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The PfAP2-G2 transcription factor is a critical regulator ...Oct 27, 2020 · PfAP2-G2...
Transcript of The PfAP2-G2 transcription factor is a critical regulator ...Oct 27, 2020 · PfAP2-G2...
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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]
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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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Page23of41
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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Page24of41
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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Page25of41
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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Page27of41
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
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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.
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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
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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
PF3D7_1008100conservedPlasmodiumprotein,unknown
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
PF3D7_1462300conservedPlasmodiumprotein,unknown
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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