.,

Collegeville,PA,USA19426

#Presentaddress:DivisionofBacterial,Parasitic,andAllergenicProducts,Officeof

VaccinesResearchandReview,CenterforBiologicsEvaluationsandResearch,Food

andDrugAdministration,SilverSpring,MD,USA

‡Towhomcorrespondenceshouldbeaddressed:[email protected]

.CC-BY-NC-ND 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprintthis version posted October 28, 2020. ; https://doi.org/10.1101/2020.10.27.355685doi: bioRxiv preprint

https://doi.org/10.1101/2020.10.27.355685

http://creativecommons.org/licenses/by-nc-nd/4.0/

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Abstract

Differentiation fromasexualbloodstagestosexualgametocytes isrequired

for transmission of malaria parasites from the human to the mosquito host.

Preventing gametocyte commitment and development would block parasite

transmission, but the underlying molecular mechanisms behind these processes

remain poorly understood. Here, we report that the ApiAP2 transcription factor,

PfAP2-G2 (PF3D7_1408200)plays a critical role in thematuration ofPlasmodium

falciparumgametocytes.PfAP2-G2bindstothepromotersofawidearrayofgenes

thatareexpressedatmanystagesoftheparasitelifecycle.Interestingly,wealsofind

binding of PfAP2-G2within the gene body of almost 3000 genes, which strongly

correlateswiththelocationofH3K36me3andseveralotherhistonemodificationsas

wellasHeterochromatinProtein1(HP1),suggestingthatoccupancyofPfAP2-G2in

genebodiesmayserveasanalternativeregulatorymechanism.Disruptionofpfap2-

g2doesnotimpactasexualdevelopment,parasitemultiplicationrate,orcommitment

to sexual development but themajority of sexual parasites are unable tomature

beyondstage III gametocytes.Theabsenceofpfap2-g2 leads tooverexpressionof

28% of the genes bound by PfAP2-G2 and none of the PfAP2-g2 bound are

downregulated,suggestingthatitisarepressor.WealsofindthatPfAP2-G2interacts

with chromatin remodeling proteins, amicrorchidia (MORC)protein, and another

ApiAP2protein(PF3D7_1139300).OverallourdatademonstratethatPfAP2-G2isan

important transcription factor thatestablishesanessentialgametocytematuration

programinassociationwithotherchromatin-relatedproteins.

Introduction

Malaria is a life-threatening disease that continues to impact the lives of

millionsofpeopleworldwide.Accordingtothe latestWHOestimates, therewere

228millioncasesofmalaria in2018,resulting in405,000deaths(WorldMalaria

Report,WHO,2019).Malariaiscausedbyunicellularprotozoanparasitesbelonging

to the genus Plasmodium. Of the six species that infect humans, Plasmodium

falciparum has the highest mortality rate (Weiss et al. 2019; WHO 2019). P.

falciparumexhibitsacomplexlifecyclewithasexualandsexualerythrocyticphases



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in the human host, followed by development in Anopheles mosquitoes which

transmittheparasitebacktoanewhost,initiatingthepre-erythrocyticliverstage

of infection. Although the cyclic 48-hour asexual blood-stage is responsible for

symptomatic disease and severe malaria, this form of the parasite cannot be

transmitted.Rather,ineachroundofreplication,afractionoftheparasites<10%

exittheasexualpathwayandundergosexualdifferentiationtoformmaleandfemale

gametocytes,whicharetransmissioncompetent(Bruceetal.1989). Inhibitionof

gametocyte development is o f great interest , because i t would prevent

malaria parasite transmission, which is one of the major goals in the effort to

achievediseaseeradication(Rabinovichetal.2017).

Gametocyte development inP. falciparum is a 9–12 day process,which is

substantiallylongerthanthatofotherhumaninfectingPlasmodiumspeciessuchas

P.vivaxandtherodentmalariaparasitesP.berghei(26–30hours)andP.yoelii(36

hours)(GautretandMotard,1999;Liu,Miao,&Cui,2011;Armisteadetal.,2018).P.

falciparum gametocyte development is divided into five (stage I to V)

morphologically distinct stages (Sinden 1982). The earliest phase of gametocyte

development, stage Ia, occurs around 24 to 30 hours post-invasion (hpi) and is

morphologically indistinguishable from the young trophozoite stage. However,

thereareseveralwell-definedmarkersofearlystageIgametocytessuchasPfs16

and Pfg27 and PfGEXP-5 (Alano, Premawansa, Bruce, & Carter, 1991; Marian C.

Bruce,Carter,Nakamura,Aikawa,&Carter,1994;Silvestrinietal.,2010;Poranetal.,

2017;Joslingetal.,2020;Llorà-Batlleetal.,2020).Inthehumanhost,stageIbto

stageIVsexually-developingparasitesaresequesteredindeeptissueslikethebone

marrowandonlystageVgametocytesfreelycirculateinthebloodandcanbepicked

upbymosquitoes(Aguilaretal.2014;Joiceetal.2014;Venugopaletal.2020)

Regulation of Plasmodium development is driven by stage-specific

transcription factors (TFs), such as thewell-studied Apicomplexan AP2 (ApiAP2)

familyofDNAbindingproteins.ApiAP2proteinsarefoundamongallmembersofthe

phylum,andeachApiAP2proteincontainsbetweenonetothreeApetala2(AP2)DNA

bindingdomains(Balajietal.2005). InP.falciparumthereare27membersof the

ApiAP2 protein family. ApiAP2 proteins have been shown to control all



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developmental transitions in Plasmodium (Jeninga, Quinn, and Petter 2019;

Modrzynskaetal.2017;Zhangetal.2017)

A master regulator of sexual (gametocyte) commitment, AP2-G, has been

identified inbothP. falciparumandPlasmodiumberghei,arodentmalariaparasite

(Kafsack et al. 2014; Sinha et al. 2014). Expression levels of PfAP2-G

(PF3D7_1222600)stronglycorrelatewiththeformationofgametocytes,andtargeted

disruptionofthepfap2-glocusresultsinacompleteblockingametocytogenesisand

downregulationofmanygametocyte-associatedgenes(Kafsacketal.2014).Another

regulatorofPlasmodiumgametocytedevelopmentisAP2-G3.Disruptionofpyap2-g3

(PY17X_1417400)inP.yoeliiresultedinsignificantreductioninthenumbersofmale

and female gametocytes, day 8 oocysts, and sporozoites, but it did not affect the

asexual growth of the parasites (Zhang et al. 2017). The P. berghei orthologue,

PBANKA_1415700,hasalsobeenknockedoutandshowntobefemale-specificand

essential for female gametocyte development, and was thus renamed PbAP2-FG

(Yudaetal.2019).TheP.falciparumorthologue,PF3D7_1317200,wasalsofoundto

beassociatedwithgametocytogenesis(Ikadaietal.2013),althoughithasnotbeen

extensivelycharacterized.

AP2-G2hasbeenshowntoplayaroleingametocytogenesisinbothP.berghei

andP.yoelii(Sinhaetal.,2014;Yuda,Iwanaga,Kaneko,&Kato,2015;Modrzynskaet

al.,2017).Knockoutofpbap2-g2doesnotinhibitsexualstageconversionbutrather

results in the nearly complete loss of gametocyte maturation and a block in

transmissiontomosquitoes(Yudaetal.2015).Yudaetal.reportedthattheP.berghei

transcription factor is bound to roughly 1,500 genes, or slightly over 1/3 of the

genomeduringasexualdevelopment,andanumberofthesegeneswereup-regulated

bymorethantwo-fold inpbap2-g2knockout lines. Inanotherstudy,disruptionof

pbap2-g2alsocausedprematureexpressionofliverstageandsporozoitestagegenes

duringasexualdevelopmentinredbloodcells(Modrzynskaetal.2017).Moreover,a

P.bergheiliver-stage(LS)transcriptomeofP.bergheireportedstrongupregulation

ofpbap2-g2inearlyLSthatisnegativelycorrelatedwiththeexpressionofliverstage-

specifictranscripts(Derbyshire2019).Together,thesefindingssuggestthatAP2-G2

actsasarepressorinboththeasexualandsexualstagesandduringearlyliver-stage



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infectioninP.berghei.Accordingly,P.yoeliiparasiteslackingthepyap2-g2genehad

greatlyreducednumbersofgametocytesandoocysts(Zhangetal.2017).Arecent

genome-wideknockoutscreensuggeststhatAP2-G2isalsonotessentialforblood-

stagedevelopmentinP.falciparum(Zhangetal.2018),andmayhavearoleinalater

stageofdevelopment.

In this study we explore the function of AP2-G2 in P. falciparum during

parasiteblood-stagedevelopment.Theperiodofgametocytematurationforrodent

malariaparasites isshorter than inP. falciparum,making itdifficult todiscernthe

developmentalphenotypesassociatedwithap2-g2knockouts.Therefore,disrupting

this gene in P. falciparum is of interest due to the longer maturation period.

Furthermore, two studies have reported differences in asexual blood-stage

development resulting from pbap2-g2 deletion. On one hand, Modrzynska et al.

observedreducedgrowthandprematureexpressionofliver-andsporozoite-stage

genes(Modrzynskaetal.2017).Conversely,Yudaetal.reportedthatpbap2-g2KO

parasites proliferate normally in blood (Yuda et al. 2015). Another discrepancy

regardstheimpactoftheKOonthesexratio.Sinhaetal.reportedadifferenceinthe

ratioofmaletofemalegametocyteswasdisruptedinpbap2-g2KOlines(Sinhaetal.

2014)whileYudaetal.observednosuchdifferences(Yudaetal.2015).

WealsofindthattheP.falciparumorthologueofAP2-G2alsoplaysacritical

roleinthematurationofgametocytes.Disruptionofpfap2-g2doesnotimpactasexual

development,parasitemultiplicationrate,orcommitmenttosexualdevelopment,but

parasites are unable to develop normally beyond stage III. Using transcriptomic

analysisandChIP-seqwehaveidentifiedanumberofcandidategenesthatarebound

and regulated by PfAP2-G2. We also identify interacting partners using protein

immunoprecipitationfollowedbymassspectrometry.Overallourworksuggeststhat

PfAP2-G2isatranscriptionalrepressorthatlikelyrecruitsadditionaltranscription

factors and chromatin remodeling machinery to the genome to control gene

expression. PfAP2-G2plays a critical role in the regulationof the development of

malaria parasites as they transition from asexual to sexual parasites, allowing for

propergametocytematuration.



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Results

PfAP2-G2 isexpressedduring the trophozoiteandschizont stagesofasexual

development

TheP.falciparumorthologueofAP2-G2,PF3D7_1408200,encodesa189kDa

protein that contains a single AP2 DNA binding domain (Figure 1A). pfap2-g2 is

maximallytranscribedattheringandearlytrophozoitestages(Bozdechetal.,2003;

Painter et al., 2018) and proteomics data indicates protein expression at the

trophozoite and schizont stages (Oehring et al. 2012). To precisely determine the

specificstageofexpressionofPfAP2-G2anditssubcellularlocalization,wetaggedthe

geneattheC-terminuswithGFP(SupplementaryFig.1).Usinglive-cellfluorescence

microscopy,weimagedahighlysynchronizedPfAP2-G2::GFPparasitepopulationfor

one complete asexual replication cycle (48-hours), every 7 h beginning with the

newly invaded ring stage (5–7 hpi) revealed that PfAP2-G2 was localized to the

nucleus, as expected, and was expressed from the early trophozoite to the late

schizontstages(Figure1B).FurtherconfirmingthenuclearlocalizationofPfAP2-G2,

nuclearfractionationoftrophozoitestageparasites(~30hpi)showedthatfulllength

PfAP2-G2::GFP(~250kDa)wasonlydetectedinthenuclearfractionoftheparasite

lysates (Figure1C).Wealsodetectedothersmaller sizedpeptides indicating that

PfAP2-G2maybeproteolyticallycleavedorisunstableinparasitelysates.

PfAP2-G2isnotrequiredforproliferationduringtheasexualstagesofthelife

cycle

Todeterminetheroleofpfap2-g2,wegeneratedageneticdisruption(KO)line

usingselectionlinkedintegration(SLI)(Birnbaumetal.2017)totruncatethepfap2-

g2 codingsequence(SupplementaryFig.2A).Asexpected,pfap2-g2KOparasites

were readily obtained. Live-cell fluorescence imaging of the transgenic parasites

showeddiffusecytoplasmicstainingsuggestingthattheremainingnonAP2domain-

containingproteinfragmentnolongerlocalizedtothenucleus(SupplementaryFig.

2B).WenextdeterminediftruncationofPfAP2-G2impactedasexualstageparasite

growthormultiplicationrate.UsingaSYBRgreengrowthassay(Vossenetal.2010),

wefindthatpfap2-g2KOparasitesdevelopsimilarlytothatofWT(Figure2A),and



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have similar multiplication rates (Figure 2B). Therefore, although PfAP2-G2 is

expressed in the asexual blood stages, it is not required for normal asexual

development.

PfAP2-G2isexpressedinthegametocytestages

RecenttranscriptomicdatahasshownthatthemRNAabundanceofpfap2-g2

ingametocytesislowrelativetotheasexualstages,withabroadpeakseenonlyin

the early gametocyte stages followed by low expression during the later stages

(SupplementaryFig.3)(Biljonetal.2019).Tocharacterizeproteinexpressionof

PfAP2-G2duringgametocytedevelopmentweimagedPfAP2-G2::GFPparasites(see

methods).PfAP2-G2wasexpressedinallstagesofgametocytedevelopment,butit

was not confined to the nucleus in later stages (Figure 3A). Indeed nuclear

fractionationof stage III gametocytesdetectedexpressionof full-lengthprotein in

bothfractions,unlikeinasexualparasites(Figure3B).Italsoseemsthatthenuclear

protein undergoes different proteolytical processing in gametocytes versus the

asexualstages.

PfAP2-G2isessentialforgametocytematuration

To determine if sexually committed PfAP2-G2 gametocytesmature fully to

Stage V, we induced PfAP2-G2 KO and WT parasite lines to undergo

gametocytogenesis and followed their development for 14 days,monitoring their

maturation via morphological analysis of Giemsa-stained thin-blood smears.

Whereas WT E5 parasites matured into Stage V gametocytes, most pfap2-g2 KO

parasites committed to sexual development could not progress beyond Stage III

(Figure 3C). PfAP2-G2 is thus a critical regulator of gametocyte maturation and

development.

PfAP2-G2makesextensivegenome-wideinteractions

Because PfAP2-G2 is predicted to be a DNA-binding protein (Campbell, de

Silva,Olszewski,Elemento,&Llinás,2010;Yudaetal.,2015)wesoughttodetermine

thegenome-widelocalizationofPfAP2-G2inbothasexualandgametocytelifecycle



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stages. To do thiswe first performedChIP-seq at the trophozoite stage using the

PfAP2-G2::GFPparasites.Peakcalling identified~5000peaks inat least2of the3

biologicalreplicates(FDR0.05)(SupplementaryFig.4A).Allreplicatesshoweda

highcorrelationwitheachotherwithrespecttotheoverallpeaksidentified(Figure

4A).Tooursurpriseonly401ofthepredictedbindingsites,correspondingto120

genes,were found in the non-codingupstream regions of genes (Supplementary

Table1),while4,600peaks,correspondingto2,932genes,werelocatedinexonic

genebodies(SupplementaryTable2).Thisdiffersfromwhathasbeenpreviously

observedforothercharacterizedP.falciparumApiAP2proteins,suchasSIP2(Flueck

etal.2010),AP2-I(Santosetal.2017)andAP2-G(Joslingetal.2020)whichwere

foundtopredominantlybind tonon-codingupstreamregions.Althoughwidescale

bindingtogenebodieswasnotpreviouslyreportedforPbAP2-G2(Yudaetal.2015),

are-analysisof theChIP-seqdata fromthisstudyusingthesamepipelinethatwe

used for our ChIP-seq data analysis, revealed that PbAP2-G2 is also broadly

distributedacrossgenebodiesintheP.bergheigenomewith4,345bindingsites.

An analysis of all the regions (both upstreamregions andORFs) bound by

PfAP2-G2, using DREME (Bailey 2011) identified two significantly enriched DNA

sequencemotifs.ThefirstisanAGAAsequencemotifwhichisrelatedtoapreviously

reportedDNAmotiffoundtoassociatewithone(ofthethree)AP2domainsofthe

ApiAP2proteinPF3D7_1139300(Campbelletal.2010).ThesecondisanACCAcore

motif (Figure 4B) closely resembling the previously identified PfAP2-G2 binding

PBMmotif.(Campbelletal.2010),demonstratingthesuccessofChIPexperiments.

In order to characterize possible regulatory targets of PfAP2-G2, we first

focused on the 120 genes that contain PfAP2-G2peaks in theirupstream regions.

Nearly87%ofthese120putativetargetgenesareboundwithin2.5kbupstreamof

the start codon (Supplementary Fig. 4B). 278 of the remaining intergenic peaks

werelocalizedtothesubtelomericendsofeachchromosome(SupplementaryFig.

4C). For further analysis we only considered genes with upstream binding

interactions less than 2.5 kb from the start codon, leaving 82 candidate genes

(SupplementaryTable1).Interestingly,PfAP2-G2attheasexualtrophozoitestage

bindstothepromotersofgenesknowntobeexpressedlaterinthegametocyteand



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mosquito lifecycle stages.These include6-cysteineprotein (p36 PF3D7_0404400),

cysteine repeat modular protein 2 (crmp2 PF3D7_0718300), sporozoite protein

essential for cell traversal (spectPF3D7_1342500), circumsporozoite protein (csp

PF3D7_0304600), cell traversal protein for ookinetes and sporozoites (celtos

PF3D7_1216600), secreted protein altered thrombospondin repeat protein (spatr

PF3D7_0212600),stearoyl-CoAdesaturase(scdPF3D7_0511200),andcap380oocyst

capsule protein (PF3D7_0320400) among others (Supplementary Table 1)

(Chattopadhyayetal.2003;vanDijketal.2010;Espinosaetal.2017;Gratraudetal.

2009;Ishinoetal.2004;Itsaraetal.2018;Singhetal.2007;Thompsonetal.2007;

Zhaoetal.2016).RepresentativePfAP2-G2peaksupstreamoftwotargetgenesare

shownin(Figure4C).WealsofoundPfAP2-G2associatedwiththeupstreamregions

ofgenesthatarehighlytranscribedintheasexualringstage(asanalyzedin(López-

Barragánet al. 2011)) including theknob-associatedhistidine richprotein (kahrp

PF3D7_0202000), Plasmodium helical interspersed subtelomeric proteins (phista

PF3D7_0115100),severalvariant familygenes includingvarsandrifins,andgenes

encodingproteinsinvolvedinegressandinvasionsuchastheRh5interactingprotein

(riprPF3D7_0323400)andserinerepeatantigen7(sera7PF3D7_0207400)(Miller

etal.2002;Peietal.2005;Sargeantetal.2006;Volzetal.2016).Therefore,PfAP2-

G2 binds to the promoters and ORFs of genes that are expressed at a different

developmentalstagethanthatinwhichAP2-G2isexpressed,suggestingthat,likein

therodentmalariaspecies,thisisatranscriptionalrepressorprotein.

We next examined the genome-wide occupancy of PfAP2-G2 in stage III

gametocytesbyChIP-seq.Weidentified947peaksincommonacrossbothreplicates,

correspondingto674genesatanFDRof0.05(Figure4D)(SupplementaryTable

3, Supplementary Table 4). PfAP2-G2 binding, once again, was found in both

upstream regions andwithin gene bodies. AlthoughPfAP2-G2 boundmany fewer

regionsingametocytescomparedtoasexualtrophozoites,whichmaybeassociated

withthedifficultyinobtaininghighamountsofchromatiningametocytes,virtually

allstageIIIPfAP2-G2targetgeneswerealsotargetsduringthetrophozoitestages

(Figure4E).Thissuggests that therearea largenumberofgenesthat arealways



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boundbyPfAP2-G2duringbothasexual and sexualparasitedevelopment (Figure

4F).DNAmotifanalysisyetagainidentifiedtheACCA(Figure4G).

Genetic disruption of PfAP2-G2 leads to aberrant gene expression in both

asexualandsexuallifecyclestages

Usingsynchronizedparasites,wecollectedseventotalRNAsamplesfromWT

orKOparasitesthroughoutthe48-hourasexuallifecyclefortranscriptomeanalysis.

In a separate experiment, total RNA samples were collected during sexual

differentiation every 12 hours for 7 continuous days starting at 2 days post-

gametocyte induction (Supplementary Fig. 5). Gene expression analysis of the

asexualstagetimecourseusingSignificanceAnalysisofMicroarray(SAM)(Tusher,

Tibshirani,andChu2001) identified327differentiallyexpressedtranscripts(>1.5

log2FC, 0.30 FDR) in the PfAP2-G2 KO line. Out of these, 237 showed enhanced

transcriptabundanceinthePfAP2G2KOlineand90showeddecreasedabundance

(Supplementary Fig.6,SupplementaryTable5). This result is quite surprising

giventhelackofanyasexualgrowthphenotypebetweenthePfAP2-G2KOandthe

WT(Figure2),implyingthatthesechangesingeneexpressiondonotimpactasexual

parasitefitnessorgametocytecommitment.Acomparisonoftranscriptabundancein

theKOandWTsexualstagesidentifiedincreasedabundancefor274transcriptsand

reducedabundance for169transcripts in thePfAP2-G2KOline(>1.5log2FC,0.30

FDR) (Supplementary Fig. 6, Supplementary Table 6). These differential

transcription patterns presumably lead to the stall in development at Stage III

gametocytesinparasiteslackingPfAP2-G2.

Todetermineatwhichstage the significantlychanginggenes (bySAM)are

maximallytranscribedinwild-typeparasites,weusedapublishedRNA-seqdataset

coveringfourdifferentasexualstages(rings,earlytrophozoite,latetrophozoite,and

schizonts),twogametocytestages(stageIIandstageV),andtheookinetestageusing

the 3D7P. falciparum strain (López-Barragán et al. 2011). Using this dataset, we

identifiedthe3D7parasitelifecyclestageatwhicheachgenehasthehighestreads

per kilobase of transcript, permillionmapped sequencing reads (RPKM). For the

asexual transcriptome,mostof thedifferentiallyexpressedgeneswemeasuredas



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differentiallyregulated inthePfAP2-G2KOarenormally transcribedmaximally in

stageVgametocyte(andnot inasexualstages), followedbytheookineteandring

stages (Figure5A).This suggests that lossofPfAP2-G2 impacts theexpressionof

genes from late-stage gametocytes, ookinetes, and asexual stages. However the

numberofgenesshowingenhancedtranscriptabundanceisstrikinglyhighforgenes

thatarenormallyexpressedinstageVgametocytes(Figure5A).Therefore,PfAP2-

G2may prevent the premature expression of late-stage gametocyte genes during

asexualdevelopment.

In the gametocyte transcriptome, we observed the opposite trend when

comparingtotheLópez-Barragándata,where70%of thetranscripts thatshowed

increasedabundanceintheKOlinewereusuallymaximallyabundantintheasexual

stages, especially at the ring stage (Figure 5A). Examples of geneswhosemRNA

abundancewashigheringametocytesincludeknob-associatedhistidine-richprotein

(kahrpPF3D7_0202000),10differentPlasmodiumhelicalinterspersedsubtelomeric

proteins (phista, phistb, phistc), 7 different serine/threonine protein kinase (FIKK

family)proteins,12genesencodingPlasmodiumexportedproteins(hyp2,hyp8,hyp9,

hyp10, hyp11, hyp12, hyp16)merozoite surface proteins (msp1,msp2,msp6,msp7,

msp11), the ApiAP2 transcription factor ap2-l (PF3D7_0730300), serine repeat

antigen(sera5,PF3D7_0207600)amongothers(SupplementaryTable6)(Peietal.,

2005;Sargeantetal.,2006;Nunes,Goldring,Doerig,&Scherf,2007;Beesonetal.,

2016).TheseresultsindicatethatPfAP2-G2repressesasubsetofgenesthroughout

bothasexualandsexualdevelopment,andtheabsenceofPfAP2-G2leadstoaberrant

timingofgeneexpression.

To identifyenrichedDNAsequencemotifs associatedwithgeneexpression

changes,weanalyzedthe5’upstreamregion(1000bpupstreamofthestartcodon)

ofthegenesshowingsignificantchangeintranscriptabundanceintheasexualand

sexualstagesusingtheFindingInformativeRegulatoryElements(FIRE)algorithm

(Elemento, Slonim, and Tavazoie 2007) . Themost significant enriched sequence

motifwastheknownDNA-interactingmotifforPfAP2-G2,TGCAACCA(p-value1.24

e-21) in genes with enhanced transcript abundance in the asexual stage

(SupplementaryFig.6).Interestingly,wealsofoundanAGAACAA(p-value3.3574e-



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15)DNA-bindingmotif that is recognizedby theApiAP2protein,PF3D7_1139300

(Supplementary Fig. 6). Similar motifs were found for genes with increased

transcript abundance in gametocytes (Supplementary Fig. 6). Intriguingly both

pfap2-g2andpf3d7_1139300(pf11_0404)areexpressedatsimilartimesthroughout

asexualdevelopmentbasedontranscriptomicdata,suggestingthatthetwoproteins

maybeactingtogetherinsomemannertoregulatetranscription(Supplementary

Fig. 7). Based on earlier evidence, genetic disruption of the pf3d7_1139300

orthologueinP.berghei(GeneID)andP.yoelii(GeneID)wasrefractoryindicatingits

essentiality (Modrzynska et al., 2017; Zhang et al., 2017). Single-cell RNA-seq has

shown a sharp upregulation of the gene encoding the ApiAP2 protein

PF3D7_1139300 when ap2-g expression peaks just before egress in committed

schizonts(Poranetal.2017).Althoughwecouldn’tdetecttheACCAPfAP2-G2binding

motif associated with genes showing decreased transcript abundance

(SupplementaryFig.8),wefoundenrichmentoftheAGACAmotif,whichhasbeen

associatedwithgametocytecommitmentanddevelopment(SupplementaryFig.8)

(Youngetal.,2005;Bischoff&Vaquero,2010;Painter,Carrasquilla,&Llinás,2017),

ingenesshowingdecreasedtranscriptabundanceingametocytes.

Genes with decreased mRNA abundance in the gametocyte transcriptome

fromthePfAP2-G2KOline(170genes)overlapsignificantlywithgenespreviously

reportedaslategametocytemarkers(VanBiljonetal.2019)(cluster9,208genes)

(Figure5B).Someexamplegenesarealphatubulin2(PF3D7_0422300),TRAP-like

protein (tlp PF3D7_0616500), secretedookineteprotein (psop13 PF3D7_0518800,

psop20 PF3D7_0715400), dynein heavy chain (PF3D7_0729900), ookinete surface

protein P28 (PF3D7_1031000), ookinete surface protein P25 (PF3D7_1031000)

(Rawlingset al., 1992;Heisset al., 2008);Ecker,Bushell,Tewari,&Sinden,2008;

Villardetal.,2007;Duffy&Kaslow,1997).Therefore,onereasonthatPfAP2-G2KO

parasitesmayfailtoprogressbeyondstageIIImaybeduetotheaberrantexpression

ofmRNAsrequiredduringthisandsubsequentstagesofgametocytedevelopment.

Theinhibitionofgametocytematurationmayalsobeduetothedecreasedexpression

of a few critical genes required for gametocyte development, such as male

development gene 1 (mdv1 PF3D7_1216500), gametocyte erythrocyte cytosolic



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protein (geco, PF3D7_1253000), gametocyte exported protein (gexp06

PF3D7_0114000),andgametocytespecificprotein(pf11-1PF3D7_1038400)during

the asexual stage. Interestingly, we also see overlap between the genes with

decreased transcript abundance inboth theasexual andgametocyte stages.These

overlapping genes are enriched mostly for members of the var, stevor, and rifin

variant gene families, Pfmc-2tm among others (Figure 5C). An early gametocyte

proteomehasrevealedthatexportedanderythrocyteremodelingproteinsarethe

most overrepresented proteins in gametocytes (Silvestrini et al. 2010). Although

members of the PfEMP1, STEVOR and RIFIN protein repertoire are expressed in

gametocytestages,theirroleisnotwellestablished(Sharpetal.,2006;Petter,Bonow,

&Klinkert,2008;Tibúrcioetal.,2012,Mwakalingaetal.,2012;Tibúrcioetal.,2013;

Neveuetal.,2018;Neveu&Lavazec,2019;),althoughdecreasedabundanceofthese

genesmaycontributetothephenotypiceffect.

PfAP2-G2 binding in the promoter and in gene bodies is associated with

differentialregulationoftranscription

Outof82geneswithupstreamPfAP2-G2peaks,only23displayedsignificantly

elevatedmRNAabundance(asanalyzedbySAM,>1.5log2FC,0.30FDR)(Figure5D).

Thissuggeststhatthetranscriptionofothergenesmaybeimpactedindirectlyinthe

PfAP2-G2knockout.Ontheotherhand,noneofthegeneswithsignificantlyreduced

mRNA abundance were bound by PfAP2-G2 in the upstream regions. However,

consideringthestageatwhichChIPwasperformedweseethatthemRNAabundance

ofmanyofthePfAP2-G2ChIP-seqtargetsdonotchangeintheabsenceofPfAP2-G2

indicating that binding of PfAP2-G2 alone might not be sufficient to affect

transcription(Figure5E).PfAP2-G2bindingtogenebodiesresultsinbothrepression

andactivation inthepfap2-g2KO.Openreading framesboundbyPfAP2-G2 inthe

gene-body ingametocytesshowedsimilareffects(SupplementaryFig.9).Overall

these results indicate that PfAP2-G2 binding to the upstream promoter region is

largelyassociatedwith repression,butPfAP2-G2alone isnot sufficient to repress

transcription.



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PfAP2-G2 shares occupancy with exonic epigenetic marks during asexual

development

Tounderstand the roleof thepervasivegenome-widePfAP2-G2binding in

exonicgenebodiesdetectedbyChIP-seq(SupplementaryTable2),weinvestigated

whetherPfAP2-G2isassociatedwithanyknownepigeneticmarks.Wecalculateda

pairwisePearsoncorrelationcoefficientbycomparingthereadcountsper1000bp

binsof thePfAP2-G2ChIP-seqresultsagainstanarrayofhistonemarks forwhich

ChIP data is available (H3K9me3,H4ac,H4K20me3,H3K26ac,H3K9ac,H3K4me3

(Karmodiyaetal.,2015),H3K36me2andH3K36me3(Jiangetal.,2013),aswellas

heterochromatinprotein1(PfHP1)(Brancuccietal.,2014)).Interestingly,wefound

thatPfAP2-G2occupancyhasthestrongestcorrelationwithH3K36me3(R=0.933),

followedbyH4K20me3(R=0.911),H3K27ac(R=0.826),andH4ac(R=0.765)(Figure

6A),which are all histonemarks that showmoderate positive correlationwithP.

falciparum transcription and are associated with transcriptionally poised genes

(Karmodiyaetal.2015).Intotal,3,633(80%)PfAP2-G2bindingsitesco-occurwith

H3K36me3marks(Figure6B,6C).The functionalroleof theassociationbetween

PfAP2-G2andH3K36me3is,andwhatthepotentialroleofPfAP2-G2inH3K36me3

recruitmentremainstobedetermined.PfAP2-G2alsoco-occurswithPfHP1towards

theendofchromosomesalthoughthiscorrelationismoderate(R=0.6)(Figure6D,

6E).

PfAP2-G2interactswiththechromatinremodelingmachinery

To identify proteins interacting with PfAP2-G2, we performed

immunoprecipitations (IP) from PfAP2-G2::GFP parasites at the trophozoite stage

using an anti-GFP antibody. Western blot analysis confirmed that the full length

PfAP2-G2proteinwaspurifiedandeluted(Figure7A),althoughsmallerGFP-positive

band(s)werealsoseen.TheresultingIPwasseparatedbySDSPAGEandtworegions

ofthegel(upperandlowerband,Figure7A)weresubjectedtoproteinanalysisby

massspectrometry.Thepurposeofanalyzingthesebandsseparatelywastoensure

thatwecoulddetectalloftheproteinsincludingthelowabundanceproteinwhich

otherwisewouldgetmaskedwhenanalyzingallofthebandstogether(Figure7B).



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Theproteomic IPdatawasanalyzedusingSAINT (Choi et al. 2011)witha

score of 0.9% and 1%FDR across all three replicates (SupplementaryTable7).

Interestingly,weidentifiedHP1asanassociatedprotein,whichisconsistentwiththe

observedco-occurrenceofPfAP2-G2peakswithHP1(Figure6D)(Figure7C).The

upperbandcontained larger chromatinremodelingproteinssuchas ISWI, SNF2L,

GCN5, FACT complex proteins (FACT-S and FACT-L), aMORC family protein, and

severalApiAP2familyproteins,includingPF3D7_1139300(PF11_0404)whosemotif

wasfoundtobeenrichedinboththetranscriptionandChIP-seqdata(Figure7D).

This suggests that direct interaction of PF3D7_1139300 and PfAP2-G2 may be

requiredfortheregulationofasubsetoftargetgenes.

Discussion

Gametocytogenesis inPlasmodium falciparum parasites is a long10-12day

developmental progression resulting in the formation of fertile male and female

gametesthatwillformanoocyteonlyupontransmissiontothemosquitohost.Recent

studieshaveshedlightonthecommitmentanddifferentiationofasexualparasites

intosexualparasites,which isdrivenbytheAP2-Gmasterregulator(Joslingetal.

2020), (Kafsack et al. 2014). However, how asexual cells are reprogrammed for

gametocytecommitment,andthedownstreampost-commitmentregulationofsexual

development is notwell characterized. There are known examples of gametocyte

stagegenessuchaspfsegxp,mdv1,andpfs16thatareexpressedveryearlyoninthe

asexualstagessuggestingthatprogrammingforgametocytogenesismaybeginearly

in asexual development, and disruption of this program could result in profound

effectspost-commitmentduringsexualdevelopment(Furuyaetal.,2005;Joiceetal.,

2014;Painteretal.,2017;Nixonetal.,2018).

InthisstudyweshowthattheApiAP2proteinPfAP2-G2isessentialforthe

development andmaturation of P. falciparum gametocytes. PfAP2-G is expressed

from the very early trophozoite stage through to stage V gametocytes. This is in

contrasttoP.berghei,wheretheproteinisexpressedfrom16hpi,duringtheschizont

stageofdevelopment(Yudaetal.2015).Livefluorescencemicroscopyandnuclear

fractionation show that PfAP2-G2 is localized to the nucleus of the trophozoite,



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schizont,andearlygametocytestages(Figure1B).InlaterstagegametocytesPfAP2-

G2proteinlevelsarereducedandexpressedthroughoutthecytoplasm(Figure3A).

This isconsistentwiththemRNAabundanceprofile,demonstratingpeakpfap2-g2

expressionintheearlyasexualstages,whereasingametocytesitsexpressionisbroad

and weak (Biljon et al. 2019). The nuclear localization and strength of protein

expressionindicatethatthemajorroleforPfAP2-G2isduringtheasexualstagesand

inearlygametocytes.

By performing genome-wide ChIP-seq analysis at the asexual trophozoite

stageandstage IIIgametocytes,weshowthatPfAP2-G2extensivelybindstoboth

intergenicandgenicregions.GenesboundbyPfAP2-G2intheupstreamregionsare

largelyexpressedlaterinthegametocyteandmosquitostages.However,PfAP2-G2

alsobindsthepromotersofgenesexpressedintheasexualringstageandgenesthat

aretranscribedinschizontstageslikemspsencodingthemerozoitesurfaceproteins,

whichareregulatedbyPfAP2-I(Santosetal.2017).OfcourseMSPsareunlikelytobe

requiredincommittedgametocytes,astheynolongeregressfromtheredbloodcell

for subsequent re-invasion. Interestingly, genes bound by PfAP2-G2 in stage III

gametocytesareessentiallythesameasthoseboundintrophozoites.Ananalysisof

theDNAmotifsenrichedintheregionsboundbyPfAP2-G2identifiedtwomotifs:an

AGAAmotifandanACCAmotif(Figure4Band5B).ThelatterresemblesthePBM-

basedpredictedmotifforPfAP2-G2,butitisdifferentfromthatidentifiedinP.berghei

(GTTGT), even though the AP2 DNA-binding domain is highly conserved (97%

identical). We also found enrichment for a second motif in the PfAP2-G2-bound

regions, which resembles the in vitro DNA sequence motif bound by the ApiAP2

protein PF3D7_1139300 (PF11_0404) (Campbell et al. 2010). Supporting the

hypothesis that these ApiAP2 proteins may function together in a complex, our

immunoprecipitation experiments against PfAP2-G2 GFP detected AP2

PF3D7_1139300(PF11_0404)asaninteractionpartner(Figure7,Supplementary

Table7).

ParasitesexpressingatruncatedPfAP2-G2versionproliferatenormallyinthe

asexual stages. These results are surprisinggiven themany global transcriptional

(327transcripts,>1.5log2FC,0.30FDR)changesoccurringintheasexualstages,and



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suggests that the parasite is tolerant to large fluctuations in gene expression.

Comparedwithwild-typeparasites,theexpressionlevelsof90genes(>1.5log2FC,

0.30FDR)weresignificantlydecreasedduringasexualdevelopmentinPfAP2-G2KO

parasites, including genes that have been reported to have functional roles in

gametocytes, perhaps leading to the developmental stall observed in Stage III

gametocytes (Painter, Carrasquilla, & Llinás, 2017; Van Biljon et al., 2019). This

suggests that the program for sexual progression is established very early, in the

asexual stage.KOofPbAP2-G2also resulted in thedownregulationof gametocyte

genes(Yudaetal.2015).GenesupregulatedinthePfAP2-G2KOweremostlythose

normallyexpressedatotherlaterdevelopmentalstagesincludingthegametocyteand

ookinete stages (Figure5A). In theupstreampromoter regionsof genes showing

increasedtranscriptabundanceinthePfAP2-G2KO,wefoundanenrichmentofthe

PfAP2-G2ACCAmotif.PfAP2-G2thereforeisinvolvedintherepressionofanumber

ofgenesthatarenotrequiredforasexualdevelopment.

Apreviousstudythatexaminedthegenome-widebindingofAP2-G2fromP.

bergheigametocytesusingChIP-seqreportedPbAP2-G2bindingatover1,500genes

thatweremostly associatedwith roles in asexual-stage proliferation (Yuda et al.

2015). Transcriptome analysis in gametocytes revealed the upregulation of 927

genesbymorethantwofoldinpbap2-g2KOlines,suggestingthatPbAP2-G2actsasa

repressor for asexual-stage genes during sexual development. However, only 397

(26.5%)ofthePbAP2-G2boundgeneswereupregulatedinthepbap2-g2KO,leading

Yuda et al. to hypothesize that relieving repression was not sufficient for the

upregulationoftherestofthegenes.Also,inourdata,notallgenesthatareboundby

PfAP2-G2showenhanced transcript abundance (only23/82geneswithupstream

PfAP2-G2peaks (28%))displayed significantlyelevatedmRNAabundance. In fact,

the PfAP2-G2motif is only present in the promoter of genes upregulated in both

developmental stages indicating that, as inP. berghei, PfAP2-G2 occupancy is not

always predictive of changes in gene expression, perhaps because it requires

interactionwithotherproteins.OncemorereflectingtherepressivenatureofPfAP2-

G2,noneofthegenesboundbyPfAP2-G2intheupstreamregionaredownregulated

intheKO.



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The effect of PfAP2-G2 truncation in transcript abundance is more

pronounced in gametocytes. Unfortunately, due to the massive dysregulation of

mRNA transcripts, it is difficult to ascertain which genes cause the stall in

developmentobservedatStageIIIgametocytes.Thesignificantlyupregulatedgenes

ingametocytesaremostlythoughttobeimportantforasexualstagedevelopment,

similartowhatwasshowninP.berghei(Yudaetal.2015).Interestingly,theApiAP2

proteinAP2-L,whichplaysacriticalroleinliver-stagedevelopment,isupregulated

inthegametocytestageinboththeP.falciparumandP.bergheiap2-g2knockouts,

andparasiteslackingPbAP2-G2areunabletocauseliverinfection.

WeobservedamoderatetostrongcorrelationbetweenPfAP2-G2occupancy

and that of H3K9me3 (R=0.933), followed by H4K20me3 (R=0.911), H3K27ac

(R=0.826), and H4ac (R=0.765) as well as PfHP1 (R=0.6) (Figure 6A). H3K36

methylationisawell-studiedmarkinmodelorganisms,andcarriesoutseveralroles.

StudiesinmetazoanshaveshownthatH3K36me3isenrichedatgeneexonsandplays

a role in the regulation of alternative splicing (Kolasinska-Zwierz et al. 2009).

AlthoughH3K36me3correlateswithactivetranscription,anotherstudyshowedthe

association of thismodificationwith facultative heterochromatin (Chantalat et al.

2011).Thus,H3K36me3isinvolvedwithbothactivelytranscribedaswellassilenced

regionsandmaycontributetotheformationofheterochromatinincombinationwith

otherhistonemodifications.Atasubsetofheterochromaticgenes,bindingtoHP1ais

requiredforenzyme-mediatedH3K36me3demethylationsuggestingthatthereare

different ways in which H3K36me3 regulates chromatin (Lin et al. 2012). In P.

falciparum, H3K36me3 is present along the entire gene body of silent var genes

includingthetranscriptionstartsite(TSS),anditisdepositedbytheP.falciparum

variant-silencingmethyltransferasecontainingaSETdomain(PfSETvs)(Jiangetal.

2013).However,inthatstudyenrichmentofH3K36me3wasfoundatthe3’endof

otherring-stageactivegenesbesidesthevariableantigenvar,rifinandstevorgenes

inPfSETKOparasites.Thisresultindicatesthereisanalternativemethyltransferase

resultinginH3K36methylation.WespeculatethatPfAP2-G2playsaroleinregulating

regionsofthegenomewhichneedtobesilencedtopreventspurioustranscription.



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We also found that PfHP1 localizedwith PfAP2-G2 towards the end of the

chromosomesandisassociatedwithbothsub-telomericandchromosome-internal

var genesandPlasmodium-exportedproteins.Adirect interactionbetweenPfHP1

andPfAP2-G2wassupportedbyIP-MS(Figure7C).AnotherassociationofPfAP2-G2

thatwassignificantiswiththeMORCprotein(Figure7D)whichhasbeenidentified

to play important roles in chromatin compaction in plants and animals and was

recentlyestablishedastheupstreamtranscriptionalrepressorofsexualcommitment

in Toxoplasma gondii (Farhat et al. 2020). This study found that MORC forms a

complexwithT.gondiiApiAP2transcriptionfactorsatsexualstagegenes.Twoofthe

T.gondiiApiAP2proteinsfoundtoassociatewithMORCbyIP-MSareTgAP2IV-2and

TgAP2IX-9,whoseAP2DNA-bindingdomainsaremostidenticaltothePfAP2-G2AP2

domain (Farhat et al. 2020). We also found the P. falciparum ISWI chromatin-

remodeling protein associated with PfAP2-G2, whichwas recently identified in a

complexwiththeMORCproteinatvargenepromoters(Bryantetal.2020)(Figure

7D).

Previousstudieshaveshownexpressionofthemastergametocyteregulator

pfap2-gpeaksat twotimesofasexualdevelopment(Poranetal.2017).The initial

expressionpeakintrophozoitesisassociatedwiththeexpressionofgenesencoding

ISWI,SNF2LexpressionandtheApiAP2encodinggenepf3d7_1222400.Thesecond

increaseinabundanceoccursjustbeforeegressincommittedschizontsandleadsto

the expression of genes encoding LSD2,HDA1 and theAP2-G2 associatedApiAP2

proteinPF3D7_1139300(Poranetal.2017).WeproposethatPF3D7_1139300may

act inacomplexwithPfAP2-G2tocompresschromatinstructure. Insummary,we

propose that PfAP2-G2 plays an important role in gene repression during the

maturation of the P. falciparum parasite sexual stages by associating with other

chromatin-relatedproteins.



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Materials&Methods:

Construction of plasmid for tagging 3’end of pfap2-g2 and pfap2-g2 gene

disruption

ThepDC2-cam-CRT-GFP(Fidock2000)plasmidwasusedtotagthe3’endof

thegenepfap2-g2withgfp.A948bphomologous fragment fromthe3’endof the

genewasamplifiedfrom3D7genomicDNAusingtheoligonucleotidepairs(Forward

PrimerP7andReversePrimerP8,seeprimerlist),whichalsoencodedrestriction

sitesBglIIandXhoI forcloning.This fragmentwas ligated intotheBglII-andXhoI-

digestedpDC2-cam-CRT-GFPresultinginthefinalvectorpDC2-cam-AP2-G2-GFP.

To create the pfap2-g2 disrupted line, the ap2-g2 gene locuswas targeted

usingtheselectionlinkedintegration(SLI)method(Birnbaumetal.2017).Todothis

a500bpfragmentofthe5’endofpfap2-g2wasamplifiedusingaforwardprimer

with a NotI restriction site aswell as a single pointmutation in the homologous

sequencetointroduceastopcodon(ForwardPrimerP9)andareverseprimerwith

anMluIsite(ReversePrimerP10)tobeligatedintothefinalvector.Thisfragment

wascloned(inframe)intopSLI-TGDusingNotIandMluIrestrictionsitesresultingin

thefinalvector,pSLI-TGD-AP2-G2.

Culturing,synchronization,transfection,andcloningofPlasmodium

falciparumparasites

Plasmodium falciparum parasites were cultured as previously described

(Trager&Jensen,1976)andmaintainedin6%O2and5%CO2andgrowninsterile

filteredRPMI1640media(Gibco)supplementedwith2MHEPES,NaHCO3(2g/L),0.1

Mhypoxanthine,0.25%(w/v)AlbumaxII(Gibco),andgentamycin(50ug/ml).The

parasites were maintained at a hematocrit of 3% and were kept between 2-5%

parasitemiaorgreaterbasedon theneedsof the individual experiments.Parasite

synchronizationwasperformedusing5%sorbitol(LambrosandVandenberg1979).

TogeneratetransgenicAP2-G2::GFPandAP2-G2KOlines,transfectionswere

carriedoutaccordingtoastandardprotocol(Deitsch,Driskill,andWellems2001).

The plasmid pDC2-cam-AP2-G2-GFP was transfected into the 3D7 strain of P.

falciparum whereas pSLI-TGD-AP2-G2 was transfected into the 3D7 clone, E5



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(Rovira-Graells et al. 2012). Inbrief,100µgofplasmidDNA (maxipreppedusing

Qiagen kit) was preloaded into O+ RBCs at 50% hematocrit in cytomix prior to

culturing with parasite-infected RBCs. After reinvasion, media containing 2.5 nM

WR99210 was added to select for transgenic parasites. Stable integrants were

maintained under constant drug pressure and were PCR-verified using specific

primers(P1,P2,P4,P5,P6)outsidethehomologyregionaftertheisolationofgDNA

(DNeasyBloodandTissuekit,Qiagen).TheresultingPfAP2G2::GFPandpfap2-g2ko

parasiteswereclonedbylimitingdilution(Rosario2008)andverifiedbydiagnostic

PCR(SupplementaryFig.1&2A)andwholegenomesequencing.Sequencinganalysis

wasdonebycreatingareferencegenomewithGFPandrestof theplasmidat the

expectedlocationandreadswerealignedtoit.

Plasmodiumfalciparumgametocyteproduction

Gametocytesweregeneratedusingthenutrientdeprivationinductionmethod

asdescribed(Miaoetal.2013).Briefly,synchronizedtrophozoiteswereculturedat

~2% parasitemia using 50% fresh RBC at 4% hematocrit. When the parasitemia

reached7–10%,gametocyteproductionwas inducedbynutrientdeprivation.The

followingday,stressedschizontsweresplit50%intotwoflasksbyaddingfreshblood

andfreshmedia.Thefollowingday,ring-stagecommittedgametocyteswerecounted

asDay1ofgametocytogenesis.Topreventthereinvasionandpropagationofasexual

parasites,parasitesweretreatedwithmediawithheparin(20U/ml)fromDay1post

induction through Day 4. The media was changed daily to ensure the proper

developmentandgrowthofthegametocytes.

Nuclear and cytoplasmic protein extraction from parasites and western

blotting

Nuclearandcytoplasmicextractsfromparasiteswerepreparedaspreviously

described(Vossetal.2002)using5-7%ofthesynchronizedparasites.Theproteins

fromthenuclearandcytoplasmicfractionswererunonagel(miniPROTEANprecast

TGXgels) and then transferred toanitrocellulosemembrane.Themembranewas

thenblockedusing5%non-fatmilkpowderin1XPBS-0.05%Tweenfor45minutes



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atroomtemperature.Themembranewasthenincubatedovernightat4oCwithan

anti-GFP (ROCHE Anti-GFP, from mouse IgG1K, Millipore Sigma) (1:1000), anti-

histone (Anti-Histone H3 antibody, AbCam) (1:3000), anti-aldolase (Anti-

Plasmodium aldolase antibody (HRP), AbCam) (1:1000) primary antibody in the

blocking buffer. After incubation, themembranewaswashed three timeswith1X

PBS-0.05%Tween andwas incubatedwith the appropriate secondary antibodies,

thenwashed.Thesecondaryantibodiesusedwereasfollows:peroxidase-conjugated

goatanti-ratHRPconjugate(Millipore)/goatanti-rabbitHRPconjugate(Millipore)

/goatanti-mouseHRPconjugate(Pierce)wasused(1:3000).Boundantibodieswere

thenvisualizedonafilmafterenhancingthesignalwithECL(Pierce)asthesubstrate.

Parasitegrowthassay

Tocomparethegrowthratesofpfap2-g2KOandWTparasitesweusedaSYBR

greengrowthassay(Vossenetal.,2010).Parasitesweresampledevery24hoursfor

10 days, starting at 0.1% parasitemia at the trophozoite stage. Both theWT and

PfAP2-G2knockoutstrainsweresynchronizedusing5%sorbitol(describedabove)

3xbeforethestartofthetimecourse.Parasiteswereseededina25cm2cultureflask

at0.1%parasitemiaand3%hematocrit.Toevaluategrowthdifferencesevery24

hoursfor10days,100μlofparasiteculturewascollectedintriplicate,transferredto

a96-wellplate,andstoredat-80oC.Followingthecompletionofthetimecourse,the

parasites were thawed at room temperature, 100 μl of SYBR green I (Molecular

Probes,EugeneOregon)wasdilutedintoparasitelysisbuffer(0.2μlofSYBRGreen

I/mloflysisbuffer)andaddedtoeachwellandmixed.Theplateswereincubatedat

37Cfor2hours.CellgrowthwasmeasuredasquantificationofDNAbySYBRgreenI

usingaTecanGENiosmicroplatedetectiondeviceattheexcitationwavelengthand

emissionwavelengthof485nmand535nm,respectively.Allvalueswereplottedas

anaverageofthreetechnicalreplicates(±S.D.)usingGraphPadPrism(version7).

RNApurificationandcDNAsynthesisforDNAmicroarrays

Total RNAwas extracted from tightly synchronized PfAP2-G2::KO andWT

parasitesevery6hours,startingat3hpi,throughoutthe48hourasexuallifecycle.A



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separatetimecoursewasdesignedtocollectRNAfromgametocytesstartingatDay1

and every 12 hours for the following 7 days. RNA was extracted from parasite-

infected RBCs collected at each timepoint using TRIzol (ThermoFisher Scientific),

followingthemanufacturer’sprotocol.cDNAsynthesisandDNAmicroarrayanalysis

wascarriedoutaspreviouslydescribed(Painteretal.2013).Two-channelAgilent

DNAmicroarrays(AMADID037237)werehybridized,washed,andscannedonan

Axon 4200A scanner. Signal intensities for each gene were extracted from the

scanned image using Agilent Feature Extraction Software version 9.5. Detailed

microarrayprotocolscanbefoundathttp://llinaslab.psu.edu/protocols/.Thedata

wereanalyzedbySignificanceAnalysisofMicroarrays(SAM)(Tusheretal.2001)for

thesignificantly(>1.5log2FC,0.30FDR)changinggenesbetweentheWTandAP2-

G2KOlinesusingtwo-classpairedt-testthroughoutthetimecourse.Allheatmaps

wereclusteredusingCluster4.0andwasvisualizedusingJavaTreeView.

Chromatin immunoprecipitation and Library preparation for sequencing

(ChIP-Seq)

ChIPwas performed as described (Josling et al. 2020) using synchronized

trophozoiteandgametocytestagePfA2-G2-GFPparasites(around7%parasitemia).

ExtractedchromatinwasshearedinSDSlysisbuffer(200µl1x109trophozoitesand

5x108gametocytes)toobtainafragmentsizeof100-150bpusinganM220focused-

ultrasonicator(CovarisInc.)usingthefollowingsettings:peakpower75W,2%duty

factor,200cyclesperburstandtotaltreatmenttimeof600s.Sonicationtimewas

optimizedforPfAP2-G2usingthecrosslinkedDNAandshearingitfor0,4,8,10,12

and 15 minutes and then running it on agarose gel for the size estimate. For

immunoprecipitations, sheared chromatin was pre-cleared using 20 μl/ml of

magneticbeadsA/G(Millipore16-663)for2hoursat4oCwithgentleagitation.The

supernatantwascollectedand50μlofthealiquotwasremovedasinput.Therestof

the chromatin was incubated overnight at 4oC with 1 μg of polyclonal anti-GFP

antibody(AbcamChIPgrade,Abcam290)or,ascontrol,thesameamountofIgG.The

immunoprecipitatedantibody/chromatincomplexwascollectedusingProteinA/G

magneticbeads(Millipore16-663).TheinputandChIPsampleswerereversecross-



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linkedovernightat45oCusing0.2MfinalconcentrationofNaCl.Thesampleswere

thentreatedwithRNAase(30minat37oC)andproteinaseK(3µLof20mg/mLwith

2 hours incubation at 45oC) and purified by using the QIAGEN MinElute PCR

purificationkit.Librarywaspreparedasdescribedearlier(Santosetal.2017).The

finallibrarywasquantifiedusingaQubitfluorometerHDDNAkitandanalyzedusing

an Agilent DNA 1000 Bioanalyzer to assess the quality, size distribution, and

detectionofanyartifacts.SequencingwasperformedusinganIlluminaHiseq2500to

obtain150bpsingle-endreads.

ChIP-seqdataanalysis

ChIP-seqdataanalysiswasdoneusingtoolsinGalaxy(usegalaxy.org).Before

startingtheanalysis,thequalityofthesequencingreadswereassessedusingFastQC

and were trimmed to remove adapter and low quality bases using Trimmomatic

(Bolger, Lohse, and Usadel 2014). The resulting reads were mapped to the P.

falciparumgenome(Pf3D7v28,obtainedfromPlasmoDB)usingBWA-MEM(Liand

Durbin2009)andduplicatesreadswereremovedusingSAMtools(Lietal.2009).

MappedsequenceswereconvertedintobigwigfilesusingbamCoverage(Ramírezet

al. 2016) and were viewed in using the Integrative Genomics Viewer (IGV)

(Thorvaldsdóttir,Robinson,andMesirov2013)foreachinputandtreatment.MACS2

(Fengetal.2012)wasusedtocallpeakswithq-valuecutoffof0.05.Thecommon

overlapping intervalsbetweenreplicateswereestablishedusingMultiple Intersect

functionofBEDtools,andtheoverlappingintervalswerecombinedintoasinglefile

usingMergeBED (Quinlan andHall2010). The genes closest to the peak summits

wereidentifiedusingclosestBED.Peaksthatwerefartherthan2.5Kbwereremoved

fromtheanalysis.Whenthepeakwaspresentbetweentwogenesthenonlythegene

closesttothepeakwasconsideredfortheanalysis.

PeaksequenceswereextractedusingextractgenomicDNAtoolusinggenomic

coordinates,andmotifsassociatedwithcalledpeakswere identifiedusingDREME

(Bailey2011)andcomparedtoidentifiedApiAP2motifsusingTOMTOM(Guptaetal.

2007).ThecorrelationheatmapofthereplicatesperformedonGFPtaggedPfAP2-G2



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ChIP-seqwasmadeusingmultiBigwigSummaryandplotCorrelationinthedeepTools

suite(Ramírezetal.2016).

CorrelationanalysisofPfAP2-G2withotherhistonemarks

CorrelationswerecalculatedbetweentheoccupancyofPfAP2-G2andthatof

various histone marks previously reported in the literature, including H3K9me3,

H4ac,H4K20me3,H3K26ac,H3K9ac,H3K4me3(Karmodiyaetal.2015),H3K36me2

and H3K36me3 (Jiang et al. 2013) and heterochromatin protein 1 (Nicolas M B

Brancucci et al. 2014) First, read counts were obtained from PfAP2-G2 and nine

histone marks in the gene body of all P. falciparum genes (n=5735; PlasmoDB

annotations,release28.0).NCIS(LiangandKeleş2012)wasusedtoscaleacontrol

input experiment to each analyzed signal ChIP-seq experiment. Pairwise Pearson

correlationcoefficientofnormalizedreadcountsacross5735regionsforPfAP2-G2

and nine histone marks. Then, proteins were ordered using complete-linkage

hierarchicalclusteringwithEuclideandistance.

PfAP2-G2-GFPImmunoprecipitationandMassSpectrometry

Nuclear extraction was performed from the PfAP2-G2::GFP parasites as

previouslydescribed(Vossetal.2002).Adaybeforetheimmunoprecipitation~1mg

ofM-270EpoxyDynabeads(Invitrogen)per100mlofculturewereconjugatedwith

polyclonalanti-GFPantibodiesatconcentrationof5ug/mg(Abcamab290)orwith

IgG(Abcamab46540)overnightat30oCaspreviouslydescribed(Joshietal.2013).

Immunoprecipitation was carried out after washing the conjugated beads with

dilutionbufferandincubatingwithdilutednuclearextractsfor90minutesat4oC.The

beadswerewashed extensivelywith dilution buffer and 1X PBS and proteinwas

elutedusing20μlloadingbufferbyshakingfor10minutesat70°Cat1000rpmand

proteinwasstoredat-20oC.Sampleswerequalitycontrolledbywesternblottingand

subjectedtoin-geltrypticdigest.

Fortrypticdigestthesamplesinpolyacrylamidegelslicesweredestainedwith

50mMAmBic/50%ACN(Acetonitrile) anddehydratedusing100%ACN.Disulfide

bondsinproteinwerereducedwith10mMDTTandreducedcysteineresidueswere



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Page26of41

then alkylated with 50mM Iodoacetamide. Finally, the processed samples were

tryptic digested (Trypsin gold, 6ng/μl) overnight at 37oC on a thermomixer, and

peptideswereextractedfromthegels.ThedriedpeptidepelletswereanalyzedbyLC-

MS/MSusingQExactivemassspectrometer(IndianaUniversityCoreProteomics).

ThedatawasprocessedusingTrans-ProteomicPipeline(TPP)aspreviously

described with a fewmodifications (Deutsch et al. 2010). Spectrawere searched

againsttheP.falciparumproteomeandcontaminantrepositorydatabase(CRAPome)

using tandem and comet searches (Mellacheruvu et al. 2013). This search was

combinedinInterProphetandtheproteinsweresearchedwithProteinProphet.Only

proteinswitherrorratelessthan0.01werereported.Theimmunopurificationassay

was performed 3 times and the data was combined to identify the significantly

enrichedproteinsbetweentheIPandcontrolusingSAINT(Choietal.2011).Proteins

withprobabilityscoreof0.99wereconsideredforanalysis.

Additionalsupplementarymaterials

SupplementaryTable1:PfAP2-G2::GFPChIP-seqdataintrophozoitestageFirst

tabofthetablecontainsalltheupstreampeakscommonintwooutofthreebiological

replicates. Second tab has only 86 genes that were considered for the analysis.

Indicatedarecoordinatesofeachpeak,theorientationofthegene,genesdownstream

tothepeaksanddistanceofpeakandATGofthegene.

SupplementaryTable2:PfAP2-G2::GFPChIP-seqdataintrophozoitestageTable

containspeaksonthegene-bodycommonintwooutofthreebiologicalreplicates.

Also,indicatedarecoordinatesofeachpeakandgenescontainingthepeaks..

SupplementaryTable3:PfAP2-G2::GFPChIP-seqdataingametocytestageIII

Tablecontainsalltheupstreampeakscommonintwobiologicalreplicates.Indicated

arecoordinatesofeachpeak,theorientationofthegene,genesdownstreamtothe

peaksanddistanceofpeakandATGofthegene.



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SupplementaryTable4:PfAP2-G2::GFPChIP-seqdataingametocytestageIII

Table containspeakson thegene-bodycommon in twobiological replicates.Also,

indicatedarecoordinatesofeachpeakandgenescontainingthepeaks.

SupplementaryTable5:Listof thedifferentially expressed genes in asexual

blood stage Significance analysis of microarray (SAM) of the asexual stage

timecourse data showed that 327 transcripts were differentially expressed (>1.5

log2FC,0.30FDR)inthePfAP2-G2KOlinecomparedtoWT.Outofthese,237showed

enhanced transcript abundance in thePfAP2G2KO lineand90showeddecreased

abundance.Thefirsttabofthesheethasupregulatedgenesandthesecondtabhas

downregulatedgenesalongwithfoldchange,q-valueandlocalfalsediscoverrate.

Supplementary Table 6: List of the differentially expressed genes in

gametocytes Significance analysis of microarray (SAM) of the gametocyte

timecourse data showed that 444 transcripts were differentially expressed (>1.5

log2FC,0.30FDR)inthePfAP2-G2KOlinecomparedtoWT.Outofthese,274showed

enhancedtranscriptabundanceinthePfAP2G2KOlineand170showeddecreased

abundance.Thefirsttabofthesheethasupregulatedgenesandthesecondtabhas

downregulatedgenesalongwithfoldchange,qvalueandlocalfalsediscoverrate.

SupplementaryTable7:ListofPfAP2-G2interactingpartnersFirsttabcontains

thelistofpeptidesobtainedintheintheupperbandofPfAP2-G2andthesecondtab

hasthelistofpeptidesobtainedintheinthelowerbandofPfAP2-G2alongwiththe

spectralcounts,averageandmaximumprobabilityandFDR.Onlythegeneswithhigh

probability of interaction measured by SAINT (pSAINT 0.9 and 1%FDR) was

consideredfortheanalysis.

Acknowledgements

ThisprojectwaslargelyfundedbyNIH/NIAID1R01AI125565(ML).NYandSMwere

supported by R01 GM121613 (SM). We thank Kelly Rios for assistance with

proteomicdataanalysis.



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Page28of41

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Anti-GFP

Anti-HistoneH3

Anti-Aldolase

B

MW Cytoplasmic Nuclear kDa AP2-G2 3D7 AP2-G2 3D7

C

Ring (2-5 h)

250 150 100

75

50

GFP Nuclear DIC Merge (green) (red)

FL (250kDa) CP (50kDa)

Figure 1 PfAP2-G2 is expressed from around 16-19 hpi in asexual stage parasite (A) Structure of PfAP2-G2 protein which contains single AP2 DNA-binding domain. (B) Live fluorescence microscopy performed on a highly synchronized population of AP2-G2::GFP parasites every 7 h of the asexual life cycle showed that PfAP2-G2::GFP colocalizes with the nucleus in both the trophozoite and schizont stages. DRAQ-5 was used as the nuclear stain. (C) Nuclear and cytoplasmic fractions from the GFP tagged PfAP2-G2 synchronized trophozoite stage parasites were subjected to western blot analysis using anti-GFP antibodies (1:1000). Full length (FL) GFP-tagged PfAP2-G2 (~250 kDa) was detected only in the nuclear fraction of the parasite lysate whereas cleavage product (CP, ~50kDa) was in both fractions. WT parasites were used as a negative control. Anti-histone H3 (1:3000) and anti-aldolase (1:1000) were used as loading controls for the nuclear and cytoplasmic fractions, respectively.

A ap2-g2gene

189kDa,1702aaAP2domain

Late Ring (9-12 h)

Early Troph (16-19 h)

Late Troph (23-26 h)

Early schizont (30-33 h)

Late schizont (37-40 h)



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0 2 4 6 8 10210

215

220

225

Days

WT (E5)

AP2-G2 KO

A

WT (E

5)

AP2-G2 K

O0

2

4

6

8WT (E5)

AP2-G2 KO

Rin

g pa

rasi

tem

ia (%

)

Fluo

resc

ence

uni

t WT (E5) AP2-G2 KO

Days

B

Figure 2 Absence of PfAP2-G2 has no effect on the growth of asexual stage parasite (A) Growth profiles of the WT and PfAP2-G2 KO parasites measured using an SYBR green assay every 24 hours over a period of 10 days starting with highly synchronous ring-stage parasites. The graph represents the plotted average +/-S.D. of biological triplicates. Fluorescence units are plotted along the Y-axis and the number of days was plotted along the X-axis for the experiment done in triplicate. (B) Bar graph representing the measurement of the multiplication rate of the WT and PfAP2-G2 KO parasites. The values are the average of three replicates, and the error bars represent the standard deviation.

WT (E5) AP2-G2 KO



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GFP Nuclear DIC Merge (green) (red)

Stage I Day 1

250 150 100 75 50

MW PfAP2-G2 GFP kDa Cytoplasmic Nuclear

Wildtype

AP2-G2 KO

GC Stage I II III IV V

A B

C

Figure 3 PfAP2-G2 is expressed throughout the gametocyte stages and is essential for maturation of gametocytes (A) Images obtained by live fluorescence microscopy performed on Stage I through Stage V gametocytes. The DIC image was merged with the DRAQ5 nuclear stain imaging to confirm the nuclear localization of the signal. The results indicate that PfAP2-G2 was expressed in all stages of the gametocytes and was localized to the nucleus. (B) Nuclear (N) and cytoplasmic (C) fractions from the stage III gametocytes were subjected to western blot analysis using anti-GFP antibodies (1:1000). The GFP-tagged AP2-G2 of expected size (~250 kDa) was detected only in the nuclear fraction of the parasite lysate. The WT parasite was used as a negative control. Anti-histone H3 (1:3000) and anti-aldolase (1:1000) were used as the loading controls for the nuclear and cytosolic fractions, respectively. (C) Giemsa-stained smears of gametocytes from the WT and PfAP2-G2 KO lines showed that the morphology of the KO gametocytes appeared normal until Stage III. The KO gametocytes were unable to develop beyond Stage III and showed severe morphological defects.

Stage II Day 3

Stage III Day 5

Stage IV Day 7

Stage IV Day 9

Stage V Day 11

Anti-GFP

Anti-HistoneH3

Anti-Aldolase

FL (250kDa)



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A B GFP 3 2 1 In 3 1 2

In 3 In 1 In 2 GFP 3 GFP 2 GFP 1 0.0 0.2 0.4 0.6 0.8 1.0

Motif enriched in PfAP2-G2 binding

regions

PBM motif

PfAP2-G2 motif 1770/5008

P-value 8.7e-088

C Input 1 AP2-G2 GFP 1 Input 2 AP2-G2 GFP 2 Input 3 AP2-G2 GFP 3 Gene Called Peaks Motif PF3D7_1216600 (CelTOS) Motif AACCA

D Input 1 AP2-G2 GFP 1 Input 2 AP2-G2 GFP 2 Gene (PF3D7_0501300) Called Peaks Motif AACCA

F

E

G

Motif enriched in PfAP2-G2 binding regions PF11_1139300 PBM motif

252/945 P-value 4.8e-034

PF3D7_1139300 1790/5008

P-value 5.2e-129

PF3D7_0304600 (CSP) Motif AACCA

2258 652

13

Genes with peaks in gametocytes

Genes with peaks in asexuals

correlation

Input Asexual AP2-G2 GFP Asexual Input Gametocytes AP2-G2 GFP Gametocytes Gene (msp2) Called peak



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Figure 4 Genome wide predictions of PfAP2-G2 targets and binding regions by ChIP seq (A) Pearson correlation analysis on three biological replicates of ChIP-seq including the Input (In) and AP2-G2 GFP (GFP) (B) DREME logo for the motif enriched in the genomic locations bound by PfAP2-G2 in the in the gene body and non-coding regions in asexual stage. Shown is the enriched PF11_1139300 motif AGAA and PfAP2-G2 motif ACCA obtained by DREME along with the PBM motif. The P-values are reported by DREME. (C) Example PfAP2-G2 peaks in asexual stages for all three replicates. Peaks are represented as red tracks below with the motif present is highlighted in blue. (D) Example PfAP2-G2 peaks in gametocytes for both replicates. (E) Venn diagram showing common genes with PfAP2-G2 peaks in asexuals and gametocytes (F) Representative peaks for the gene msp2 enriched with PfAP2-G2 in both asexual and gametocyte stages. (G) DREME logo for the motifs enriched in the genomic locations bound by PfAP2-G2 in the in the gene body and non-coding regions in gametocytes. The P-values are reported by DREME.



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A

B C

Significantly changing genes in asexual stages

alpha tubulin 2, dynein heavy chain, pfs25, pfs28, pf11-1

Pfmc-2tm, unknown exported proteins (phist and hyp), pfemp1, rifins, stevors

Late gametocyte genes (208)

Decreased abundance in gametocytes (170)

increased abundance decreased abundance

166 128 42

Decreased abundance in gametocytes (170)

Decreased abundance in asexuals (90)

126 46 44

2642

2714 10

78

4233

9 5 1 616 16

020406080100

12

89

29 33

1030 33

52

7 7 315

52

30

020406080

100

Significantly changing genes in gametocyte stages

(van Biljon et al., 2019) ( this study)

FPK

M

FPK

M



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D

E sera7scdmsp2Riprrh4spectcrmp2cap380kharpP36csppfemp1

(Transcript abundance)

20-2

TP 3 9 15 21 27 33 39 45

log2 FC (KO/WT)

82gen

es

PF3D7_0212600 secreted protein with altered thrombospondin repeat domain PF3D7_0207400 serine repeat antigen 7 PF3D7_0216800 TMEM121 domain-containing protein, putative PF3D7_0202000 knob-associated histidine-rich protein PF3D7_0200100 erythrocyte membrane protein 1, PfEMP1 PF3D7_0323400 Rh5 interacting protein PF3D7_0320400 oocyst capsule protein Cap380 PF3D7_0304600 circumsporozoite (CS) protein PF3D7_0301800 Plasmodium exported protein, unknown function PF3D7_0511200 stearoyl-CoA desaturase PF3D7_0632800 erythrocyte membrane protein 1, PfEMP1 PF3D7_0702100 Plasmodium exported protein (PHISTb), unknown function, pseudogene PF3D7_0800100 erythrocyte membrane protein 1, PfEMP1 PF3D7_0931300 conserved Plasmodium protein, unknown function PF3D7_0936100 early transcribed membrane protein PF3D7_1016500 Plasmodium exported protein (PHISTc), unknown function PF3D7_1102000 DnaJ protein, putative PF3D7_1100100 erythrocyte membrane protein 1, PfEMP1 PF3D7_1252900 Plasmodium exported protein, unknown function PF3D7_1327100 conserved protein, unknown function PF3D7_1447500 inner membrane complex protein, putative PF3D7_1455600 ferlin, putative PF3D7_1477800 acyl-CoA binding protein

Genes with enhanced transcript abundance in

asexual stages (236) ChIP targets in asexual stages (82) 23 213 59

List of the target PfAP2-G2 genes in asexual stages with enhanced transcript abundance

Expression fold

change for PfAP2-G2

targets



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Figure 5 Transcriptional changes in absence of PfAP2-G2 (A) Classification of significantly changing genes in asexual and gametocyte stages based on its maximum transcription in either asexual, gametocytes or mosquito stages, using RNA-seq data (López-Barragán et al., 2011). (B) Overlap between the genes with decreased transcript abundance (>1.5 log2FC, 0.30 FDR) in gametocytes and earlier established late gametocyte markers (van Biljon et al., 2019). (C) Venn diagram showing overlap between genes with decreased transcript abundance in gametocytes and asexual stages. (D) Venn diagram showing number of overlapping genes with PfAP2-G2 enrichment in the upstream regions and also increased transcript abundance (analyzed by SAM, >1.5 log2FC, 0.30 FDR) in absence of PfAP2-G2. (E) Expression fold change (log2 KO versus WT) for the genes that are bound by PfAP2-G2 in the upstream regions in asexual stages. The genes are clustered based on Pearson’s correlation. Boxed is the timepoint at which ChIP-seq was performed.



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A

PCC

H3K9me3

HP1

AP2-G2

H4ac

H4K20me3

H3K36me3

H3K27ac

H3K9ac

H3K4me3

H3K36me2

H3K

9me3

H

P1

AP2

-G2

H4a

c H

4K20

me3

H

3K36

me3

H

3K27

ac

H3K

9ac

H3K

4me3

H

3K36

me2

P value < 0.001

AP2-G2 Input

AP2-G2 GFP

Input

H3K36me3

H4K20me3

Chr 4

B

C

AP2-G2Input AP2-G2 GFP

Input

PfHP1

Chr 4

D

E

H3K36me3 binding locations

8099 (Karmodiya et al., 2015)

PfAP2-G2 binding locations in gene bodies

4600

PfHP1 binding locations 737

(Brancucci et al., 2014) 475

Pear

son

corr

elat

ion 3633

PfAP2-G2 binding locations in gene bodies

4600



https://doi.org/10.1101/2020.10.27.355685


Figure 6 PfAP2-G2 shows high correlation with H3K36me3 repressive histone modifications (A) Heatmap showing correlation between occupancy of PfAP2-G2 and nine histone modifications in the gene body of all P. falciparum genes (n=5735). PfAP2-G2 strongly correlates with H3K36me3 and H4K20me3 (B) Venn diagram comparing the peaks obtained by ChIP-seq on H3K36me3 marks (oange) (Jiang et al., 2013) and PfAP2-G2-GFP (green) shows that 80% of times PfAP2-G2 shows co-occupancy with H3K36me3 marks. (C) IGV screenshot showing co-occupancy of PfAP2-G2 with H3K36me3 and H4K20me3. First gray track represents Input for PfAP2-G2 and second green track shows the regions bound by PfAP2-G2. Third gray track represents input for H3K36me3 and H4K20me3 and the last two orange tracks represent the regions bound by H3K36me3 and H4K20me3 as indicated in the figure. Blue track shows the genes. (D) IGV screenshot showing co-occupancy of PfAP2-G2 with PfHP1. First gray track represents Input for PfAP2-G2 and second green track shows the regions bound by PfAP2-G2. Third gray track represents input for PfHP1 and the last two pink tracks represent the regions bound by PfHP1 as indicated in the figure. Blue track shows the genes. (E) Venn diagram comparing the peaks obtained by ChIP-seq on PfHP1 marks (pink) (Brancucci et al., 2014) and PfAP2-G2-GFP (green) shows that 80% of times PfAP2-G2 shows co-occupancy with H3K36me3 marks.



https://doi.org/10.1101/2020.10.27.355685


Input GFP IgG Elution

250 kDa

50 kDa

A

C

B

Number of PfAP2-G2 peptides detected in IP vs control 9|5|16 0|0|0

Upper band ~1940 aa

Number of PfAP2-G2 peptides detected in IP vs control 9|5|16 0|0|0

Lower band ~400 aa

400 aa

GeneID GeneDescription PeptidesinreplicateIPs

Peptidesincontrol

AverageProbability

PF3D7_1105100 histoneH2B(H2B) 24|11|14 0|2|1 1

PF3D7_1441400FACTcomplexsubunitSSRP1,

putative(FACTS) 17|11|11 0|2|2 1

PF3D7_0617900histoneH3variant,putative

(H3.3) 16|8|9 0|0|0 1

PF3D7_0917900 heatshockprotein70(HSP70) 13|8|12 0|0|0 1

PF3D7_0517400FACTcomplexsubunitSPT16,

putative(FACTL) 19|8|12 0|0|0 1

PF3D7_135880040SribosomalproteinS15

(RPS15) 5|5|5 0|0|0 0.9997

PF3D7_1105000 histoneH4(H4) 37|14|22 0|3|6 0.9993

PF3D7_1220900heterochromatinprotein1

(HP1) 13|5|6 0|1|0 0.999

PfAP2-G2 interacting partners in the lower band



https://doi.org/10.1101/2020.10.27.355685


GeneID GeneDescription PeptidesinreplicateIPs

Peptidesincontrol

AverageProbability

PF3D7_0624600 SNF2helicase,putative(ISWI) 28|14|27 0|1|5 1

PF3D7_1212700eukaryotictranslationinitiationfactor3subunit

A,putative(EIF3A) 13|17|22 0|0|1 1

PF3D7_0607000 translationinitiationfactorIF-2,putative 11|5|14 0|0|0 1

PF3D7_0711000 AAAfamilyATPase,CDC48subfamily(Cdc48) 7|10|10 0|2|0 0.9997

PF3D7_0710200conservedPlasmodiumprotein,unknown

function 6|6|23 0|0|0 0.9997

PF3D7_1104200 chromatinremodelingprotein(SNF2L) 15|7|12 0|3|0 0.9993

PF3D7_1007700 transcriptionfactorwithAP2domain(s)(ApiAP2) 13|6|6 0|0|1 0.9993

PF3D7_0517400 FACTcomplexsubunitSPT16,putative(FACT-L) 7|4|8 0|0|0 0.999

PF3D7_1468100 MORCfamilyprotein,putative 14|8|17 0|1|3 0.9983

PF3D7_1433500 DNAtopoisomerase2(TOP2) 9|8|16 0|0|4 0.9983


function 4|5|14 0|0|0 0.998

PF3D7_0823300 histoneacetyltransferaseGCN5(GCN5) 5|5|8 0|1|0 0.998

PF3D7_0500800matureparasite-infectederythrocytesurface

antigen(MESA) 36|28|42 0|8|7 0.9977

PF3D7_0811200ERmembraneproteincomplexsubunit1,

putative(EMC1) 5|3|7 0|0|0 0.9977


function 4|7|12 0|0|0 0.9973

PF3D7_0610900transcriptionelongationfactorSPT5,putative

(SPT5) 5|7|8 0|1|0 0.9957

PF3D7_1023900chromodomain-helicase-DNA-bindingprotein1

homolog,putative(CHD1) 30|12|33 0|3|4 0.9933

PF3D7_1139300 transcriptionfactorwithAP2domain(s)(ApiAP2) 2|2|3 0|0|0 0.967

D

Figure 7 Identification of potential interacting partners of PfAP2-G2 (A) SDS PAGE showing immunoprecipitation (without crosslinking) of the PfAP2-G2 GFP parasite line using antibodies against GFP. Full length PfAP2-G2 and the cleaved product was analyzed separately by mass spectrometry to identify the interacting partners of PfAP2-G2. (B) Schematic showing the peptides recovered from upper and lower band. Also shown are the number of PfAP2-G2 peptides in both the bands and in the controls. (C) List of PfAP2-G2 interacting partners in the lower band with high probability of interaction measured by SAINT (pSAINT 0.9 and 1%FDR). (D) List of PfAP2-G2 interacting partners in the upper band with high probability of interaction measured by SAINT (pSAINT 0.9 and 1%FDR) in the upper band.

PfAP2-G2 interacting partners in the upper band.CC-BY-NC-ND 4.0 International licenseavailable under a

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The PfAP2-G2 transcription factor is a critical regulator ...Oct 27, 2020 · PfAP2-G2 (PF3D7_1408200) plays a critical role in the maturation of Plasmodium falciparum gametocytes