METTL3 safeguards cell identity and epigenome of human trophoblast stem cells

Domain: biology
Published: 2025-12-16
Editorial outcome: major_revision

Abstract
N6-methyladenosine(m6A)isthemostprevalentRNAmodificationandplays
diverserolesinmammaliancells.However,itssignificanceinhumantropho-
blastlineagecommitmentandplacentadevelopmentremainspoorlyunder-
stood.HereweshowthatMETTL3,theprimaryenzymeresponsibleform6A
depositions,specificallymarkscytotrophoblastsinhumanplacenta,and
exhibitsdefectiveexpressionincytotrophoblastsfrompreeclampsiapatients.
Throughin-depthinvitroinvestigation,weidentifycriticalrolesofMETTL3in
generatinghumantrophoblaststemcells(hTSCs)fromhumanexpanded
potentialstemcells,andinmaintaininghTSCs’self-renewalandidentity.In
hTSCs,METTL3lossinducespro-inflammatoryresponsesandpromotespre-
maturesenescence,leadingtopreeclampsia-associatedtranscriptomicsig-
nature.Mechanistically,METTL3-dependentm6Amethylationdirectly
regulateshistonemodifierssuchasEP300andEZH2tomaintainepigenetic
homeostasis,aswellasvarioustranscriptionfactors,includingJUND.Targeted
inhibitionofEP300inhTSCssuccessfullyamelioratepreeclampsiasignature
causedbyMETTL3deficiency.ThesefindingsrevealMETTL3-dependentm6A
methylationasacriticalregulatorofhumantrophoblastdevelopmentandits
potentialinvolvementinpregnancy-relateddiseases.

Introduction
Theplacentaisatransientorganthatconnectsthedevelopingembryo
tothematernalsystemsduringgestation.Itsupportsfetalgrowthby
facilitatingnutrientsexchangeandoxygensupplywhileprotectingthe
fetusfromthematernalimmunesystem.Defectiveplacentafunction
and development canresult in pregnancy-associated complications,
suchaspreeclampsia(PE)1,2,fetalgrowthrestriction3,andintrauterine
lethalityand miscarriage4. The functions ofthehumanplacenta are
mediatedbyextensiveproliferationanddifferentiationoftrophoblast
cells.Threemaintypesoftrophoblastcellsintheplacentahavebeen
identified:cytotrophoblasts(CTBs),syncytiotrophoblasts(STBs),and
extravilloustrophoblasts(EVTs). CTBs are multipotent progenitors
that can differentiate into both STBs and EVTs within the rapidly
developingplacenta5.STBsaremultinuclearandareresponsiblefor
hormone secretion, while EVTs invade the maternal decidua and
remodel the maternal vasculature5. Normal pregnancy involves
senescenceintheplacentaasaphysiologicalprocess6.Forinstance,
EVTsacquireasenescencephenotypeduringplacentationduetotheir
intrinsicgenomeamplificationfeature7.
Recentadvanceshaveestablishedhumantrophoblaststemcells
(hTSCs)derivedfromhumanembryoandplacentatissues8,aswellas
humanpluripotentstemcells(hPSCs)9–12orreprogrammingsomatic
cells13.hTSCsexhibitcharacteristicsofinvivoCTBsandserveasan
in vitro model for studying human placenta development and the
pregnancycomplications8.Notably,bonafidehTSCscanbederived
from human expanded potential stem cells (hEPSCs)11, providing a
valuable resource for studying human trophoblast lineage
differentiation14,15 and clinical applications such as viral infection12,
diseasemodelling16,anddrugscreening17.
The importance of RNA modifications in regulating chromatin
status,genetranscription,andtranslationfordiversecellularactivities
has been well documented18–20. N6-methyladenosine (m6A)
methylation is primarily catalyzed by the methyltransferase like 3
(METTL3) and 14 (METTL14) methyltransferase writer complex18,21,
playing a critical role in RNA stability22,23, chromatin state24–29, and
translationefficiency23,30.m6Amodificationiscriticalfordevelopment
suchasmaternal-to-zygotictransition31,32,stemcelldifferentiation33,34
and hematopoiesis35–37. Dysregulation of m6A modification is impli-
catedinobesity,cancer,andotherdiseases38.METTL3hasalsobeen
reportedtoregulateagingandsenescenceprocessinhumancellsinan
m6A dependent or independent manner39–41. However, its role in
human trophoblast lineage development under physiological and
pathologicalconditionsremainspoorlyunderstood.
In this study, we systematically investigated the functions of
METTL3andRNAm6Amethylationinthehumantrophoblastdevel-
opmentfromhEPSCs,hTSCs,andthehumanplacenta.Single-nucleus
RNAsequencedataandimmunostainingresultsrevealedthatCTBsin
the human placenta are specifically marked by high METTL3 and
METTL14expressions.Adoxycycline-inducibleknockout(iKO)system
demonstratedtheessentialrolesofMETTL3inhTSCgeneration,self-
renewal,andmaintenanceofcellularidentity.Notably,METTL3actsas
agatekeeperinhTSCsforcellsurvivalandforpreventingpremature
senescence.ItsdepletioninhTSCsleadstoPE-likefeatures,andinhi-
bitingEP300efficientlymitigatestheobservedMETTL3inactivation
phenotypes.Mechanistically,METTL3-dependentRNAm6Amethyla-
tiondirectlyregulateshistonemodifierssuchasEP300andEZH2to
maintainepigenetichomeostasis,aswellasvarioustranscriptionfac-
tors,includingJUND.Additionally,invivoCTBsfromPEpatientsshow
reduced METTL3 expression alongside elevated H3K27ac level. Col-
lectively,theseresultshighlightthecrucialrolesofMETTL3andRNA
m6A methylation in human placentation and possibly in the patho-
genesisofpregnancy-relateddisorders.

Results
METTL3specificallymarkscytotrophoblastsinhumanplacenta
InordertoinvestigateMETTL3expressionduringpregnancy,were-
analyzedthepublishedsingle-nucleusRNAsequence(snRNA-seq)data
ofhumanplacenta42.Thereporteduniformmanifoldapproximation
andprojection(UMAP)wassuccessfullyreproducedfortheearlyand
latepregnancywiththepreviouslydefined11cellclusters42(Fig.1aand
Supplementary Fig. 1a). These single-nucleus data captured tropho-
blastcellsfromCTBstomatureSTBs,profilingthetrophoblastsyn-
cytialization and placentation process (Fig. 1a and Supplementary
Fig.1a).TheanalysisrevealedthatMETTL3andMETTL14wereexclu-
sivelyexpressedinCTBsandproliferation-associatedCTBs(pCTBs),as
identified by the CTB marker PEG1043 and the absence of the STB
markergeneSDC18(Fig.1b,candSupplementaryFig.1b).Interestingly,
both METTL3 and METTL14 expressions significantly declined when
CTBstransitionedtothecompetencestageforsyncytialization,cor-
respondingtoclusters11and9inearlyandlatepregnancy,respec-
tively (Fig. 1b and Supplementary Fig. 1b). Conversely, m6A
demethylasesFTOandALKBH5showedanincreasedexpressiondur-
ingSTBmaturation,particularlyin late pregnancy(Fig.1bandSup-
plementaryFig.1b).Invivohumanplacentalvilliat8weeksexhibited
METTL3andMETTL14proteinsexclusivelyinCTBs,co-localizedwith
PEG10butnothCG(Fig.1d,e).Inlatepregnancy,METTL3remained
exclusively expressed in CTBs alongside PEG10, even though the
overall CTB population was markedly diminished (Supplementary
Fig.1c,d).ThesefindingsdemonstratethatMETTL3specificallymarks
CTBsinhumanplacentas,suggestingitspotentialrolesinregulating
CTBscellularidentityandtheirdifferentiationcapacity.

METTL3andm6AmethylationdecreaseduringhTSCstoSTBs
differentiationinvitro
hTSCs,asaninvitromodelofCTBs,candifferentiateintoSTBsand
EVTs8,11,12.Inlinewiththeinvivoobservation,immunofluorescenceand
western blotting analysis confirmed elevated METTL3and METTL14
expression levels in hTSCs compared to the differentiated STBs
(Fig. 2a–c). Re-analysis of the published RNA-seq data on hTSCs to
STBs differentiation12 also revealed a decline in the expression of
METTL3andMETTL14duringtheprocess(Fig.2d).Additionally,sev-
eralm6Areadergenes,includingYTHDC2,YTHDF2/3,andIGF2BP1/3,
andm6AerasergeneFTOshoweddecreasedexpression(Fig.2d).Asa
result, m6A level markedly decreased during STBs differentiation
(Fig.2e).Thesefindingssuggestadownregulationofthewholem6A
methylationpathwayduringSTBsdevelopmentfromhTSCs.
Tofurtherelucidatem6Adynamicsduringthisprocess,weper-
formed methylated RNA immunoprecipitation sequencing (MeRIP-
seq) on hTSCs originally derived from in vivo placental villi44 (CT-
hTSCs)andtheirdifferentiatedday6STBs.Theresultsdemonstrateda
substantialdecreaseinm6A signalson the 3’ UTRsof transcripts in
STBs compared to hTSCs (Fig. 2f). Specifically, 15,725 m6A-modified
peakswereidentifiedinhTSCs,whereas12,449peaksweremodified
withm6AinSTBs,with5660and2384peaksbeingspecifictohTSCs
andSTBs,respectively(Fig.2gandSupplementaryTables3and4).De
novomotifanalysisofthesepeaksrevealedtheclassicalm6A“RRACH”
motif(RdenotesAorG,andHdenotesA,C,orU)inbothcells(Fig.2f).
Additionally,4463peakslostm6AmodificationduringhTSCsdiffer-
entiation to STBs, while 511 peaks gained m6A (Fig. 2h and Supple-
mentaryData1).Forexample,markergenesforhTSCs,suchasPEG3,
PEG10,andMKI67,exhibitedm6AmodificationinhTSCsbutshoweda
substantialdecreaseinSTBs(Fig.2h,i).Severalkeytrophoblastmaster
regulatorgenes,includingGATA2,TFAP2A,andTFAP2C,alsodisplayed
m6A methylation in hTSCs, with no significant m6A level changes
observedupondifferentiation(Fig.2h,j).Althoughmosttranscriptsin
STBslostm6Amodification,anotablenumberoftranscripts,suchas
TNFRSF10B,GADD45G,andH3C4,gainedm6Amodification(Fig.2h,k).
Interestingly,transcriptsofCGAandCGBclusters,whichencodethe
hormone hCG secreted by STBs, lacked m6A modification in both
hTSCsandSTBs(Fig.2l),suggestinghCGregulationmaynotinvolve
m6Amethylationduringpregnancy.Theseresultsdemonstratethat,
despitethedistinctm6AmethylationprofilesobservedinhTSCsand
STBs, both METTL3 expression level and overall m6A methylation
declineduringthedifferentiationprocess.

METTL3isrequiredinhTSCsgenerationfromhEPSCs
ToexploretheroleofMETTL3inhumantrophoblastlineagesdevel-
opment,weestablishedaninvitrocellularmodeltogeneratehTSCs
fromhEPSCs,whichpossessesbothembryonicandextraembryonic
lineage potentials11,12,45. To this end, a doxycycline (DOX)-inducible
METTL3expressionsystemwasdevelopedinMETTL3-knockout(KO)
hEPSCs(SupplementaryFig.2a). Ourpreviousstudysuggestedthat
withdrawal of DOX in hEPSCs led to METTL3 transgene expression
ceasing,resultingincellproliferationdefectsandfailuretoundergo
properdifferentiation14.Thedefectivetrophoblastgenerationwasalso
confirmed by immunofluorescence and western blotting, which
revealedthedecreasedexpressionoftrophoblastmasterregulators
GATA2 and GATA3, while NANOG, a pluripotency factor, remained
expressed(SupplementaryFig.2b–d),consistentwiththeconstitutive
Nanog expression in Mettl3-KO mouse embryonic stem cells33,46.
Additionally,hCG,secretedbymaturetrophoblasts,wassignificantly
reduced (Supplementary Fig. 2e). These results underscore the
necessityofMETTL3inhTSCsgenerationfromhEPSCsandsuggest
thatlossofMETTL3causestrophoblastdifferentiationdefects,pos-
siblyresultingfromthefailureofpluripotencyexit.

METTL3lossleadstodefectivehTSCs
METTL3expressionissignificantlyhigherinhTSCsthaninthediffer-
entiated trophoblasts (Fig. 2a–d). We next investigated whether
METTL3is required in the establishedhTSCs.Multipleindependent
hTSClinesweresuccessfullyderivedfromtheMETTL3-iKOhEPSCsin
the presence ofDOX(SupplementaryFig. 3a). In contrast,nohTSC
lines were derived from the iKO hEPSCs where DOX was removed
(SupplementaryFig.3a).TheestablishedhTSCsexhibitedtypicalepi-
thelialmorphologywhenculturedintheTSCmediumcontainingDOX
(Fig.3a).BytitratingtheDOXconcentration,weconfirmedthat5ng/
ml DOX maintained a comparable exogenous METTL3 protein
expression as found in normal hTSCs (thereafter referred as
P0(+DOX)) (Supplementary Fig. 3b). Furthermore, 50ng/ml DOX
inducedMETTL3overexpression(SupplementaryFig.3b).UponDOX
removal, METTL3 protein level decreased substantially, becoming
barely detected at passage 1 and not detectable in passage 2 and
passage3(thereafterreferredtoP1(-DOX),P2(-DOX)andP3(-DOX))
(Supplementary Fig. 3c). Notably, the protein level of another
methyltransferase METTL14, which is stabilized by METTL347, was
sharplydecreaseduponMETTL3loss,whereasYTHDC1,anm6Areader
protein,wassubstantiallyupregulated(SupplementaryFig.3c).This
compensatory effect may reflect a response to m6A loss in hTSCs.
Consistent with METTL3 loss, RNA m6A level decreased after DOX
withdrawal(SupplementaryFig.3d).Interestingly,METTL3losssub-
stantially decreased hTSC proliferation, whereas its overexpression
promoted cellproliferation (Fig. 3a,b). Eventually, P3(-DOX) hTSCs
became flattened, enlarged, non-proliferative and multinucleated
(Fig.3a),reminiscentofSTBdifferentiation12,suggestingatendencyof
cellfusionfollowingMETTL3loss(Fig.3c,d).
Subsequently,weinvestigatedthesubcellularchangesinMETTL3-
deficienthTSCsusingtransmissionelectronmicroscopy(TEM),which
revealedsubcellularalterationslinkedtocelldeath,suchasapoptosis
andnecroptosis,asevidencedbythepresenceofapoptoticbodiesand
swollenmitochondriaresemblingnecrosisinP3(-DOX)hTSCs(Fig.3e).
Consistently,celldeath-associatedproteinsRIPK1,RIPK3,andMLKL
wereactivateduponMETTL3loss(Fig.3f),inlinewiththeincreased
TUNELsignalsinP3(-DOX)hTSCs(SupplementaryFig.3e).Notably,a
significantincreaseinthenumberoflysosomeswasobservedinP3(-
DOX)hTSCs(Fig.3e).Elevatedlysosomalmasshas beenassociated
withsenescence-associatedβ-galactosidase(SA-β-gal)activity,awell-
known hallmark of senescence6,48. As expected, P3(-DOX) hTSCs
exhibited enhanced β-gal signals (Fig. 3g, h). Additionally, we con-
firmedthepresenceofhigherlevelsofDNAdamagemarkersγH2AX
and 53BP1 in P3(-DOX) hTSCs (Fig. 3i, j). Collectively, our findings
indicatethatlossofMETTL3inhTSCsleadstoseveredefectsinself-
renewalcapacityandcellsurvival,resultingincellularsenescence.

METTL3safeguardscellularidentityanddifferentiationpoten-
tialofhTSCs
ToinvestigatethetranscriptomicchangesinhTSCsuponMETTL3loss,
we performed bulk RNA-seq to analyze the global gene expression
profiles in P0(+DOX), P2(-DOX), and P3(-DOX) of two independent
hTSClines.FunctionalenrichmentsofgenesupregulatedinP2(-DOX)
andP3(-DOX)hTSCsrevealedpathwaysrelatedtohormoneactivity
andinflammatoryresponse,whiledownregulatedgeneswererestric-
tedinpathwaysassociatedwithcellcyclecheckpointandDNArepli-
cation(SupplementaryFig.3f).Specifically,aseriesofgenesinvolved
inmaturetrophoblastlineages,includingSTBmarkergenessuchas
ERVW-1,CGA,CGBs,andPSGsand EVTmarkergeneHLA-G,showed
significant increased expression in P2(-DOX) and P3(-DOX) hTSCs
(Fig.3kandSupplementaryFig.3g).Inaddition,genesetenrichment
analysis(GSEA)revealedsignificantenrichmentofpathwaysincluding
thesenescence-associatedsecretoryphenotype(SASP),inflammatory
response, TNFα signaling via NF-κB, and gene signature for STB43 in
P2(-DOX) compared to P0(+DOX) hTSCs (Fig. 3l). We validated the
increasedexpressionofERVW-1andhCG,anddecreasedexpressionof
Ki67inP2(-DOX)andP3(-DOX)hTSCsatproteinlevels(Supplementary
Fig.3h–j).Wefurtheremployedthetrophoblastorganoid(TO)model,
whichbettermimicsinvivoconditionsthanmonolayerhTSCs49,and
obtainedconsistentresults:TOsderivedfromP2(-DOX)hTSCsexhib-
ited impaired cellular proliferation (Supplementary Fig. 3k) and ele-
vated expression of STB genes (Supplementary Fig. 3l). To examine
whetherthefunctionsofMETTL3inhTSCsrelyonm6ARNAmethyla-
tionandtofurtherreinforceourconclusions,wetreatedmultiplewild-
typehTSClines,includingtwodistincthEPSC-derivedhTSClinesand
onehTSClineoriginallyderivedfrominvivoplacentalvilli44,withthe
specificMETTL3inhibitorSTM2457,whichinhibitsthecatalyticactivity
of METTL350. The results demonstrated that, similar to the genetic
knockoutofMETTL3,STM2457treatmentinmultiplehTSClinescon-
sistentlyledtoimpairedproliferation,increasedcell-cellfusionevents,
elevatedDNAdamageaccumulation,andhormonesecretion (Fig.3f
andSupplementaryFig.4a–n).Notably,inhibitionofMETTL3didnot
affecttheexpressionofkeytrophoblastmasterregulators,including
GATA2,GATA3,andTFAP2C(SupplementaryFig.4f–j).
Cellularsenescenceislinkedtoinflammation,stemcellexhaus-
tion, and tissue dysfunction51. Despite loss of METTL3 in hTSCs
increased expression of genes of mature trophoblasts, particularly
STBs(Fig.3k,l),thesegeneswerestillexpressedatmuchlowerlevels
compared to those in normal mature STBs (see below), suggesting
precociousdifferentiationofthemutanthTSCsratherthangenuine
STB development. To test this hypothesis, we examined gene
expression changes in STBs derived from P0(+DOX) and P2(-DOX)
hTSCs,aswellasfromhTSCsoverexpressingMETTL3(hTSCstreated
with 50ng/ml DOX) (Fig. 3m). Key STB marker genes, including
ERVW-1,SDC1,CGA,CGBs,andPSGs,werenotfullyactivatedinSTBs
derived from P2(-DOX) hTSCs compared to those from P0(+DOX)
hTSCs (Supplementary Fig. 4o). Western blotting analysis further
confirmed the inefficient expression of hCG hormone in STBs from
P2(-DOX) hTSCs (Fig. 3n, o), supporting the idea that METTL3 loss
impairs stem cell identity and promotes precocious differentiation
towardSTBs.ThesefindingsunderscorethecriticalrolesofMETTL3in
maintaininghTSCstemcellidentityandnormalSTBdifferentiation.

METTL3regulatesgeneexpressioninhTSCsthroughboth
m6A-dependentandm6A-independentmechanisms
ToexploretheunderlyingmechanismsofhowMETTL3maintainsthe
stemcellidentityofhTSCs,weperformedMeRIP-seqtoprofilem6A
modificationinP0(+DOX)andP2(-DOX)hTSCs.InP0(+DOX)hTSCs,
m6Amodificationwasmainlyfoundin3’UTRandexonregions,whilein
P2(-DOX) cells, significant decreases were observed in the 3’UTR
regions (Fig. 4a and Supplementary Fig. 5a). These modifications in
P0(+DOX)hTSCswereprimarilyassociatedwiththeclassical‘GGACU’
consensusmotif(SupplementaryFig.5a).InP2(-DOX)cells,weiden-
tified6,077geneswithdecreasedm6Amethylation(Fig.4b).Wealso
observedasmallfractionoftranscriptsshowingincreasedm6Amod-
ificationfollowingMETTL3deletion(Fig.4b),whichislikelyattributed
tothepresenceofotherm6Amethyltransferases.Wenextfocusedon
theregionswithdecreasedm6AmethylationafterMETTL3loss,asthey
arelikelyMETTL3-dependent.Asexpected,theregionswithdecreased
m6A modification in P2(-DOX) cells were found around the 3’UTR
regions and mapped to the classical ‘GGACU’ motif (Fig. 4c). Func-
tionalenrichmentofthesepeaksrevealedthattheaffectedgenesand
pathways were relevant to protein and histone modification, utero
embryonicandplacentadevelopment,andDNAreplicationandrepair
(SupplementaryFig.5b).Notably,severaltrophoblastmasterregulator
genes, such as GATA2/3, TFAP2C, TEAD1/4, displayed METTL3-
dependent m6A methylation in hTSCs (Fig. 4b, d, e), but did not
showsignificantdifferencesinexpression(Fig.4d).Furtheranalysis
confirmedthatlossofMETTL3didnotaffecttheirsplicingisoformsor
proteinlevels(SupplementaryFig.5c–e).Interestingly,twoimprinting
genes PEG352 and PEG1053, essential in placenta development, dis-
playedm6Amodificationandtheirexpressiondrasticallydecreasedin
METTL3-deficient hTSCs (Fig. 4d, e and Supplementary Fig. 5f).
Nevertheless,forcedexpressionsofPEG3andPEG10inMETTL3-defi-
cienthTSCsdidnotrescuetheproliferationdefectorthepremature
expression of STB marker genes caused by METTL3 loss (Supple-
mentaryFig.5g–i).
Next,weprioritizedtheanalysisofthreegenesets:genesignature
forSTB43,SASP54andthehallmarkofinflammatoryresponse(MSigDB),
due to their consistent upregulation upon METTL3 depletion, as
observedinFig.3l.ByintegratingRNA-seqandMeRIP-seqdata,the
analysisrevealedthatonlyaminorproportionofupregulatedgenes
withinthesethreegenesetsweremodifiedwithm6A(Fig.4f).Speci-
fically,only10–20%ofthetotalmRNAinthesegenesetsexhibitedm6A
modification(Fig.4gandSupplementaryData2).Additionally,upon
METTL3depletion,thefoldchangesinexpressionform6A-modified
transcriptswerecomparabletoorevensmallerthanthoseobserved
fornon-m6A-modifiedtranscripts(SupplementaryFig.5j),indicating
that m6A methylation is unlikely to be a driving factor behind the
upregulation of these genes. For example, the STB marker genes
ERVW-1andCGAwerenotmodifiedwithm6Abutwereupregulated
uponMETTL3loss(Fig.4d,h).Ontheotherhand,severalupregulated
genesdidcarrym6Amodification,suchastheSASPgeneGDF15andthe
inflammation-relatedgeneIL4R(Fig.4handSupplementaryData2).
These findings indicate that METTL3 modulates gene expression in
hTSCsthroughbothm6A-dependentandm6A-independentpathways.
Giventherelativelylowproportionofm6A-modifiedmRNA,thereg-
ulatoryeffectsofMETTL3maybepredominantlymediatedviaindirect
mechanismsratherthandirectm6Amodificationoftargettranscripts.

METTL3orchestratestrophoblastgenenetworkthroughboth
epitranscriptomicandhistoneepigeneticmechanisms
RNAm6Amethylationscaninfluencegeneexpressionbypromotingor
repressingtranscriptstabilityinmammaliancells,dependingonthe
m6Areadersthatrecognizethesemodifications23,55.Tofurthereluci-
datetheroleofMETTL3inregulatinggeneexpressionsinhTSCs,we
conductedanRNAstabilityassay.Inthisassay,weusedActinomycinD
to transiently inhibit global transcription, followed by RNA-seq
experimentsat0h,2h,4h,and6htoassesstranscriptiondynamicsin
P0(+DOX)andP2(-DOX)hTSCs(SupplementaryFig.6a).Theresults
demonstratedthat,ingeneral,transcriptsinhTSCstendedtobemore
stablewhenMETTL3wasabsent(SupplementaryFig.6b),suggesting
thatMETTL3-dependentm6ARNAmethylationmaycontributetoRNA
decayinthesecells.RegardingmRNAstabilitychangeswithinthree
genesets,theanalysisindicatedthattheirmRNAstendedtobemore
stable (Supplementary Fig. 6b). However, only the STB gene set
exhibitedstatisticallysignificantdifferences(SupplementaryFig.6b),
suggesting alternative mechanisms beyond m6A modification reg-
ulating their expressions. We further categorized all m6A modified
transcriptsintotwoclusters(cluster1andcluster2)basedontheir
half-lifetimes(SupplementaryFig.6c).Genescategorizedincluster1
exhibitedlongerhalf-lifetimesafterMETTL3lossandwereenrichedin
pathwaysrelatedtoinuteroembryonicandplacentadevelopment,as
wellashistonemodificationandacetylation(SupplementaryFig.6c).
In contrast, genes categorized in cluster 2 exhibited shorter half-
lifetimesafterMETTL3lossandwereenrichedinpathwaysassociated
with DNA repair and replication, and cell cycle (Supplemen-
taryFig.6c).
ToinvestigatetherelationshipbetweenRNAstabilitywithtran-
scriptomicchanges,weanalyzedtheoverlapgenesincluster1with
thoseshowingupregulatedexpression,andthegenesincluster2with
thoseexhibitingdownregulatedexpressioninP2(-DOX)hTSCsfrom
bulk RNA-seq results (Fig. 5a). The identified differential expressed
genes (DEGs) (168 up vs. 38 down) were considered to be directly
regulated by m6A modification through RNA decay or stabilization
(Fig. 5a, b and Supplementary Data 3). The upregulated gene list
includedgenesencodinghistonemodifierEP300forhistoneacetyla-
tion and several enhancer-binding transcription factors (TFs) like
JUND,FOSL2,andCEBPA(Fig.5b),whichdisplayedMETTL3-dependent
m6AmodificationandexhibitedhigherRNAstabilityinP2(-DOX)cells
(Fig.5c,d).Incontrast,thedownregulatedgenelistincludedEZH2,
whichcatalyzesH3K27me3,anditstranscriptsexhibitedreducedsta-
bilityinP2(-DOX)hTSCs(Fig.5bandSupplementaryFig.6d,e).Fur-
thermore,employingasingle-baseelongationandligationbasedPCR
amplification method (SELECT)56, we confirmed EP300 and EZH2
transcriptsweremodifiedwithm6AincontrolHUES8-hTSCs,witha
reductioninm6AlevelfollowingSTM2457treatment(Supplementary
Fig. 6f). Western blotting confirmed elevated protein expression of
EP300aswellasreducedproteinexpressionofEZH2inP2(-DOX)and
P3(-DOX) hTSCs (Fig. 5e, f). These findings align with the observed
changes in histone marks:METTL3-deficienthTSCs exhibited higher
H3K27ac and lower H3K27me3 levels compared to P0(+DOX) cells
(Fig.5g,h),incontrastwiththerapiddeclineofH3K27acinnormal
hTSCs to STB differentiation (Supplementary Fig. 6g, h). Western
blottinganalysisfurtherconfirmedincreasedlevelsofEP300,c-JUN,
JUND,andH3K27ac,aswellasdecreasedlevelsofEZH2andH3K27me3
uponSTM2457treatmentinnormalhTSCs(SupplementaryFig.6i,j).
Thesedatasuggestthatdysregulatedepigeneticdynamicsmaycon-
tributetothedefectsinMETTL3-deficienthTSCs.
WefurtheranalyzedtheepigeneticlandscapeofP0(+DOX)and
P3(-DOX) hTSCs by comparing H3K27ac and H3K27me3 profiles
through CUT&Tag. Our findings revealed a significant elevation in
H3K27aclevelandacorrespondingreductioninH3K27me3levelin
P3(-DOX)cellsrelativetoP0(+DOX)cells(Fig.5i),whichalignswith
westernblottingresults(Fig.5g,h).Additionally,anelevatednumber
ofbindingsitesforEP300andJUNDwasobservedinP3(-DOX)cells
(Fig.5i).IntegrativeanalysisofRNA-seqandCUT&Tagdatasetsindi-
catedthatH3K27acenrichmentandEP300occupancyexhibitaposi-
tive correlation with gene expression level, whereas H3K27me3
depositiondemonstratesaninversecorrelationwithgeneexpression
inP3(-DOX)comparedtoP0(+DOX)hTSCs(SupplementaryFig.7a–f).
As anticipated, H3K27ac, EP300, and JUND displayed increased
enrichmentacrosstheSTB,SASP,andinflammationgenesetsupon
METTL3 depletion, whereas H3K27me3 exhibited an inverse trend
(Fig.5j),indicatingthepivotalrolesofTFsandhistonemodificationsin
regulatinggeneexpressioninhTSCsthroughMETTL3.
Tofurtherevaluatethedirectregulatoryroleofm6Amodification
inhTSCs,weanalyzedtheoverlapofgeneswithintheSTB,SASP,and
inflammationgenesetsthatwereco-modifiedbym6A,H3K27ac,and
H3K27me3. The results demonstrated that nearly all m6A-modified
genesinthesegenesetsalsoexhibitedconcurrentmodificationsby
H3K27ac and/or H3K27me3 (Supplementary Fig. 7g). Notably,
approximately two-thirds of the m6A-modified genes were simulta-
neouslyco-regulatedbybothH3K27acandH3K27me3(Supplemen-
taryFig.7g),suggestingacomplexinterplayofepitranscriptomicand
histoneepigeneticmechanismsingeneregulationwithinthesepath-
ways. For instance, STB genes within the PSG clusters and sPE
inflammation-associatedgenessuchasTNFRSF1Bdidnotexhibitm6A
modification but were upregulated following METTL3 depletion
(SupplementaryData2).Thesegenesshowedincreasedenrichmentof
H3K27ac,EP300,andJUND,concomitantwithdecreasedH3K27me3
depositioninP3(-DOX)hTSCsrelativetoP0(+DOX)hTSCs(Fig.5k,l).A
similar pattern was also observed in SASP-related genes, including
SERPINE1andVGF,aswellasinflammation-associatedgenessuchasIL6
(Supplementary Fig. 7h, i), indicating an indirect regulatory effect
mediatedthroughm6Amethylation.Conversely,GDF15,amarkergene
for STB and a component of SASP that was modified with m6A,
exhibitedincreasedmRNAstabilityuponMETTL3KO(Supplementary
Data2),alongwithelevatedenrichmentofH3K27ac,EP300,andJUND
(Fig.5m).Acomparabletrendwasalsoobservedintheinflammation-
relatedgeneRIPK2(SupplementaryFig.7j).Collectively,theseresults
indicate that METTL3 orchestrates trophoblast gene regulation via
both direct m6A-dependent mechanisms and indirect epigenetic
pathways involving EP300-mediated H3K27ac and EZH2-mediated
H3K27me3, with the histone epigenetic pathways playing a more
prominentrole.

EfficientrescueofMETTL3-KOhTSCdefectsbychemical
manipulationofhistonemodifications
We further asked whether correcting the dysregulated epigenetic
modifications,specificallyH3K27me3andH3K27ac,couldcorrectthe
defective gene expression in METTL3-KO hTSCs (Fig. 6a). As antici-
pated,treatmentwiththeEP300inhibitorA-48557inP3(-DOX)hTSCs
reduced H3K27ac level and significantly decreased the abnormal
expressionofSTBproteins,includinghCGandSDC1(Fig.6b–d).Pre-
vious studies have shown that PRC2 mediated H3K27me3 acts as a
barriertotrophoblastlineageinductionfromhumanembryonicstem
cells (hESCs)58,59, but the role of H3K27me3 in trophoblast lineage
maintenanceisyetunderstood.Initially,wetreatednormalhTSCswith
theEZH2inhibitors,UNC199959orEPZ643860,forseveralpassages,and
theresultsweresimilartothoseobservedinhTSCsuponMETTL3loss
(Supplementary Fig. 8). This included decreased proliferation,
increased hCG hormone secretion, and enhanced cell-cell fusion
(SupplementaryFig.8a–e),indicatingtheessentialrolesofH3K27me3
forpreservingcellidentityofhTSCs.SinceefficientEZH2activators
wereunavailable,weusedaKDM6Binhibitor,GSK-J161,toincreasethe
H3K27me3 level in P3(-DOX) hTSCs (Fig. 6a). Immunostaining and
westernblottinganalysisdemonstratedthatGSK-J1treatmentinP3(-
DOX)hTSCsledtoaslightlyhigherH3K27me3levelandsignificantly
suppressed the expression of the STB proteins (Fig. 6b–d). These
findingscollectivelydemonstratethatchemicalinhibitionofH3K27ac
orreinforcementofH3K27me3couldefficientlyrescuethedefective
geneexpressioninMETTL3-KOhTSCs.

METTL3-KOhTSCsexhibitpreeclampsiamolecularsignatures,
andinhibitionofEP300alleviatesthesefeatures
PrematuretrophoblastsanddefectiveCTBsintheplacentaareasso-
ciatedwithpreeclampsia(PE)62–64.Previousstudyhasshownawide-
spreadincreaseinH3K27acoccupanciesinCTBsfrompatientswith
severePE(sPE)65.Therefore,wetestedwhetherMETTL3lossinhTSCs
would induce PE-like signatures. Analysis of the published data64
revealedthatgeneshighlyexpressedintheCTBsfromsPEpatients
alsoshowedhigherexpressionlevelsinP3(-DOX)vs.P0(+DOX)hTSCs
(Fig. 6e). Furthermore, following METTL3 KO in hTSCs, LTR10A, a
transposable element (TE) associated with PE due to its
hyperactivation66,exhibitedincreasedenhanceractivityindicatedby
theTEenhancerpredictinglinearregressionmodel67(Fig.6f).Notably,
LTR10AlociinP3(-DOX)hTSCsexhibitedincreasedchromatinacces-
sibility, elevated H3K27ac level, and increased JUND occupancies
(Fig.6g).HigherH3K27aclevelandhyperactivationofLTR10Ainboth
sPECTBsandMETTL3-deficienthTSCspromptedustospeculatethat
the inhibition of EP300-mediated H3K27ac might ameliorate the PE
signaturecausedbyMETTL3loss.TreatmentwithA-485inP3(-DOX)
cells effectively mitigated the PE signature, as evidenced by the
reduced expression of genes that are enriched in CTBs from sPE
(Fig. 6e, h). We further genetically deleted specific LTR10A loci in
hTSCs and evaluated the nearby PE related gene expression. Genes
suchasENG65,66and ATG9B68,whichcontainthe LTR10Aelementin
their enhancer regions (Fig. 6i) and are associated with PE, were
selected for analysis. Due to the challenges in clonally expanding
hTSCsfromsinglecells,weemployedapopulation-wideapproachand
demonstratedover50%deletionefficiencyacrossvarioustargetsand
experiments by examining LTR10A expression (Fig. 6j). Excising
LTR10AlocisignificantlyreducedtheexpressionofENGandATG9Bin
P3(-DOX)hTSCs(Fig.6k).Insummary,ourstudyindicatesthatlossof
METTL3inhTSCsinducesPEtranscriptomicsignature,whichcanbe
effectivelyalleviatedbyinhibitingEP300-mediatedH3K27ac,offering
apotentialtherapeutictargetforPEtreatment.

TheMETTL3-EP300-H3K27acaxisisakeyregulatoroftropho-
blastdevelopmentandisinvolvedinthepathogenesisofPE
duringplacentation
WefinallyinvestigatedthecorrelationsbetweenMETTL3andhistone
modificationsH3K27acandH3K27me3inhumanplacentasinvivo.The
results demonstrated that CTBs expressing METTL3 exhibited
significantly lower level of H3K27ac compared to METTL3-negative
trophoblasts (Fig. 7a, b). Notably, a subset of mature STBs lacked
H3K27acexpressiondespitealsobeingnegativeforMETTL3(Fig.7a),
indicatingthatH3K27acmayhavestage-specificregulatoryrolesduring
trophoblastdifferentiation.Conversely,nosignificantcorrelationwas
observedbetweenMETTL3expressionandH3K27me3level(Fig.7a,b).
OurinvitrodatademonstratedthatMETTL3-KOhTSCsacquired
PE-associated signatures at the transcriptomic level. Given the sig-
nificantlyelevatedH3K27aclevelobservedinCTBsofPEpatients65
andtheroleofMETTL3inregulatingEP300-mediatedH3K27ac,we
hypothesized that METTL3 deficiency may exist in PE CTBs. To
investigatethis,weanalyzedtwoPEplacentasfrom35-and37-week
pregnanciesandincludedtwogestationalage-matchednormalpla-
centas from 38-week as controls. The results demonstrated a sig-
nificantreductionintheproportionofMETTL3-positivetrophoblasts
in PE placentas compared to normal controls (Fig. 7c, d). More
importantly,METTL3expressionwasmarkedlydecreasedinPEG10-
positive CTBs within PE placentas relative to those in normal pla-
centas (Fig. 7c, e). Notably, a substantial subset of PEG10-positive
CTBsinPEplacentaslackeddetectableMETTL3expression(Fig.7c).
Consistentwiththepreviousstudy65,H3K27aclevelwassignificantly
elevated in PE CTBs (PEG10-positive cells) compared to normal
controls (Fig. 7f, g). These findings suggest that the abnormal
increaseinH3K27aclevelinPECTBsmayresultfromdeficienciesin
METTL3expression,indicatingapotentialmechanisticlinkbetween
METTL3 downregulation and epigenetic dysregulation in PE
pathology.

Methods
Ethicalstatement
The em1-hEPSC line was derived from human preimplantation
embryos donated for research45. All the donors gave written
informedconsentsforderivationofEPSCfromtheirspareembryos
after completed family through assisted reproduction. The deriva-
tionprotocolswereapprovedbytheInstitutionalReviewBoardsof
the University of Hong Kong/Hospital Authority Hong Kong West
Cluster (IRB number:UW18-017) and the Councilof Reproductive
Technology, Hong Kong (research licence number: R5004). The
human placental specimens used in this study of immunohis-
tochemistry (IHC) were collected from two early pregnant women
(8 weeks of pregnancy), two healthy women with normal-term
pregnancies(38weeks)andtwopregnantwomendiagnosedwithPE.
ForthePEpatients,placentaltissueswerecollectedfromtermpla-
centa of 35-week and 37-week respectively. PE was diagnosed
accordingtothefollowingcriteria:new-onsethypertension(systolic
bloodpressureof140mmHgormoreordiastolicbloodpressureof
90mmHgormoreontwooccasionsatleast4hapartafter20weeks
of gestation), and proteinuria: 300mg or more per 24h urine col-
lection (or this amount extrapolated from a timed collection). All
placentalspecimenswerecollectedfromWeifangPeople’sHospital.
Experiments using the placental specimens were reviewed and
approved by the Ethics Committee of Weifang People’s Hospital
under research license KYLL20230106-1 and KYLL20250826-4.
Donorshavesignedtheinformedconsent.

CultureofhEPSCsandhTSCs
Em1-hEPSCsweremaintainedonSNL76/7feederlayersandenzyma-
ticallypassagedwithTrypLE(ThermoFisherScientific,12604013)ata
four-dayintervalata1:4to1:8ratio.ThehEPSCmedium(hEPSCM)was
prepared based on a previous study with slight modification11. This
medium is N2B27-based, supplemented with four inhibitors: 2.5μM
XAV939 (Tocris, 3748), 2.5μM endo-IWR1 (Tocris, 3532), 1μM
CHIR99021 (Tocris, 4423), and 0.1μM A419259 (Tocris, 3914). The
N2B27basalmediumconsistsofa1:1mixtureofDMEM/F12(Thermo,
21331020) and Neurobasal medium (Thermo, 21103049), supple-
mentedwith200×N2and100×B27,1%ITS-X(ThermoFisherSci-
entific, 5150056), 50μM β-mercaptoethanol, 1% Penicillin-
Streptomycin-Glutamine (Thermo Fisher Scientific, 15140163), 1%
non-essential amino acid, 50μg/ml Vitamin C (Sigma, A8960), and
human leukaemia inhibitory factor (hLIF, 10ng/ml). Primed state
HUES8 hESCs were cultured in mTeSR1 (STEMCELL Technologies,
85850) medium on plates pre-coated with 1% Matrigel (Corning,
354230).ToconvertthesecellsintohEPSCs,primedHUES8cellswere
passaged into hEPSCM and maintained for more than 10 passages,
during which undifferentiated colonies were manually selected to
establishstablehEPSClines.
hTSCs derived from hEPSCs (em1-hTSCs and HUES8-hTSCs) or
frominvivoplacentavilli44(CT-hTSCs)aremaintainedinhTSCmed-
ium (hTSCM) as described in the published study with slight
modification8. hTSCM is made up of DMEM/F12 containing 0.5%
penicillin-streptomycin,1%foetalbovineserum,1%ITS-X,0.3%bovine
albumin fraction V (Thermo Fisher Scientific, 15260037), supple-
mentedwith50μg/mlVitaminC,50μg/mlhumanEGF(Peprotech,AF-
100-15), 2μM CHIR99021, 0.5μM A-83-01 (Tocris, 2939), 1μM
SB431542(Tocris,1614),10μMvalproicacid(Tocris,2815),and2.5μM
Y27632 (Selleckchem, S1049). Routine passage of hTSCs was con-
ducted every four days by enzymatic digestion using TrypLE. Cells
wereresuspendedandseededinhTSCMoncellcultureplatecoated
with1%Matrigelatasplitratioof1:3.hTSCMwasrefreshedeverytwo
days during culture. All cell cultures were maintained at 37°C with
5%CO .
2

GeneeditinginhEPSCsandhTSCs
TogenerateMETTL3-iKOhEPSCs,an exogenous METTL3expression
cassettewasfirstconstructedbyinsertingMETTL3codingsequence
into Piggybac Tet-On plasmid. This plasmid was transfected into
hEPSCsviaelectroporationusingNeontransfectionsystem(Thermo
FisherScientific)withfollowingparameters:1100V,30ms,and1pulse.
SinglecolonyformationwasperformedtoobtainhEPSClineswiththe
exogenousMETTL3expressioncassette.Subsequently,ansgRNAtar-
geting exon 1 of METTL3 was cloned into the plasmid pKLV2-
U6gRNA5(BbsI)-PGKpuro2ABFP-W (Addgene #67974) and co-
transfected with the plasmid pKLV2-EF1a-Cas9Bsd-W (Addgene
#68343),encodingCas9,intohEPSCsusingthesameelectroporation
parameters. Transfected cells were cultured in hEPSCM with DOX
unlessotherwisespecified.Onedaypost-electroporation,cellswere
selectedwith10µg/mlblasticidinfor24h,followedbyselectionwith
both 10µg/ml blasticidin and 2µg/ml puromycin for 48h. Selected
cells were then subjected to single colony formation to establish
individualMETTL3-iKOhEPSClines.ThelossofMETTL3intheselected
iKOcelllineswasverifiedbysangersequencingofMETTL3allelesand
bywesternblottingafterculturinginhEPSCswithoutDOX.DOXwas
usedataconcentrationof5ng/mlunlessotherwisespecified.Primers
usedforsgRNAsarelistedinSupplementaryTable1.
TooverexpressPEG3andPEG10inMETTL3-iKOhTSCs,cDNAof
PEG3andPEG10wasamplifiedthroughPCRandclonedintopiggybac
backboneandtransfectedintohTSCsviaelectroporationusingNeon
transfectionsystemwithfollowingparameters:1200V,40ms,and1
pulse.ExpressionofexogeneousPEG3andPEG10wasdrivenbyCAG
promoter.Theemptypiggybacbackbonewastransfectedasthecon-
trol.At24haftertransfection,drugselectionwasperformedwith5µg/
ml blasticidin for two days. Selected cells were then used for sub-
sequentexperimentalanalysis.
ToexcisespecificLTR10AlociinhTSCs,correspondingsgRNAs
wereclonedintotheplasmidpKLV2-U6gRNA(BbsI)-PGKpuro2ABFP-W
(Addgene #67974). Next, plasmids encoding the sgRNAs were co-
transfected with the plasmid pKLV2-EF1a-Cas9Bsd-W (Addgene
#68343) which encodes the Cas9 protein via electroporation using
Neon transfection system (Thermo Fisher Scientific) with following
parameters:1200V,40ms,and1pulse.Non-targetsgRNAwastrans-
fectedside-by-sideasthecontrol.At24haftertransfection,cellswere
selected with 1µg/ml puromycin and 5µg/ml blasticidin for 2 days.
DNAandRNAwereextractedfromtheresultantcellpopulationsand
genotyped for the presence of excisions. Successful excision pools
werefurtheranalyzedforthepercentofexcisedallelesusingRT-qPCR.
UponDOXremovalfor3passages,hTSCswithLTR10Aexcisionwere
harvested for the expression analysis of genes nearby the targeted
LTR10AusingRT-qPCR.PrimersofsgRNAsandprimersusedforgen-
otypingandRT-qPCRarelistedinSupplementaryTable1.

Differentiationoftrophoblastlineageandderivationofstable
hTSClinesfromhEPSCs
Fortrophoblastlineagedifferentiation,METTL3-iKOhEPSCswithDOX
orwithoutDOXwereharvestedusingTrypLEfor3minandseededin
hTSCMon cellculture platepre-coatedwith1%Matrigelwith acell
densityof10,000cellsper6-wellplate.Cellswerecollectedatindi-
catedtimepointsfordownstreamexperimentsandanalysis.
For the derivation ofstablehTSClines,5ng/mlDOXwascon-
tinuouslyadded tothehTSCMthroughouttrophoblastdifferentia-
tion and establishment of hTSCs. By approximately day 8–10 of
differentiation from hEPSCs, characteristic hTSC-like epithelial
coloniesweremanuallyselectedandtransferredtoindividualwells
ofa96-wellplatepre-coatedwith1%Matrigel,designatedaspassage
1(P1).Somecolonieswereabletoadheretotheplateandproliferate,
while others did not. The proliferating hTSC colonies were dis-
sociated into single cells using TrypLE and passaged at a ratio of
1:1–1:2 for early passages. The established hTSC lines were further
characterized through gene expression analysis and assessment of
theirdifferentiationpotential.DOXwasusedataconcentrationof
5ng/mlunlessotherwisespecified.

DifferentiationofSTBsfromhTSCs
DifferentiationofSTBsfromhTSCswaspreformedaccordingtothe
publishedstudywithminormodifications8.hTSCswereseededina
6-wellplatepre-coatedwith1%Matrigelatadensityof2×105cellsper
wellandculturedin2mlofSTBmedium:DMEM/F12supplemented
with 1% ITS-X, 0.5% penicillin-streptomycin, 0.3% bovine albumin
fractionV,0.1mMβ-mercaptoethanol,4%KnockOutserumreplace-
ment,2.5μMY27632,and2μMForskolin(Tocris,1099).Themedium
wasrefreshedevery2days,andcellswereanalysedatindicatedtime
points.

Cellproliferationassay
2×105 METTL3-iKO hTSCs (routinely cultured with 5ng/ml DOX and
referredasP0)wereseededin6-wellplatesandculturedwith50ng/ml
DOX,5ng/mlDOXorwithoutDOX.Cellswerecountedevery4daysand
passagedintonew6-wellplateswitharatioof1:3.Numberofcellswas
counted3timesatpassage1,2,and3(P1,P2,andP3)foreachgroup.

Reversetranscription-quantitativepolymerasechainreaction
(RT-qPCR)
TotalRNAwasextractedusingTRIzolreagent(ThermoFisherScien-
tific,15596026)followingthemanufacturer’sprotocolandconverted
tocDNAusingPrimeScript™RTMasterMix(Takara,RR036A).Quan-
titativePCR(qPCR)wasperformedusingiTaq™UniversalSYBR®Green
Supermix(Biorad) with CFX Opus Real-TimePCR Systems (Biorad).
The2−ΔCtmethodwithGAPDHastheendogenouscontrolwasusedto
calculatetherelativegeneexpression.PrimersofRT-qPCRarelistedin
SupplementaryTable1.

Immunofluorescencestaining
Cells were fixed with freshly prepared 4% paraformaldehyde (Sigma,
158127)solution,permeabilizedin5%FBS/PBSsupplementedwith0.1%
TritonX-100(Sigma,X100)for30minandblockedwith5%FBS/PBSfor
2h.Primaryantibodiesweredilutedinblockingbufferaccordingtothe
manufacturer’s protocols and incubated overnight at 4°C. Signals of
primary antibodies were detected using AlexaFluor-conjugated sec-
ondaryantibodies,andcellnucleiwerecounterstainedbyDAPI.Images
were acquired using a Zeiss LSM800 confocal laser scanning micro-
scope.AntibodiesusedinthisstudyarelistedinSupplementaryTable2.

Immunohistochemistry(IHC)
The collected samples were immediately fixed in 4% paraformalde-
hydefor24hoursat4°C.Subsequently,thesamplesweredehydrated
through a graded ethanol series (70%, 80%, 90%, 100%), cleared in
xylene,andembeddedinparaffinthroughtwocyclesofimmersionin
moltenwax(60°C,1heach).5μm-thicksectionswerecutfromthe
paraffinblocks,followedbydeparaffinizationinxyleneandrehydra-
tionthroughadescendingethanolgradient(100%,95%,80%,70%).
Antigenretrievalwasperformedbyheat-mediatedepitoperestoration
incitratebuffer(pH6.0,BeyotimeBiotechnology,Cat#P0083)usinga
pressurized decloaking chamber (121°C, 2min). Sections then were
blocked with 5% bovine serum albumin (BSA) and incubated with
correspondingprimaryantibodyat4°Covernight.Afterincubation,
sectionswerewashedthreetimes(5mineach)withPBS,followedby
incubationwithfluorescence-conjugatedsecondaryantibodyfor1hat
room temperature, and cell nuclei were counterstained by DAPI.
ImageswereacquiredusingaZeissLSM800confocallaserscanning
microscope.AntibodiesusedinthisstudyarelistedinSupplementary
Table2.

Westernblot
Forwesternblotanalysis,onemillioncellswerelysedusingRIPAbuffer
(Thermo Fisher Scientific, 89900) supplemented with a protease
inhibitor cocktail (Roche, 11697498001). SDS-PAGE was performed
using10%TGXpolyacrylamidegels(Biorad,1610173)withathickness
of 1.0mm, followed by transfer onto Immun-Blot PVDF Membrane
(Biorad, 1620177) using Trans-Blot Turbo Transfer System (Biorad).
Membranewasblockedwitheither5%non-fatdrymilk(CellSignalling
Technology,#9999)or5%BSAinTBSTfor2-hourincubationatroom
temperaturewithgentleshaking.Transferredmembraneswereincu-
bated with primaryantibodies overnight at4°C and were detected
using HRP-conjugated antibodies. Images were developed using
SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo
FisherScientific)andcapturedusingiBright™FL1500ImagingSystem
(ThermoFisherScientific).Antibodiesusedinthisstudyarelistedin
SupplementaryTable2.Unprocessedscansofalltheblotswerepro-
videdintheSourcedata.

Transmissionelectronmicroscopy(TEM)
Approximately300,000cellspermloftrypsinizedP0(+DOX)andP3(-
DOX)hTSCswerefixedusingelectronmicroscopyfixative(4%paraf-
ormaldehyde) at 4°C overnight. The sample preparation was con-
ductedintheElectronMicroscopyLaboratoryatQueenMaryHospital.
Following sample preparation, the specimens were examined using
thePhilipsCM100transmissionelectronmicroscope,andimagesat
variousmagnificationswerecapturedforsubsequentanalysis.

Senescence-associated-β-galactosidase(SA-β-gal)staining
SA-β-gal staining was performed according to manufacturer’s
instruction (CST, #9860). Briefly, cultured cells were washed twice
withPBSandfixedatroomtemperaturefor5minin2%formaldehyde
and0.2%glutaraldehyde.FixedcellswerestainedwithSA-β-galstain-
ing solution at 37°C overnight, and images were captured using a
microscopedigitalcamera(Olympus).

RNAm6Amethylationquantification
TRIzolreagentwasused to extractthe totalRNA. m6Acontentwas
assayedusingthem6ARNAmethylationassaykit(EpigenTek,P-9005).
Briefly, 80μl of binding solution and 200ng of sample RNA were
addedintoeachdesignatedwellandincubatedat37°Cfor90minfor
RNAbinding.1ngm6A-methylatedRNAprovidedbytheassaykitwas
used as positive control. Wells were washed three times with wash
buffer,followedbytheadditionof50μlofdilutedcaptureantibody
andincubationatroomtemperaturefor60min.Subsequently,wells
were incubated with detection antibody and enhancer solution at
room temperature for 30min. The reaction was developed with
developersolutioninthedarkfor1–10minat25°C,stoppedwithstop
solution,and measured using a microplate reader at 450nm within
2–10min.

RNAstabilityassay
AssayforRNAstabilityanalysiswasperformedaspreviouslydescribed
in ref. 24 with some modifications. P0(+DOX) and P2(-DOX) hTSCs
wereculturedinsix-wellcultureplatesto∼80%confluence.Actino-
mycinD(ActD)(Thermo,11805017)wasaddedatafinalconcentration
of5μg/ml.Cellswerecollectedatfourtimepoints(0,2,4,6h)after
theadditionofActDandtotalRNAwasextractedwithTRIzolreagent,
thenperformedRNAsequencing.

Single-baseelongation-andligation-basedqPCRamplification
(SELECT-qPCR)
SELECTwasappliedtodetectm6Asites56.Briefly,1.5μgtotalRNAwas
mixed with 100nM m6A or control primersand 5μM dNTP with 1×
CutSmartbufferin17μLreaction.TheprimerswereannealedtoRNA
usingfollowingPCRsteps:90°Cfor1min,80°Cfor1min,70°Cfor
1min,60°Cfor1min,50°Cfor1min,40°Cfor6min,30°Cfor1min,
20°Cfor1min,and10°Cfor1min.Then,a3μLmixtureof0.01UBst
DNApolymerase,0.5USplintRligase,and10nMATPwasaddedtothe
RNA/primerssampleandsubsequentlyincubatedat40°Cfor20min
and 80°C for 20min. Next, 4μL of mixture was used for RT-qPCR
reaction in triplicate with SYBR Green Real-Time PCR Master Mix
(Appliedbiosystems).PrimersusedinthisstudyarelistedinSupple-
mentaryTable1.

RNAsequencing
Total RNA was extracted using TRIzol reagent. Preparation of the
libraryforRNAsequencingwasperformedaccordingtothemethod
previously described82. mRNA was enriched from total RNA using
magnetic beads with poly-A oligos attached. Divalent cations were
usedforfragmentationunderelevatedtemperaturewithFirst-Strand
Synthesis Reaction Buffer from New England Biolabs. First-strand
cDNA was synthesized using M-MLV reverse transcriptase without
RNaseHinthepresenceofrandomhexamerprimers.Second-strand
cDNAsynthesiswasperformedusingDNApolymeraseIandRNaseH.
SynthesizedDNAfragmentswereligatedtoNextAdaptorsfromNew
EnglandBiolabsandamplifiedbyPCRusingPhusionhigh-fidelityDNA
polymerase(Vazyme).Libraryquality wasassessed using anAgilent
Bioanalyzer2100system,andsequencingwasperformedontheIllu-
minaNovaSeq6000System.

Cleavageundertargetsandtagmentation(CUT&Tag)
CUT&Tag was performed according to the manufacturer’s protocol
(Vazyme,TD904).Briefly,50,000cellswereharvestedfornucleiiso-
lation.Isolatednucleiwereincubatedwithprimaryantibodiesover-
night at 4°C, followed by capture using corresponding secondary
antibodies.UnboundantibodieswereremovedusingDig-Washbuffer,
andapA-Tn5adaptorcomplexwaspreparedandaddedtothenuclei
with gentle vortexing. Nuclei were then subjected to tagmentation,
andtheresultantDNAwaspurifiedforlibrarypreparationandampli-
fication.Thespike-inDNAwasaddedalongwithstopbuffertoensure
normalization across samples. Sequencing was performed on the
IlluminaNovaSeq6000system.

MethylatedRNAimmunoprecipitation(MeRIP)sequencing
TotalRNAfromCT-hTSCsandtheirderivedday6STBs,andP0(+DOX)
andP2(-DOX)hTSCswasextractedusingTRIzol.MeRIP-seqwasper-
formedbyShanghaiJiayin BiotechnologyCompanyLimited.Briefly,
total RNA was fragmented into ~100 nucleotide long fragments.
Approximately5%ofthefragmentedRNAwasreservedasinputRNA,
whiletheremainingRNAwasimmunoprecipitatedusingan affinity-
purifiedanti-m6Apolyclonalantibody(Millipore,ABE572).The
SMARTerStrandedTotalRNA-SeqKit-version2(PicoInputMamma-
lian,Takara/Clontech,634413)wasusedtoconstructlibrariesforboth
theinputandIPsamples.SequencingwasperformedusinganIllumina
NovaSeq6000platform.

Sequencingdataanalysis
Generalpre-processingofreads:theRNA-seq,MeRIP-seq,andCUT&-
TagwereperformedusingIlluminaNovaSeq6000(Novogene).Raw
reads were trimmed using Trim Galore (v0.6.10) (https://www.
bioinformatics.babraham.ac.uk/projects/trim_galore/), filtering out
readswithaqualityscorelowerthan20andreadlengthsshorterthan
20basepairs.Alignmenttothehumangenome(GRCh38assembly)
wasperformedusingHisat2(v2.2.1)forRNA-seqandMeRIP-seq,and
Bowtie2 (v2.5.3) for CUT&Tag. featureCounts (v2.0.6) was used to
calculatethegenecountswithgeneannotationfromGencode(v45).
Thealignedreadsweresplitintostrand-specificBAMfilesandsorted
usingsamtools(v1.10).
ForRNA-seq,thebatcheffectbetweenhTSC3#andhTSC4#cell
lineswasremovedusingComBat-seqinsvaRpackage(v2.48.0).The
expression of transcripts was quantified as reads per kilobase of
transcriptspermillionmappedreads(TPM).Differentiallyexpressed
genes(DEGs)wereidentifiedusingDESeq2(v1.40.2)withacutoffof|
log₂(fold change)|>1 and an adjusted p value<0.05. For splicing
isoformanalysis,theassemblyofmappedreadsfromeachsample
wasperformedutilizingStringTie(v2.0.4)aspartofanapproachthat
was based on reference data. Expression quantification was calcu-
latedasfragmentsperkilobasepermillionmappedreads(FPKM)for
each annotated genomic region to evaluate transcript abundance
and differential expression. Enhancer predictions for transposable
elements were based on previous study67. Deviations in gene
expression between P0(+DOX) and P2(-DOX) hTSCs for protein-
codinggenesweremodelledasalinearcombinationofoccurrences
ofnearbyTEsubfamilyintegrants(within50kbofthetranscription
start site).
ForCUT&Tag,duplicatedreadswereremovedusingMarkDupli-
catesfromPicard(v3.0.0).Readcountswerenormalizedbasedonthe
spike-inchromatintoaccountfortechnicalvariation.Peakscallingwas
performed using SEACR (v3.1), with options: 0.05 non-stringent.
Strand-specificbigwigfilesforvisualization,heatmaps,andaggrega-
tionplotsweregeneratedusingbamComparefromdeepTools(v3.5.1).
Fordifferentialpeakanalysis,aconsensussetofpeakswascreatedfor
eachcondition(P0andP3).Readcountsforthesepeakswerequan-
tifiedfromthenormalizedBAMfilesandanalyzedusingDESeq2.
ForMeRIP-seq,samtools(v1.10)wasusedtoseparatetheplusand
minusstrandedreadswith“−dXS:+”and“−dXS:-”.Forpeakscalling
and differential peaks analysis, exomePeak2 (v1.12.0)83 was used to
determinetheread-enrichedregionsfromMeRIP-seqdata.Basedon
theuniquelymappedreads,thepvalueforagivenregionwascom-
putedutilizingadynamicPoissondistribution.Whenthepvaluewas
<1×10−10,theregionwasdesignatedasapeak.Peakswereannotatedto
theirassociatedgenesbasedongenomicpositionandgeneannotation
data. In addition, the distribution of peaks on different functional
regions,includingthe3’UTR,5’UTR,andCDS,wasfurtherassessed.
Homer(v4.11)wasusedtodetecttheenrichedmotifswithinm6Apeaks
withtheparameter“-rna-len5,6,7,8”.Identificationofm6A-modified
genes required both a detected m6A modification and reduced
expressionintheP2(-DOX)grouprelativetoP0(+DOX).

Geneenrichmentanalysis
CuratedgenesetsofKEGGandBiologicalProcesses(BP)weredown-
loadedfromtheMolecularSignatureDatabase(MSigDB,v7.5.1,http://
software.broadinstitute.org/gsea/msigdb/index.jsp)andwereusedto
calculate enrichment scores. Significantly enriched gene sets were
identified with a Benjamini–Hochberg adjusted p≤0.05. For the
identifiedpathways,weselectedDEGsandperformedGOandKEGG
enrichmentanalysisusingtheclusterProfiler(v4.8.3)package.Public
genesetswereacquiredfromGSEAdatabase.Genesetswereusedto
score each input cell with the Seurat function “AddModuleScore”.
Changesinthescoresacross+DOXand-DOXgroupwerecalculatedby
ggpubrpackage(v0.6.0)usingWilcoxontest.

HalflifetimeRNA-seqanalysis
RawreadsweretrimmedwithTrimmomatic(v0.39),thenalignedto
humangenomeandtranscriptome(hg38)andexternalRNAControl
Consortium (ERCC) RNA spike-in control (Thermo Fisher Scientific)
usingHisat2(v2.1.0).ReadsoneachGENCODEannotatedgenewere
countedusingfeatureCountsandthennormalizedtocountspermil-
lion(CPM)usingedgeRpackagesinR.CPMwasconvertedtoattomole
bylinearfittingoftheRNAERCCspike-in.HalflifetimeofRNAwas
estimatedusingformulalistedinpreviousstudy23.Specifically,asActD
treatment results in transcription stalling, the change of RNA con-
centrationatagiventime(dC/dt)isproportionaltotheconstantof
RNAdecay(K )andtheRNAconcentration(C),leadingtothefol-
decay
lowingequation:
d d C t = (cid:1)K decay C ð1Þ
Thus,theRNAdegradationrateK wasestimatedby:
decay
(cid:1) (cid:3)
C
ln = (cid:1)K t ð2Þ
C0 decay
To calculate the RNA half-life (t ), when 50% of the RNA is
1/2
decayed(thatis,C/C0=1/2),theequationwas:
(cid:1) (cid:3)
ln 1 = (cid:1)K t ð3Þ
2 decay 2 1
Fromwhere:
ln2
t
2 1
=
K decay
ð4Þ
Thefinalhalf-lifewascalculatedbyusingtheaveragevalueof0h,
2h,4h,and6h.

SinglenucleusRNAsequence(snRNA-seq)analysis
The published snRNA-seq data42 of human placenta tissue at early
pregnancy(8–10weeks)andlatepregnancy(term)weredownloaded
(GSE247038),withtheembeddedUMAPandcellannotations.Thecell
clusteringwasreproduced,andthescaledaverageexpressionofgenes
ofinterestwasvisualized.

Statisticalandreproducibility
Nostatisticalmethodswereusedtopredeterminesamplesizes,and
samplesizeswerechosenbasedonpreviouslypublishedstudiesin
the field. All experiments, including transmission electron micro-
scopy, RT-qPCR, western blotting, and immunofluorescence, for
quantitative analysis and representative images were performed
usingatleastthreebiologicalreplicates,exceptforRNA-seq,ATAC-
seq, MeRIP-seq, and CUT&Tag experiments, all of which had two
biological replicates. The statistical analysis was conducted with
Microsoft Excel or R (v 4.2.3). p values were calculated using
unpairedStudent’st-testorWilcoxontest.Thethresholdforstatis-
ticalsignificancewasp<0.05.Fortheidentificationofdifferentially
expressedgenes,theWaldtestwasappliedusingtheDESeq2pack-
age in R. The number of measurements and independent experi-
ments,andthestatisticaltestusedforeachanalysisperformedwere
describedinthefigurelegends.

Reportingsummary
Further information on research design is available in the Nature
PortfolioReportingSummarylinkedtothisarticle.

Dataavailability
AllrawdatageneratedinthisstudyareavailableattheGeneExpres-
sionOmnibus(GEO)undertheaccessionnumberGSE280039.There
arenorestrictionsondataavailabilityoruse.ThesnRNA-seqdataof
humanplacentafromthisstudy42isavailableintheGEOdatabasewith
the accession GSE247038 and the array data from this study64 is
available in the GEO database under the accession code
GSE40182.Sourcedataareprovidedwiththispaper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors investigate the role of METTL3-mediated m6A RNA methylation in hTSCs generation from hEPSCs, hTSC self-renewal and cellular identity maintenance, and the underlying mechanism. While the study is interesting and comprehensive, specific concerns should be addressed to strengthen its scientific rigor.\nMajor concerns\n1. The DOX-inducible METTL3 depletion system is well-designed to avoid potential confounding effects of long-term knockout and to capture dynamic cellular transitions within 1-3 passages. However, the following issues must be addressed:\ni. Almost all experiments rely on the same cell line. How are cell line-specific biases eliminated?\nii. For key phenotypes (cell identity/histone modifications), ±DOX controls must be included to rule out nonspecific effects of DOX itself.\niii. Complementary approaches (e.g., METTL3 knockdown and/or STM2457 treatment) are required to rigorously establish the causality between of METTL3/m6A depletion and the phenotype.\niv. Since the main findings are derived from this in vitro system, in vivo validation or correlation analysis between METTL3/m6A with H3K27ac/H3K27me3 in human placental tissues could be performed to establish physiological relevance.\n2. The conclusion that METTL3 suppresses STB genes in hTSCs indirectly of m6A methylation remains questionable. While RNA levels of trophoblast master regulators show minimal changes upon METTL3 loss, m6A is known to regulate splicing and translation efficiency. Have the authors analyzed the splicing isoforms and the protein level of these genes? In addition, why not show the results of forced expression of PEG3 and PEG10 in P3 hTSCs?\n3. The proposed mechanism - wherein METTL3 depletion alters EP300 and EZH2 expression, leading to H3K27ac/H3K27me3 changes at inflammatory/SASP genes and hTSC defects- requires clarification:\nAre inflammatory/SASP genes modified by m6A? Provide the MeRIP-seq data for these genes. Is the half-life of these genes altered in METTL3 KO cells? Are trophoblast master regulators, STB specific genes, and PEG3/PEG10 direct targets of EP300? If so, do their promoters/enhancers exhibit altered H3K27ac or H3K27me3 levels upon METTL3 KO? What exactly is the mechanism that causes hTSC defects?\n4. To confirm that EP300/EZH2 expression is controlled by m6A, the authors should validate m6A changes on these genes using an independent method and determine whether STM2457 treatment changes the expression of these two genes.\nMinor concerns:\n1. Fig. 1D. The claim that METTL3 and METTL14 are specifically enriched in CTBs requires quantification, rather than just a single image.\n2. Since that the role of METTL3 is studied in the generation process of hTSC from hEPSC, the expression level of METTL3 and METTL14 in hEPSC should as well be shown in Figure 1.\n3. Is LTR10A RNA modified by m6A?", "reviewer_2": "The study presents several findings regarding the role of METTL3-mediated m6A RNA methylation in human trophoblast development. METTL3 and METTL14 specifically marked cytotrophoblast cells (CTBs) in the human placenta, with expression declining during differentiation into syncytiotrophoblasts (STBs). METTL3 was demonstrated to be essential for generating human trophoblast stem cells (hTSCs) from human expanded potential stem cells (hEPSCs), with the knockout preventing proper differentiation. The loss of METTL3 in established hTSCs resulted in impaired self-renewal and proliferation, increased cell death (apoptosis and necroptosis), premature senescence, dysregulated differentiation towards STBs, upregulation of inflammatory response genes, and the senescence-associated secretory phenotype (SASP). Mechanistically, METTL3-dependent m6A modifications were shown to regulate the transcript stability of key epigenetic modifiers: EP300 (histone acetyltransferase) - stabilized in METTL3-KO cells; EZH2 (H3K27 methyltransferase) - destabilized in METTL3-KO cells. This led to increased H3K27ac and decreased H3K27me3 histone marks. METTL3-deficient hTSCs exhibited preeclampsia (PE)-like molecular signatures. Chemical intervention targeting the dysregulated epigenetic machinery rescued some defects: EP300 inhibitor (A-485) reduced abnormal STB gene expression and mitigated PE signatures; KDM6B inhibitor (GSK-J1) increased H3K27me3 levels and suppressed STB gene expression. This work provides new insights into the epigenetic regulation of human trophoblast development, highlighting m6A RNA methylation's critical role.\nThe characterization of METTL3/METTL14 expression in trophoblast cells is supported by scRNA-seq, immunofluorescence, and western blotting. The requirement of METTL3 for hTSC generation and maintenance is demonstrated using a doxycycline-inducible knockout system and complementary approaches. MeRIP-seq, RNA stability assays, and histone mark profiling support the mechanism linking METTL3 to epigenetic regulation. The connection to preeclampsia would benefit from additional validation in clinical samples beyond transcriptomic signatures.\nThe study employs appropriate cell models (hEPSCs, hTSCs) and a controlled genetic system (doxycycline-inducible METTL3 KO). Multiple complementary approaches validated key findings, including genetic (METTL3 KO) and pharmacological (STM2457 inhibitor) manipulations. State-of-the-art omics techniques (RNA-seq, MeRIP-seq, CUT&Tag) provided comprehensive molecular profiling. Rescue experiments with epigenetic inhibitors strengthened mechanistic conclusions.\nPoints to consider:\nThe connection to preeclampsia relies primarily on transcriptomic signatures rather than functional assessment of PE features like abnormal placentation or hypertension. The rescue experiments with epigenetic inhibitors show partial reversal of phenotypes, suggesting additional mechanisms may be involved. While the paper proposes a linear pathway from METTL3 loss to epigenetic changes to cellular phenotypes, the actual molecular networks are likely more complex. Revisions acknowledging limitations in the clinical relevance and considering alternative mechanisms would strengthen the paper\nIn the pie chart (Fig. 4a), the authors showed nearly 900 upregulated mRNAs on P2 out of 5409 m6A-modified RNAs, of which only 168 demonstrated increased stabilization upon treatment with Actinomycin D on P2 versus P0. This suggests that changes in m6A due to METTL3 loss and increased RNA stability cannot account for the upregulation of the majority of genes. They conducted a meRIPseq analysis of total RNA and gene expression from mRNAseq. Based on these data, which classes of RNAs were m6A modified and how many are mRNA?\nIt is unclear when METTL3 expression is first reduced after Dox removal. However, to conclude that changes in mRNA stabilization of EP300 and EZH2 lead to changes in histone marks, mRNA stabilization should be demonstrated at an earlier time point after METTL3 loss and before the observed widespread changes in the transcriptome, including EZH2 and EP300 on P2, earlier.\nIn Fig. 4e, EZH2 and EP300 protein levels remain unchanged at P2. In contrast, H3K27ac levels increase significantly along with a moderate decrease in H3K27me3 (Fig. 4g). Thus, the authors need to demonstrate that changes in EZH2 and EP300 occupancy at the target regulatory regions or genes before P2 explain the increased histone modifications. Additionally, it is unclear which day the tracks in Fig. 4i are from. The authors should include a discussion of the possible contribution of other histone modifications to these changes in gene expression. Did H3K4me3 levels change at the DEGs during this time?\nOn page 9, the description of the results from Fig. S6 refers to Fig. S3. This needs to be corrected.", "reviewer_3": "Fu et al have carried out an interesting and thorough analysis on the role of METTL3 in hTSCs induced in culture from hEPSCs. Posttranscriptional regulatory mechanisms are an understudied facet of reproductive and developmental biology and the placenta is an understudied but essential tissue so this study is timely and of wide interest.\nIt would be great to see this study published but I think there are a few outstanding experiments required.\n1) The authors very convincingly show that METTL3 is required for survival of hTSCs and maintenance of the stem cell state. They observe senescence and cell death but also precocious differentiation towards STB. One key missing part of the study is a description of global and transcript-specific m6A levels in normal STB differentiation. As both immunofluorescence (as shown in figure S3d) and meRIP experiments seem to be working efficiently it should be straightforward to complete these. These experiments would strengthen the conclusions about the role of METTL3 and m6A in maintenance of the hTSC state and in differentiation and rule out any possibility that what is being described is an artefact of cells dying.\n2) The authors show that key hTSC transcription factors like GATA2 and TFAP2C are modified by m6A and that this is lost upon METTL3 depletion. This loss does not appear to have an effect on the transcript abundance. However, it may have an effect on translation, so it is necessary to look at the protein level to rule out their depletion in the precocious STB differentiation.\n3) The authors describe an indirect mechanism for regulating STB differentiation genes via histone modifiers EP300 and EZH2. One missing analysis is a global comparison of the H3K27ac and H3Kme3 cut and run peaks with the RNA-seq data set. An analysis similar to the meRIP -RNA-seq comparison in Figure 3d would be ideal. There are some examples shown but a more unbiased analysis is necessary. Are all the misexpressed genes now explained by either m6A or epigenetic regulation or are there still STB genes up-regulated with no clear mechanism?\nA few minor comments:\n1) The use of P0, P2 etc is not the most easy nomenclature to follow. Suggest changing to P0 (5ng/ml), P2-DOX etc or similar\n2) Some detail needs to be added to the text about the nature of the passaging. If cells are not proliferating then how are they being passaged 1:2, 1:4?. Is the ratio the same for all conditions? What length of time between passages?\n3) Figure 5 j and k. Please check the labelling is correct. The graphs seem to be very similar and it’s difficult to see what is different.\n4) Detail needs to be added to the text on the presence of 2 bands on the Ezh2 Western", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "The authors investigate the role of METTL3-mediated m6A RNA methylation in hTSCs generation from hEPSCs, hTSC self-renewal and cellular identity maintenance, and the underlying mechanism. While the study is interesting and comprehensive, specific concerns should be addressed to strengthen its scientific rigor.\nMajor concerns\n1. The DOX-inducible METTL3 depletion system is well-designed to avoid potential confounding effects of long-term knockout and to capture dynamic cellular transitions within 1-3 passages. However, the following issues must be addressed:\ni. Almost all experiments rely on the same cell line. How are cell line-specific biases eliminated?\nThank you for the question. We would like to clarify that our main conclusions in the original manuscript, which utilized inducible METTL3-KO in hTSCs, were based on two independent cell lines, TSC#3 and TSC#4 (e.g., in the original Fig. 2k, 5e, S3g, and now shown in updated Fig. 3k, 6e, S3g). Although the overall trends in transcriptomic changes following METTL3 loss are consistent across both cell lines, there are some subtle differences. Additionally, we used wild-type hTSCs treated with the METTL3 inhibitor STM2457 and reproduced our key findings (original Fig. S3m-q and now updated Fig. S4a-f).\nGiven that these hTSC lines are all derived from hEPSCs with same background (em-1 hEPSCs)1, to strengthen our conclusions, we further conducted experiments using hTSCs derived from a different hEPSC line (HUES8) and from in vivo placenta villi (CT-hTSCs)2. The new results are as follows:\n1. For HUES8-hTSCs, we treated the cells with the METTL3 inhibitor STM2457 (10 μM) for two passages and performed RNA-seq analysis. The results indicated that STM2457 treatment led to a significant upregulation of several mature syncytiotrophoblast (STB) genes, including ERVW-1, CGA, CGB3, CGB5, and SDC1 (updated Fig. S4g, h). In contrast, expressions of some key master regulator genes such as GATA2/3, TFAP2A/C, and TEAD1/4 showed no significant changes (updated Fig. S4g, h). This further supports our conclusion that m6A loss in hTSCs induces STB gene upregulation independently of these key master regulators of hTSCs. We confirmed these findings at the protein level as well (updated Fig. S4i, j).\nAdditionally, we observed that METTL3 inhibition in HUES8-hTSCs leads to increased expression levels of EP300, c-JUN, JUND, and H3K27ac, while expression levels of EZH2 and H3K27me3 are decreased (updated Fig. S6i, j).\n2. For cytotrophoblast-derived hTSCs (CT-hTSCs), we treated them with the METTL3 inhibitor STM2457 (10 μM) for two passages and conducted immunofluorescence experiments. This treatment leads to decreased proliferation capacity, increased hormone expression, and a higher fusion index, while GATA2 expression remained unaffected. These findings are presented in the updated Fig. S4k-n.\nTogether, our new data from multiple cell lines further support the main conclusion of this study, that loss of METTL3 in hTSCs induces the upregulation of mature STB genes primarily through epigenetic mechanisms, rather than by altering known trophoblast master regulators. We have incorporated these new results into the manuscript in the main text in line 246-254 and 354-356, and in the Methods section in line 30-36.\nii. For key phenotypes (cell identity/histone modifications), ±DOX controls must be included to rule out nonspecific effects of DOX itself.\nThank you for the comment. In this study, we established an inducible knockout system in which exogenous METTL3 expression controlled by DOX treatment, was introduced, followed by deletion of the endogenous METTL3. Upon removal of DOX, METTL3 protein levels were rapidly decreased (original Fig. S3b, c and now updated Fig. S3b, c). We also titrated DOX concentration and found that 5 ng/ml DOX induces exogenous METTL3 expression to levels comparable to the endogenous METTL3 in normal hTSCs (original Fig. S3b and now updated Fig. S3b). Therefore, unless otherwise specified, we used 5 ng/ml DOX to maintain inducible METTL3-KO hTSCs, as described in Methods section in line 92. For all experiments utilizing this inducible KO system, +DOX (5 ng/ml, designated as P0) served as the control group. We analyzed the phenotype and mechanism in KO cells from different passages (-DOX P1, P2, and P3).\nAlthough 5 ng/ml DOX is an extremely low concentration, as suggested, we also evaluated its potential effects on wild type hTSC cultures. Specifically, wild type hTSCs were cultured with 5 ng/ml DOX for three passages, followed by removal of DOX for an additional three passages. RT-qPCR and western blot analyses were performed to assess certain STB gene markers (e.g., CGA, CGB, and SDC1), pan-trophoblast markers (e.g., GATA2 and GATA3) and histone modifications (H3K27ac and H3K27me3). The results, shown below, indicate that this low concentration of DOX does not exert substantial nonspecific effects in our system: there was no obvious upregulation of STB marker genes nor changes in histone modifications.\nConsidering the low DOX concentration employed in this study, and the validation of our primary findings across multiple cell lines using the specific METTL3 inhibitor STM2457, we would not include this data in the manuscript.\niii. Complementary approaches (e.g., METTL3 knockdown and/or STM2457 treatment) are required to rigorously establish the causality between of METTL3/m6A depletion and the phenotype.\nThank you for the suggestion regarding genetic inactivation and chemical inhibition. In fact, we have already included several results on METTL3 inhibition using STM2457 treatment in the original manuscript (e.g., in the original Fig. 2f and S3m-q, now shown in the updated Fig. 3f and S4a-f). These results demonstrate that STM2457 treatment and METTL3 knockout have similar phenotypes, including reduced cell proliferation, increased DNA damage, and elevated STB-related hormone secretion.\nAs advised by your first comment, we also performed METTL3 inhibition using STM2457 in two additional hTSC lines derived from different backgrounds. Similar to the genetic METTL3 KO, pharmacological inhibition of METTL3 in wild type hTSCs led to decreased cell proliferation, increased cell fusion, altered hormone secretion, and histone modification changes. These new findings are presented in the updated Fig. S4g-n and S6i, j. We have incorporated these new results in the main text in line 246-254, and 354-358.\niv. Since the main findings are derived from this in vitro system, in vivo validation or correlation analysis between METTL3/m6A with H3K27ac/H3K27me3 in human placental tissues could be performed to establish physiological relevance.\nThank you for the valuable advice. Additionally, as also suggested by the second reviewer, we conducted comprehensive immunofluorescence analysis using samples collected from 8-week human placentas (early pregnancy), 38-week normal term placentas (late pregnancy) from two donors, and term placentas diagnosed with preeclampsia (PE) from two patients (35- and 37-week). The results are summarized and presented below:\n1. We re-stained METTL3 and METTL14 with the CTB marker PEG10 and the STB marker hCG in 8-week normal placental tissue and performed statistical analysis. The results, now presented in the updated Fig. 1d and e, indicate that METTL3 and METTL14 are specifically localized in CTBs but not in STBs.\n2. We performed staining for METTL3 and H3K27ac/me3 in placental tissues from 8-week, and H3K27ac/me3 levels were quantified by distinguishing METTL3-positive and METTL3-negative cells across all trophoblast cell types, including both CTBs and STBs. The results, now presented in the updated Fig. 7a and 7b, demonstrate that CTBs expressing METTL3 exhibit significantly lower levels of H3K27ac compared to METTL3-negative trophoblasts. This finding is consistent with our in vitro data, indicating that METTL3 negatively regulates EP300-mediated H3K27ac. Notably, we also observed a subset of mature STBs that lack H3K27ac expression despite being negative for METTL3 (highlighted with the yellow dashed-line region in the updated Fig. 7a), suggesting that H3K27ac may play stage-specific regulatory roles during trophoblast differentiation. In contrast, no significant correlation was observed between METTL3 expression and H3K27me3 levels. We have incorporated these new results in the main text in line 443-451.\n3. Given the significantly elevated H3K27ac levels observed in CTBs of preeclampsia (PE) patients3 and the role of METTL3 in regulating EP300-mediated H3K27ac, we hypothesized that METTL3 deficiency may exist in PE CTBs. To investigate this, we analyzed samples from two PE placentas of 35- and 37-week pregnancies respectively and included samples from two gestational age-matched normal placentas of 38-week pregnancy as controls. The results, now presented in updated Fig. 7c-g, demonstrate a significant reduction in the proportion of METTL3-positive trophoblasts in PE placentas compared to normal controls (Fig. 7c, d). More importantly, METTL3 expression was markedly decreased in PEG10-positive CTBs within PE placentas relative to those in normal placentas (Fig. 7c, e). Notably, a substantial subset of PEG10-positive CTBs in PE placentas lacked detectable METTL3 expression (Fig. 7c, white arrows). Consistent with the previous study3, H3K27ac levels were significantly elevated in PE CTBs (PEG10-positive cells) compared to normal controls (Fig. 7f, g). These findings suggest that the abnormal increase in H3K27ac levels in PE CTBs may result from deficiencies in METTL3 expression, indicating a potential mechanistic link between METTL3 downregulation and epigenetic dysregulation in PE pathology. We have incorporated these new results in the main text in line 453-467, and Method section in line 8-18.\nTaken together, our new in vivo data demonstrated that METTL3 is exclusively expressed in CTBs during normal pregnancy but is reduced in CTBs from PE placentas. METTL3 expression negatively correlates with H3K27ac expression, but not with H3K27me3 expression, during normal placentation. Furthermore, in the context of PE, decreased METTL3 levels in CTBs may lead to increased H3K27ac expression, aligning with our in vitro findings that METTL3-KO hTSCs acquire in vivo PE-associated signatures with increased H3K27ac.\n2. The conclusion that METTL3 suppresses STB genes in hTSCs indirectly of m6A methylation remains questionable. While RNA levels of trophoblast master regulators show minimal changes upon METTL3 loss, m6A is known to regulate splicing and translation efficiency. Have the authors analyzed the splicing isoforms and the protein level of these genes? In addition, why not show the results of forced expression of PEG3 and PEG10 in P3 hTSCs?\nAs suggested, we analyzed the splicing isoforms of several key trophoblast master regulator genes, including GATA2, GATA3, TFAP2A, TFAP2C, TEAD1, and TEAD4, using our RNA-seq data. The results indicate that loss of m6A modification on these transcripts does not affect their splicing isoforms. The updated findings are now presented in Fig. S5c and in the main text (line 289-290), and the splicing isoform analysis has been added to Methods section in line 237-241.\nFurthermore, we measured the protein levels of these master regulators in two cell models - inducible METTL3-KO hTSCs (with and without DOX) and METTL3-inhibited (DMSO- and STM2457-treated) wild-type hTSCs - and observed no significant differences in the protein levels of these regulators. The new data are presented in the updated Fig. S4f, i, j, l and Fig. S5d, e. Based on these results, we conclude that METTL3 suppresses STB genes in hTSCs indirectly, not through m6A methylation of these known trophoblast master regulator genes, but likely primarily via histone epigenetic mechanisms. We have incorporated these new results in the main text in line 252-254, and 289-290.\nAdditionally, we now included the PEG3/PEG10 overexpression data in the updated Fig. S5g–i. The results demonstrate that forced expression of PEG3 and PEG10 in METTL3-KO hTSCs could not rescue the proliferation defect nor the premature expression of STB marker genes caused by METTL3 loss. These findings have been incorporated in the main text in line 293-295 and in the Methods section in line 62-67.\n3. The proposed mechanism - wherein METTL3 depletion alters EP300 and EZH2 expression, leading to H3K27ac/H3K27me3 changes at inflammatory/SASP genes and hTSC defects- requires clarification:\nAre inflammatory/SASP genes modified by m6A? Provide the MeRIP-seq data for these genes. Is the half-life of these genes altered in METTL3 KO cells? Are trophoblast master regulators, STB specific genes, and PEG3/PEG10 direct targets of EP300? If so, do their promoters/enhancers exhibit altered H3K27ac or H3K27me3 levels upon METTL3 KO? What exactly is the mechanism that causes hTSC defects?\nThanks for these insightful comments. To address these questions and further clarify the underlying mechanisms of how METTL3 regulates inflammatory/SASP/STB genes in hTSCs, we re-analyzed our MeRIP-seq and CUT&Tag data and conducted additional EP300 CUT&Tag in P0 and P3 hTSCs. The new results are shown below:\n1. To enhance clarity regarding the data and conclusions, we now included the STB gene set4 in the new analysis instead of focusing on individual STB marker gene as in the original manuscript. The transcriptomic analysis demonstrated that the STB gene set was significantly activated upon METTL3 knockout in hTSCs (updated Fig. 3l).\n2. To clarify the underlying mechanisms, we re-analyzed our RNA-seq and MeRIP-seq data. The analysis revealed that only a minor proportion of upregulated genes within these three gene sets were modified with m6A (updated Fig. 4f). Specifically, only 10–20% of the total mRNA in these gene sets exhibited m6A modifications (updated Fig. 4g). Additionally, upon METTL3 depletion, the fold changes in expression for m6A-modified transcripts were comparable to or even smaller than those observed for non-m6A-modified transcripts (updated Fig. S5j), indicating that m6A methylation is unlikely to be a key driving factor behind the upregulation of these genes. For example, the STB marker genes ERVW-1 and CGA were not modified with m6A but were upregulated upon METTL3 loss (updated Fig. 4h). On the other hand, several upregulated genes did carry m6A modifications, such as the SASP gene GDF15 and the inflammation-related gene IL4R (updated Fig. 4h). These findings indicate that METTL3 modulates gene expression in hTSCs through both m6A-dependent and m6A-independent pathways. Given the relatively low proportion of m6A-modified mRNA, the regulatory effects of METTL3 may be predominantly mediated via indirect mechanisms rather than direct m6A modification of target transcripts. All information regarding m6A modifications and gene expressions within these three gene sets is provided in the updated Table S2. We have incorporated these new results in the main text in line 297-313.\n3. Furthermore, by re-analyzing our Actinomycin D treatment RNA-seq data using inflammatory, SASP, and STB gene sets, we confirmed that although transcripts within these three gene sets tended to be more stable, only the STB gene set exhibited a significant increase in stability, while the remaining two gene sets showed no significant change (updated Fig. S6b). These results also suggest that alternative mechanisms may contribute to gene expression regulation in hTSCs upon METTL3 loss, independently of m6A. We have incorporated these new results in the main text in line 325-328. All information regarding RNA stability within these three gene sets is provided in the updated Table S2.\n4. In our new EP300 CUT&Tag results, we observed an overall increase in EP300 binding sites following METTL3 KO (updated Fig. 5i), consistent with our western blotting data (updated Fig. 5e, f). Regarding trophoblast master regulators such as GATA2, GATA3, TFAP2A and TFAP2C, we found that EP300 binds to their promoters and enhancers, along with H3K27ac depositions, in P0 hTSCs, with further increased binding detected in P3 hTSCs. These findings are illustrated below. However, both our original and new data confirm that the transcription and protein levels of these regulators remain unchanged upon METTL3 knockout or inhibition (see the second comment above). Based on these results, we conclude that the phenotypes observed upon METTL3 deficiency in hTSCs are not due to alterations in these master regulators. Therefore, we decided not to include the IGV tracks in the manuscript.\nSimilarly, for PEG3 and PEG10, our data suggest that EP300 can bind to their promoters and enhancers in P0 hTSCs, with increased binding in P3 hTSCs (shown below). However, our original data suggested that transcription and protein levels of PEG3 and PEG10 both decrease upon METTL3 KO (now in Fig. 4d, S5f). Importantly, overexpression of PEG3 and PEG10 could not rescue the dysregulated expression of STB genes or impaired growth capacity in P3 hTSCs (updated Fig. S5g-i). Thus, we conclude that the phenotypes resulting from METTL3 KO in hTSCs are not primarily driven by PEG3 and PEG10. Thus, we decided not to include the IGV tracks shown below in the manuscript.\n5. To further elucidate the mechanisms underlying hTSC defects caused by METTL3 loss, we integrated RNA-seq, MeRIP-seq, and CUT&Tag datasets. Our new results demonstrate that:\na. H3K27ac enrichment and EP300 occupancy exhibit a positive correlation with gene expression levels, whereas H3K27me3 deposition demonstrates an inverse correlation with gene expression in P3 compared to P0 hTSCs (updated Fig. S7a-f). Additionally, H3K27ac, EP300, and JUND displayed increased enrichment across the STB, SASP, and inflammation gene sets upon METTL3 depletion, whereas H3K27me3 exhibited an inverse trend (updated Fig. 5j), indicating the pivotal roles of transcription factors and histone modifications in regulating gene expression in hTSCs through METTL3.\nb. Nearly all m6A-modified genes in these three gene sets also exhibit concurrent modifications by H3K27ac and/or H3K27me3 (updated Fig. S7g). Notably, approximately two-thirds of the m6A-modified genes are simultaneously co-regulated by both H3K27ac and H3K27me3 (updated Fig. S7g), suggesting a complex interplay of epitranscriptomic and histone epigenetic mechanisms in gene regulation within these pathways. For instance, STB genes within the PSG clusters and inflammation-associated genes such as TNFRSF1B did not exhibit m6A modifications but were upregulated following METTL3 depletion (Table S2). These genes showed increased enrichment of H3K27ac, EP300, and JUND, concomitant with decreased H3K27me3 deposition in P3 hTSCs compared to P0 hTSCs (updated Fig. 5k, l). A similar pattern was also observed in SASP-related genes, including SERPINE1 and VGF, as well as inflammation-associated genes such as IL6 (updated Fig. S7h, i and Table S2), indicating an indirect regulatory effect mediated through m6A methylation. Conversely, GDF15, a marker gene for STB and a component of SASP that was modified with m6A, exhibited increased mRNA stability upon METTL3 KO (Table S2), along with elevated enrichment of H3K27ac, EP300, and JUND (updated Fig. 5m). A comparable trend was also observed in the inflammation-related gene RIPK2 (updated Fig. S7j and Table S2).\nCollectively, these new results indicate that METTL3 orchestrates trophoblast gene regulation via both direct m6A-dependent mechanisms and indirect epigenetic pathways involving EP300-mediated H3K27ac and EZH2-mediated H3K27me3, with the histone epigenetic pathways playing a more prominent role. Following METTL3 loss in hTSCs, a small subset of genes directly affected by m6A regulation (which are also co-regulated by H3K27ac and/or H3K27me3). However, most dysregulated genes are not m6A-modified and are indirectly regulated through changes in H3K27ac and H3K27me3 modifications. Detailed information on m6A, H3K27ac, H3K27me3 modifications, RNA stability, and gene expression for these gene sets is provided in the updated Table S2. These findings have been incorporated in the main text in line 365-394.\n4. To confirm that EP300/EZH2 expression is controlled by m6A, the authors should validate m6A changes on these genes using an independent method and determine whether STM2457 treatment changes the expression of these two genes.\nThank you for your comment. We now employed a single-base elongation- and ligation-based PCR amplification method (termed ‘SELECT’)5 to independently detect specific m6A modifications on EP300 and EZH2 transcripts in wild-type hTSCs (HUES8-hTSC line) treated with DMSO and STM2457 for two passages. The new data are presented in the updated Fig. S6f, which indicates that EP300 and EZH2 transcripts are modified with m6A in control hTSCs, with a reduction in m6A levels following STM2457 treatment. We also incorporated this data in the main text in line 347-349 and in Methods section in line183-191.\nAdditionally, consistent with the METTL3 knockout results, STM2457 treatment in wild type hTSCs leads to upregulation of EP300 and downregulation of EZH2 proteins levels. We have incorporated these new results in the updated Fig. S6i, j and in the main text in line 354-356.\nMinor concerns:\n1. Fig. 1D. The claim that METTL3 and METTL14 are specifically enriched in CTBs requires quantification, rather than just a single image.\nWe have now improved our data accordingly. Specifically, we re-stained METTL3 and METTL14 with the STB marker hCG and the CTB marker PEG10 in 8-week and 38-week human placental tissue using immunofluorescence, followed by quantification, and replaced the previous H&E staining figure. In vivo human placental villi at 8 weeks exhibited exclusive METTL3 and METTL14 proteins expression in CTBs, co-localized with PEG10 but not hCG (updated Fig. 1d, e). In late pregnancy, METTL3 remained exclusively expressed in CTBs alongside PEG10, even though the overall CTB population was markedly reduced compared to early pregnancy (updated Fig. S1c, d). These findings demonstrate that METTL3 specifically marks CTBs in human placentas, suggesting its potential role in regulating CTBs cellular identity and their differentiation capacity. We have incorporated this data in the main text in line 135-141.\n2. Since that the role of METTL3 is studied in the generation process of hTSC from hEPSC, the expression level of METTL3 and METTL14 in hEPSC should as well be shown in Figure 1.\nWe have now included hEPSCs as a control and incorporated these results into the updated Fig. 2a-c, as suggested by the third reviewer. The updated western blotting, immunostaining, and quantification data show that STB cells have markedly lower levels of METTL3 and METTL14 proteins compared to both hTSCs and hEPSCs. Additionally, METTL3 and METTL14 levels are slightly higher in hEPSCs than in hTSCs, although the difference in METTL14 expression is not statistically significant. The manuscript has been revised accordingly in lines 144-152 to accurately present these findings.\n3. Is LTR10A RNA modified by m6A?\nBy re-analyzing our MeRIP-seq data, we concluded that LTR10A is not modified by m6A, as no m6A signals could be mapped to LTR10A consensus sequence. As a positive control, we analyzed another transposable element, SVA_D, which was confirmed to be m6A-modified in hTSCs using the same pipeline (shown below). Based on these results, we speculate that METTL3 regulates LTR10A indirectly, potentially through transcription factors such as EP300 and JUND, which contain an enriched motif within LTR10A and are upregulated upon METTL3 loss (data shown in the updated Fig. 6g, i). Since the regulation of LTR10A by m6A is not the focus of this study, we did not include this data in the manuscript.\nReviewer #2 (Remarks to the Author):\nThe study presents several findings regarding the role of METTL3-mediated m6A RNA methylation in human trophoblast development. METTL3 and METTL14 specifically marked cytotrophoblast cells (CTBs) in the human placenta, with expression declining during differentiation into syncytiotrophoblasts (STBs). METTL3 was demonstrated to be essential for generating human trophoblast stem cells (hTSCs) from human expanded potential stem cells (hEPSCs), with the knockout preventing proper differentiation. The loss of METTL3 in established hTSCs resulted in impaired self-renewal and proliferation, increased cell death (apoptosis and necroptosis), premature senescence, dysregulated differentiation towards STBs, upregulation of inflammatory response genes, and the senescence-associated secretory phenotype (SASP). Mechanistically, METTL3-dependent m6A modifications were shown to regulate the transcript stability of key epigenetic modifiers: EP300 (histone acetyltransferase) - stabilized in METTL3-KO cells; EZH2 (H3K27 methyltransferase) - destabilized in METTL3-KO cells. This led to increased H3K27ac and decreased H3K27me3 histone marks. METTL3-deficient hTSCs exhibited preeclampsia (PE)-like molecular signatures. Chemical intervention targeting the dysregulated epigenetic machinery rescued some defects: EP300 inhibitor (A-485) reduced abnormal STB gene expression and mitigated PE signatures; KDM6B inhibitor (GSK-J1) increased H3K27me3 levels and suppressed STB gene expression. This work provides new insights into the epigenetic regulation of human trophoblast development, highlighting m6A RNA methylation's critical role.\nThe characterization of METTL3/METTL14 expression in trophoblast cells is supported by scRNA-seq, immunofluorescence, and western blotting. The requirement of METTL3 for hTSC generation and maintenance is demonstrated using a doxycycline-inducible knockout system and complementary approaches. MeRIP-seq, RNA stability assays, and histone mark profiling support the mechanism linking METTL3 to epigenetic regulation. The connection to preeclampsia would benefit from additional validation in clinical samples beyond transcriptomic signatures.\nThe study employs appropriate cell models (hEPSCs, hTSCs) and a controlled genetic system (doxycycline-inducible METTL3 KO). Multiple complementary approaches validated key findings, including genetic (METTL3 KO) and pharmacological (STM2457 inhibitor) manipulations. State-of-the-art omics techniques (RNA-seq, MeRIP-seq, CUT&Tag) provided comprehensive molecular profiling. Rescue experiments with epigenetic inhibitors strengthened mechanistic conclusions.\nThank you for your appreciation of our work and for providing us with the opportunity to improve it.\nPoints to consider:\nThe connection to preeclampsia relies primarily on transcriptomic signatures rather than functional assessment of PE features like abnormal placentation or hypertension. The rescue experiments with epigenetic inhibitors show partial reversal of phenotypes, suggesting additional mechanisms may be involved. While the paper proposes a linear pathway from METTL3 loss to epigenetic changes to cellular phenotypes, the actual molecular networks are likely more complex. Revisions acknowledging limitations in the clinical relevance and considering alternative mechanisms would strengthen the paper.\nThank you very much for your insightful comments regarding the clinical relevance of RNA methylation in preeclampsia and the exploration of alternative mechanisms. To enhance our study and also suggested by other two reviewers, we conducted additional experiments and analyses and present the summarized results below.\n1. We performed immunofluorescence staining for METTL3 and H3K27ac/me3 in placental tissues from 8-week, and H3K27ac/me3 levels were quantified by distinguishing METTL3-positive and METTL3-negative cells across all trophoblast cell types, including both cytotrophoblasts (CTBs) and syncytiotrophoblasts (STBs). The results, now presented in the updated Fig. 7a and 7b, demonstrate that CTBs expressing METTL3 exhibit significantly lower levels of H3K27ac compared to METTL3-negative trophoblasts. This finding is consistent with our in vitro data, indicating that METTL3 negatively regulates EP300-mediated H3K27ac. Notably, we also observed a subset of mature STBs that lack H3K27ac expression despite being negative for METTL3 (highlighted within the yellow dashed-line region in the updated Fig. 7a), suggesting that H3K27ac may play stage-specific regulatory roles during trophoblast differentiation. In contrast, no significant correlation was observed between METTL3 expression and H3K27me3 levels. We have incorporated these new results in the main text in line 443-451.\n2. Given the significantly elevated H3K27ac levels observed in CTBs of preeclampsia (PE) patients3 and the role of METTL3 in regulating EP300-mediated H3K27ac, we hypothesized that METTL3 deficiency may exist in PE CTBs. To investigate this, we analyzed samples from two PE placentas of 35- and 37-week pregnancies respectively and included samples from two gestational age-matched normal placentas of 38-week as controls. PE was diagnosed according to the following criteria: new-onset hypertension and proteinuria (details see Method section in line 9-19). The results, now presented in updated Fig. 7c-g, demonstrate a significant reduction in the proportion of METTL3-positive trophoblasts in PE placentas compared to normal controls (Fig. 7c, d). More importantly, METTL3 expression was markedly decreased in PEG10-positive CTBs within PE placentas relative to those in normal placentas (Fig. 7c, e). Notably, a substantial subset of PEG10-positive CTBs in PE placentas lacked detectable METTL3 expression (Fig. 7c, white arrows). Consistent with the previous study3, H3K27ac levels were significantly elevated in PE CTBs (PEG10-positive cells) compared to normal controls (Fig. 7f, g). These findings suggest that the abnormal increase in H3K27ac levels in PE CTBs may result from deficiencies in METTL3 expression, indicating a potential mechanistic link between METTL3 downregulation and epigenetic dysregulation in PE pathology. We have incorporated these new results in the main text in line 453-467.\n3. To further clarify the underlying mechanisms, we conducted additional CUT&Tag for EP300 and re-analyzed our multi-omics data focusing on three gene sets: SASP, inflammation and the signature for STB cells, as these three gene sets were upregulated upon METTL3 KO in hTSCs (updated Fig. 3l). The new analysis revealed that only a minor proportion of upregulated genes within these three gene sets were modified with m6A (updated Fig. 4f). Specifically, only 10–20% of the total mRNA in these gene sets exhibited m6A modifications (updated Fig. 4g). Additionally, upon METTL3 depletion, the fold changes in expression for m6A-modified transcripts were comparable to or even smaller than those observed for non-m6A-modified transcripts (Fig. S5j), indicating that m6A methylation is unlikely to be a driving factor behind the upregulation of these genes. Further analysis suggested that H3K27ac enrichment and EP300 occupancy exhibit a positive correlation with gene expression levels, whereas H3K27me3 deposition demonstrates an inverse correlation with gene expression in P3 compared to P0 hTSCs (Fig. S7a–f). Additionally, H3K27ac, EP300, and JUND displayed increased enrichment across the STB, SASP, and inflammation gene sets upon METTL3 depletion, whereas H3K27me3 exhibited an inverse trend (Fig. 5j), indicating the pivotal roles of transcription factors and histone modifications in regulating gene expression in hTSCs. Interestingly, we noticed nearly all m6A-modified genes in these three gene sets also exhibit concurrent modifications by H3K27ac and/or H3K27me3 (Fig. S7g). Notably, approximately two-thirds of the m6A-modified genes are simultaneously co-regulated by both H3K27ac and H3K27me3 (Fig. S7g), suggesting a complex interplay of epitranscriptomic and histone epigenetic mechanisms in gene regulation within these pathways. Collectively, these new results indicate that METTL3 orchestrates trophoblast gene regulation via both direct m6A-dependent mechanisms and indirect epigenetic pathways involving EP300-mediated H3K27ac and EZH2-mediated H3K27me3, with the histone epigenetic pathways playing a more prominent role. Detailed information on m6A, H3K27ac, H3K27me3 modifications, RNA stability, and gene expression for these gene sets is provided in the updated Table S2. These findings have been incorporated in the main text in lines 297-313, 365-394.\n4. As noted by the reviewer, our rescue experiments using epigenetic inhibitors resulted in only partial reversal of the observed phenotypes, including the aberrant epigenetic states and associated gene expression changes. These interventions did not fully restore the proliferation and apoptosis deficits caused by METTL3 deficiency. This indicates that additional mechanisms beyond the METTL3-histone epigenetic-gene expression axis are involved, and the underlying molecular networks are likely more complex. For instance, a recent study demonstrated that m6A RNA methylation can regulate mitochondrial function by promoting the translation of nuclear-encoded mitochondrial complex subunit RNAs in neuronal cells6, which aligns with our observation that METTL3 loss in hTSCs leads to mitochondrial impairment (updated Fig. 3e). Whether METTL3 directly protects mitochondrial functions in hTSCs to preserve their self-renewal and differentiation capacities warrants further investigation. We have also incorporated this point into the discussion section in line 522-526.\nIn the pie chart (Fig. 4a), the authors showed nearly 900 upregulated mRNAs on P2 out of 5409 m6A-modified RNAs, of which only 168 demonstrated increased stabilization upon treatment with Actinomycin D on P2 versus P0. This suggests that changes in m6A due to METTL3 loss and increased RNA stability cannot account for the upregulation of the majority of genes. They conducted a meRIPseq analysis of total RNA and gene expression from mRNAseq. Based on these data, which classes of RNAs were m6A modified and how many are mRNA?\nThank you for your comments.\n1. The RNAs with m6A modifications in our MeRIP-seq data included both mRNA and lncRNA classes. We would like to clarify that our RNA m6A MeRIP experiment begins with total RNA. After RNA fragmentation and immunoprecipitation using an m6A antibody, we employed the SMARTer Stranded Total RNA-Seq Kit - version 2 (Pico Input Mammalian, Takara/Clontech, 634413) to construct libraries for both the input and IP samples. This kit includes a rRNA depletion step prior to library construction. We have updated the details in Methods section accordingly for the MeRIP-seq procedure in line 221. As a result, the MeRIP-seq data should encompass both mRNA and lncRNA classes.\n2. Upon re-examining our MeRIP-seq dataset, we identified a few genes that were initially listed as separate entries due to discrepancies in their Ensembl identifiers, despite representing the same gene symbols. This duplication arose from inconsistencies in the Ensembl annotation database, where some genes are associated with multiple identifier versions. To ensure accuracy and consistency, we consolidated these entries based on unified gene symbols. Consequently, the total number of m6A-modified genes was revised from 5,409 to 5,407. The updated Venn diagram in Fig. 5a has been revised accordingly to reflect these adjustments for improved clarity and precision.\n3. As suggested, we analyzed the types of m6A-modified RNAs and the proportion of mRNAs. The results, summarized below, indicate that out of a total of 5,407 m6A-modified RNAs, 4,407 (81.5%) are mRNAs, while 1,000 (18.5%) are non-coding RNAs. For the original Fig. 4a, we are sorry that we labelled the number incorrectly which in fact included overlaps. We have now corrected the label in the updated Fig. 5a that each number exclusively represents the corresponding portion of the Venn diagram.\nNow focusing on the 741 upregulated genes with m6A modifications, we found that only 168 exhibited increased half-lives, indicating m6A-dependent stabilization of these transcripts. The remaining genes may be regulated indirectly by m6A. To clarify this, we identified transcription factors (TFs) within the 168 genes and found 30 TFs, notably enhancer-associated factors such as CEBPA, EP300, FOSL2, and JUND. Computational analysis predicted that these TFs target 236 genes, representing 41.2% of the remaining 573 m6A-modified upregulated genes, suggesting an m6A-driven regulation mode via TFs. These findings support a model in which METTL3 primarily stabilizes specific enhancer-associated TFs, which then modulate the epigenetic landscape and reconfigure gene networks in human trophoblast stem cells.\nIt is unclear when METTL3 expression is first reduced after Dox removal. However, to conclude that changes in mRNA stabilization of EP300 and EZH2 lead to changes in histone marks, mRNA stabilization should be demonstrated at an earlier time point after METTL3 loss and before the observed widespread changes in the transcriptome, including EZH2 and EP300 on P2, earlier.\nThanks for pointing out these. In our original manuscript, we presented western blotting data showing that METTL3 and METTL14 proteins substantially decreased after DOX removal at Passage 1, with levels becoming nearly undetectable at Passage 2 and 3 (original Fig. S3c). To validate these findings, we repeated the experiment and performed long-exposure western blotting, which is now shown in the updated Fig. S3c. The new data consistently indicates that, although METTL3 expression significantly declined without DOX at Passages 1 to 3, METTL3 protein remains detectable at Passage 1 (3 days after DOX withdrawal).\nAs suggested, we also assessed and quantified the expression levels of EP300 and EZH2 at an earlier time point (Passage 1). The revised data are presented in the updated Fig. 5e and 5f, which show that, although EP300 exhibited a tendency toward upregulation and EZH2 toward downregulation from P0 to P3 in hTSCs, there were no statistically significant differences at P1 compared to P0, likely due to residual METTL3 and the short duration after DOX withdrawal.\nIn Fig. 4e, EZH2 and EP300 protein levels remain unchanged at P2. In contrast, H3K27ac levels increase significantly along with a moderate decrease in H3K27me3 (Fig. 4g). Thus, the authors need to demonstrate that changes in EZH2 and EP300 occupancy at the target regulatory regions or genes before P2 explain the increased histone modifications. Additionally, it is unclear which day the tracks in Fig. 4i are from. The authors should include a discussion of the possible contribution of other histone modifications to these changes in gene expression. Did H3K4me3 levels change at the DEGs during this time?\nThank you for these insightful comments.\n1. As per your previous suggestion, we re-examined the expression levels of EP300 and EZH2 across Passages 0, 1, 2, and 3 after DOX withdrawal, and performed proper statistical analyses. The updated results show that, although EP300 tended to be upregulated and EZH2 tended to be downregulated from P0 to P3 in hTSCs, there were no statistically significant differences at P1 compared to P0 cells. EP300 significantly increased and EZH2 decreased in P2 and P3 hTSCs compared to P0 cells. Consistent with the histone modification changes depicted in the original Fig. 4f, g (now presented in the updated Fig. 5g, h), we conclude that METTL3 knockout in hTSCs impacts EP300 and EZH2 expression, which subsequently leads to alterations in H3K27ac and H3K27me3 levels.\n2. In original Fig. 4i and j, the CUT&Tag data for H3K27ac, H3K27me3, and JUND are from P0 and P3 hTSCs. The ChIP-seq data for GATA3 and the ATAC-seq data are also from P0 and P3 hTSCs. Since the GATA3 ChIP-seq and ATAC-seq data are not directly relevant to our study, we have decided to exclude these datasets and instead include CUT&Tag data for EP300 in the updated manuscript. The detailed information has been incorporated into updated Fig. 5k-m, along with corresponding figure legends.\n3. We analyzed H3K4me3 levels in P0, P2, and P3 hTSCs, and the results shown below suggest that overall H3K4me3 levels do not significantly change upon METTL3 knockout. However, we cannot exclude the possibility that H3K4me3 modifications at specific DEGs are affected. To investigate this further, H3K4me3 CUT&Tag could be employed in future studies to profile H3K4me3 binding patterns across the genome.\n4. We have now included a discussion of the potential contribution of other histone modifications, specifically H3K4me3 and H3K9me3, to the gene expression changes observed upon METTL3 loss in hTSCs in the main text in line 502-505. Notably, H3K9me3-mediated heterochromatin has been reported to be regulated by METTL37, and its potential role should be considered in hTSCs.\nOn page 9, the description of the results from Fig. S6 refers to Fig. S3. This needs to be corrected.\nThanks for pointing out and we have made the correction (line 406).\nReviewer #3 (Remarks to the Author):\nFu et al have carried out an interesting and thorough analysis on the role of METTL3 in hTSCs induced in culture from hEPSCs. Posttranscriptional regulatory mechanisms are an understudied facet of reproductive and developmental biology and the placenta is an understudied but essential tissue so this study is timely and of wide interest.\nThank you for your appreciation of our work and for providing us with the opportunity to improve it.\nIt would be great to see this study published but I think there are a few outstanding experiments required.\n1) The authors very convincingly show that METTL3 is required for survival of hTSCs and maintenance of the stem cell state. They observe senescence and cell death but also precocious differentiation towards STB. One key missing part of the study is a description of global and transcript-specific m6A levels in normal STB differentiation. As both immunofluorescence (as shown in figure S3d) and meRIP experiments seem to be working efficiently it should be straightforward to complete these. These experiments would strengthen the conclusions about the role of METTL3 and m6A in maintenance of the hTSC state and in differentiation and rule out any possibility that what is being described is an artefact of cells dying.\nThank you for your insightful suggestions. We have now conducted a MeRIP-seq experiment in normal hTSCs to STBs differentiation, using the CT-hTSC line directly derived from in vivo placental villi2. Additionally, we performed m6A ELISA assay to quantify m6A dynamics during this process. The new results are now presented in the updated Fig. 2e-l. As also suggested by the first reviewer, we relocated the original Fig. 1e and f into the revised Fig. 2a-d, which now included the in vitro hTSCs to STBs differentiation data and incorporated hEPSCs as controls.\nThe new results suggest that, in line with our in vivo observation, METTL3 and METTL14 expression levels are higher in hTSCs compared to STBs (updated Fig. 2a-c). Re-analysis of our published RNA-seq data on hTSCs to STBs differentiation8 also revealed a substantial decline in the expression of METTL3 and METTL14, and of several m6A reader genes including YTHDC2, YTHDF2/3 and IGF2BP1/3 during the process (updated Fig. 2d). As a result, m6A levels markedly decreased during hTSC to STB development (updated Fig. 2e). These findings indicate a downregulation of the m6A methylation pathway during STB development from hTSCs.\nConsistently, the MeRIP-seq results demonstrated a substantial decrease in m6A signals on the 3’UTRs of transcripts in STBs compared to hTSCs (updated Fig. 2f). Specifically, 15,725 m6A-modified peaks were identified in hTSCs, whereas 12,449 peaks were modified with m6A in STBs, with 5,660 and 2,384 peaks being specific to hTSCs and STBs, respectively (updated Fig. 2g and Table S1). De novo motif analysis of these peaks revealed the classical m6A ‘RRACH’ motif (R denotes A or G, and H denotes A, C, or U) in both cells (updated Fig. 2f). Additionally, 4,463 peaks lost m6A modification during hTSCs to STBs, while 511 peaks gained m6A (updated Fig. 2h and Table S1). For example, marker genes for hTSCs, such as PEG3, PEG10, and MKI67, exhibited m6A modifications in hTSCs but showed a substantial decrease in STBs (updated Fig. 2h, i). Several key trophoblast master regulator genes, including GATA2, TFAP2A, and TFAP2C, also displayed m6A methylation in hTSCs, but with no significant changes in STBs (updated Fig. 2h, j). Although many transcripts lost m6A modifications in STBs, a notable number of transcripts, such as TNFRSF10B, GADD45G, and H3C4, gained m6A modifications (updated Fig. 2h, k). Interestingly, transcripts within the CGA and CGB clusters, which encode the hormone hCG secreted by STBs, lacked m6A modifications in both hTSCs and STBs (updated Fig. 2l). These findings are consistent across both WT- and KO- hTSCs and suggest that hormone secretion by STBs is not regulated via m6A pathways directly but may involve other mechanisms, such as histone epigenetic regulation based on our study.\nTaken together, our new results demonstrate that, despite the distinct m6A methylation profiles observed in hTSCs and STBs, both METTL3 expression levels and overall m6A methylation decline during the differentiation process. These new data not only elucidate the global and transcript-specific m6A dynamics between hTSCs and STBs but also help explain why METTL3-KO hTSCs acquire gene expression signatures characteristic of STBs. These findings have been incorporated in the main text in line 143-174.\n2) The authors show that key hTSC transcription factors like GATA2 and TFAP2C are modified by m6A and that this is lost upon METTL3 depletion. This loss does not appear to have an effect on the transcript abundance. However, it may have an effect on translation, so it is necessary to look at the protein level to rule out their depletion in the precocious STB differentiation.\nThanks for pointing this out. We have now included data on the protein level changes of GATA2, GATA3 and TFAP2C in two cell models- inducible METTL3-KO hTSCs (with and without DOX) and METTL3-inhibited (DMSO- and STM2457-treated) wild-type hTSCs- and observed no significant differences in the protein levels of these regulators. The new data are presented in the updated Fig. S4f, i, j, l and Fig. S5d, e. Furthermore, as suggested by the first reviewer, we analyzed the splicing isoforms of several key trophoblast master regulator genes, including GATA2, GATA3, TFAP2A, TFAP2C, TEAD1, and TEAD4, using our RNA-seq data. The results indicate that the loss of m6A modifications on these transcripts does not affect their splicing isoforms. The updated findings are now presented in Fig. S5c. These new results demonstrated that although these transcription factors are m6A methylated, their protein levels do not appear to differ significantly upon METTL3 loss or inhibition in hTSCs. Therefore, we conclude that the premature STB differentiation observed with METTL3 deficiency in hTSCs is unlikely to be driven by these key transcription factors, given that their expression levels remain unchanged. These findings have been incorporated in the main text in line 252-254, 289-290.\n3) The authors describe an indirect mechanism for regulating STB differentiation genes via histone modifiers EP300 and EZH2. One missing analysis is a global comparison of the H3K27ac and H3Kme3 cut and run peaks with the RNA-seq data set. An analysis similar to the meRIP -RNA-seq comparison in Figure 3d would be ideal. There are some examples shown but a more unbiased analysis is necessary. Are all the misexpressed genes now explained by either m6A or epigenetic regulation or are there still STB genes up-regulated with no clear mechanism?\nThanks for the insightful suggestion and comments. To elucidate the underlying mechanisms, we performed additional CUT&Tag experiments targeting EP300 in P0(+DOX) and P3(-DOX) hTSCs and re-analyzed our multi-omics datasets. The updated results are presented below.\n1. As suggested, we compared the global H3K27ac and H3K27me3 peaks with RNA-seq data. Additionally, in response to the first reviewer's comment, we incorporated EP300 CUT&Tag data to further validate our findings. The updated results are shown in Fig. S7a-f and demonstrate that, at the genome-wide level, H3K27ac and EP300 binding positively correlate with gene expression, whereas H3K27me3 shows a negative correlation. These findings suggest that the increased H3K27ac and EP300 binding, along with the decreased H3K27me3 in METTL3-KO hTSCs, may contribute to the dysregulated gene expression observed.\n2. To enhance clarity regarding the data and conclusions, we included the STB gene set4 in the new analysis instead of focusing on individual STB marker genes as in the original manuscript. The transcriptomic analysis showed that expression of the STB gene set was significantly upregulated following METTL3 knockout in hTSCs, accompanied by activation of SASP and inflammation pathways (updated Fig. 3l). We then focused on these three gene sets. By re-analyzing our RNA-seq and MeRIP-seq data, the new results revealed that only a small proportion of upregulated genes within these three gene sets were modified with m6A (updated Fig. 4f). Specifically, only 10–20% of the total mRNA in these gene sets exhibited m6A modifications (updated Fig. 4g). Additionally, upon METTL3 depletion, the fold changes in expression for m6A-modified transcripts were comparable to or even smaller than those observed for non-m6A-modified transcripts (Fig. S5j), indicating that m6A methylation is unlikely to be the only key driving factor behind the upregulation of these genes. For example, the STB marker genes ERVW-1 and CGA were not modified with m6A but were upregulated upon METTL3 loss (updated Fig. 4h). On the other hand, several upregulated genes did carry m6A modifications, such as the SASP gene GDF15 and the inflammation-related gene IL4R (updated Fig. 4h). These findings indicate that METTL3 modulates gene expression in hTSCs through both m6A-dependent and m6A-independent pathways. Given the relatively low proportion of m6A-modified mRNA, the regulatory effects of METTL3 may be predominantly mediated via indirect mechanisms rather than direct m6A modification of target transcripts. All information regarding m6A modifications and gene expressions within these three gene sets is provided in updated Table S2. We have incorporated these new results in the main text in line 297-313.\n3. To further elucidate the mechanisms underlying hTSC defects, we integrated RNA-seq, MeRIP-seq, and CUT&Tag datasets. Our new results demonstrate that:\na. H3K27ac, EP300, and JUND displayed increased enrichment across the STB, SASP, and inflammation gene sets upon METTL3 depletion, whereas H3K27me3 exhibited an inverse trend (updated Fig. 5j), indicating the pivotal roles of transcription factors and histone modifications in regulating gene expression in hTSCs through METTL3.\nb. Nearly all m6A-modified genes in these three gene sets also exhibit concurrent modifications by H3K27ac and/or H3K27me3 (updated Fig. S7g). Notably, approximately two-thirds of the m6A-modified genes are simultaneously co-regulated by both H3K27ac and H3K27me3 (updated Fig. S7g), suggesting a complex interplay of epitranscriptomic and histone epigenetic mechanisms in gene regulation within these pathways. For instance, STB genes within the PSG clusters and inflammation-associated genes such as TNFRSF1B did not exhibit m6A modifications but were upregulated following METTL3 depletion (Table S2). These genes showed increased enrichment of H3K27ac, EP300, and JUND, concomitant with decreased H3K27me3 deposition in P3(-DOX) hTSCs relative to P0 (+DOX) hTSCs (updated Fig. 5k, l). A similar pattern was also observed in SASP-related genes, including SERPINE1 and VGF, as well as inflammation-associated genes such as IL6 (updated Fig. S7h, i and Table S2), indicating an indirect regulatory effect mediated through m6A methylation. Conversely, GDF15, a marker gene for STB and a component of SASP that was modified with m6A, exhibited increased mRNA stability upon METTL3 KO (Table S2), along with elevated enrichment of H3K27ac, EP300, and JUND (updated Fig. 5m). A comparable trend was also observed in the inflammation-related gene RIPK2 (updated Fig. S7j and Table S2).\nCollectively, these new results indicate that METTL3 orchestrates trophoblast gene regulation via both direct m6A-dependent mechanisms and indirect epigenetic pathways involving EP300-mediated H3K27ac and EZH2-mediated H3K27me3, with the histone epigenetic pathways playing a more prominent role. Following METTL3 loss in hTSCs, a small subset of genes is directly affected by m6A regulation (which are also co-regulated by H3K27ac and/or H3K27me3). However, most dysregulated genes are not m6A-modified and are indirectly regulated through changes in H3K27ac and H3K27me3 modifications. Detailed information on m6A, H3K27ac, H3K27me3 modifications, RNA stability, and gene expression changes for all genes across these three gene sets is provided in the updated Table S2. These findings have been incorporated in the main text in line 365-394.\nA few minor comments:\n1) The use of P0, P2 etc is not the most easy nomenclature to follow. Suggest changing to P0 (5ng/ml), P2-DOX etc or similar\nThanks for the advice. We have changed the P0 to P0(+DOX), and P2 to P2(-DOX) in the updated manuscript and figures. We have clarified in Methods section in line 92 that unless otherwise specified, 5 ng/ml DOX is used to maintain METTL3-inducible knockout hTSCs.\n2) Some detail needs to be added to the text about the nature of the passaging. If cells are not proliferating then how are they being passaged 1:2, 1:4?. Is the ratio the same for all conditions? What length of time between passages?\nThanks for the questions. In our original Methods part of Culture of hTSCs, we clarified that routine passage of hTSCs was conducted every four days, and cells were resuspended and seeded in hTSC medium on cell culture plate coated with 1% Matrigel at a split ratio of 1:3. For most experiments in this study, including those involving METTL3 knockout and inhibition, we strictly adhered to this protocol to ensure consistent comparison between experimental and control groups.\nAdditionally, we would like to clarify that, as shown in the updated Fig. 3a, b and Fig. S4a, k, although METTL3-KO (at early passage) and -inhibited hTSCs do not proliferate robustly, they still exhibit proliferation. Only in the absence of DOX from P2 to P3 did the cell number tend to decline, primarily due to apoptosis and senescence. In response to your suggestion, we have added a detailed description of the growth curve in the updated Methods section in line 103.\n3) Figure 5 j and k. Please check the labelling is correct. The graphs seem to be very similar and it’s difficult to see what is different.\nThanks for the comment. We have improved the labeling in Fig. 5j and 5k, which are now presented as updated Fig. 6j and 6k. In this experimental design, we aimed to excise the specific LTR10A loci near the ATG9B and ENG genes respectively- positions where LTR10A functions as an enhancer- in METTL3-KO hTSCs. We then assessed the expression of the nearby ATG9B and ENG genes by RT-qPCR. Due to challenges associated with clonally expanding hTSCs from single cells, we employed a population-wide approach. Specifically, we electroporated plasmids carrying sgRNAs targeting the respective LTR10A loci, along with Cas9, into DOX treated hTSCs, with non-targeting sgRNA serving as the control. 24 hours post-transfection, cells were selected with puromycin and blasticidin for two days, then cultured continuously without DOX. P3(-DOX) cells were harvested to evaluate LTR10A knockout efficiency and the expression levels of adjacent ATG9B and ENG genes. As shown in updated Fig. 6j, the qPCR results indicate a relatively high efficiency of LTR10A deletion near ATG9B or ENG locus. Furthermore, Fig. 6k demonstrates that the expression of ATG9B and ENG was significantly reduced following deletion of their respective nearby LTR10A elements. We also updated the details in Methods section (Gene editing in hTSCs, line 72).\n4) Detail needs to be added to the text on the presence of 2 bands on the Ezh2 Western\nThank you for pointing this out. In our original study, we used a polyclonal antibody against EZH2 (Abclonal, A11085), which may recognize multiple splicing variants of EZH2. To enhance specificity and validate our main conclusions, we have now switched to a monoclonal antibody (Abcam, ab307646). The original western blotting images have been replaced with new data and now presented in the updated Fig. 5e. Additionally, we have updated the catalog number of the EZH2 antibody in Table S4.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you for your review comments and for helping us improve our work."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have fully addressed all my concerns through extensive new experiments, data, and analysis. These additions have significantly strengthened the strengthened the manuscript, and I recommend it for publication.", "reviewer_2": "The authors provide detailed and constructive answers and address requests for additional data, clarifying methodological details, and acknowledging the study's limitations.\nAdditional Experimental Evidence\n• The authors performed new immunofluorescence experiments on placental tissues to compare METTL3 and H3K27ac/me3 in cytotrophoblasts and syncytiotrophoblasts. They demonstrated lower H3K27ac in METTL3-positive cells and added these results to revised figures, supporting the negative regulation of H3K27ac by METTL3.\n• They analyzed preeclampsia (PE) placental samples and found significantly reduced METTL3 expression in PE cytotrophoblasts, plus elevated H3K27ac levels, providing a more direct link to PE pathology and addressing requests for clinical relevance.\nMechanistic Clarifications\n• Revisiting their multi-omics data, the authors clarified that only a minority of upregulated inflammation, SASP, and STB genes are directly modified by m6A, and histone modifications play a more prominent regulatory role than m6A methylation in gene expression changes following METTL3 loss. They highlighted the interplay between epigenetic and epitranscriptomic mechanisms.\n• Rescue experiments with epigenetic inhibitors partially reversed the phenotype, indicating additional mechanisms exist. The authors also referenced other studies on METTL3's role in mitochondrial regulation and suggested this as prospective research directions.\nClarification of Methodological Details\n• The MeRIP-seq approach includes both mRNA and lncRNA, and the authors corrected counts and clarified annotation errors in the revised figures. Most m6A-modified RNAs are mRNAs.\n• They provided computational predictions for transcription factor regulation and described how METTL3 stabilizes TFs which then modulate gene networks in trophoblast cells.\nResponses to Timing and Histone Modification Questions\n• Earlier time point assessments revealed METTL3 protein persists initially after Dox withdrawal; significant changes in EP300/EZH2 and related histone marks occur by passage 2. They provided updated experiments and clarified which datasets and days their figures refer to, making corrections where needed.\n• Analysis showed no significant changes in global H3K4me3 upon METTL3 knockout, but suggested future studies could examine this at specific DEGs. The authors incorporated a discussion on H3K4me3 and H3K9me3 in the manuscript, as suggested.\nGeneral Corrections and Acknowledgements\n• The authors made corrections to figure references and labels as pointed out, improved precision in reporting, and acknowledged the complexity and the need for further studies on alternative pathways.\nAdditional questions that could be addressed to provide a more precise understanding of the biological mechanisms and the translational relevance of the findings but are perhaps beyond the scope for the current manuscript.\n• Functional Clinical Validation: Can METTL3 expression or H3K27ac signatures be correlated with concrete clinical outcomes or functional PE phenotypes (e.g., abnormal placentation, maternal hypertension) rather than just transcriptomic or molecular markers?\n• Rescue Experiment Complexity: What additional cellular or molecular pathways are implicated in the partial rescue that occurs with epigenetic inhibitors, and can direct mitochondrial functional assays clarify the role of METTL3 in trophoblast bioenergetics or metabolism?\n• Epigenetic Interactions: Is the role of other histone marks (such as H3K4me3 and H3K9me3) in METTL3-deficient cells mapped at genomic loci corresponding to key differentially expressed genes. Would further CUT&Tag/ChIP-seq studies resolve patterns at single-cell or region-specific resolution?\n• Longitudinal Analysis: What is the chronology of changes following METTL3 depletion, especially at intermediate time points (prior to passage 2), and do these correspond to initial drivers of cellular phenotype or later downstream effects?\n• Translational Potential: Are there therapeutic implications for targeting METTL3/epigenetic regulation in PE, and what are the safety and efficacy considerations in relevant clinical or animal models?", "reviewer_3": "The authors have done a thorough job addressing all my points of concern so I am happy to recommend that the manuscript be published.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "The authors have fully addressed all my concerns through extensive new experiments, data, and analysis. These additions have significantly strengthened the manuscript, and I recommend it for publication.\nThank you for your advice and suggestions.\nReviewer #2 (Remarks to the Author):\nThe authors provide detailed and constructive answers and address requests for additional data, clarifying methodological details, and acknowledging the study's limitations.\nAdditional Experimental Evidence\n• The authors performed new immunofluorescence experiments on placental tissues to compare METTL3 and H3K27ac/me3 in cytotrophoblasts and syncytiotrophoblasts. They demonstrated lower H3K27ac in METTL3-positive cells and added these results to revised figures, supporting the negative regulation of H3K27ac by METTL3.\n• They analyzed preeclampsia (PE) placental samples and found significantly reduced METTL3 expression in PE cytotrophoblasts, plus elevated H3K27ac levels, providing a more direct link to PE pathology and addressing requests for clinical relevance.\nMechanistic Clarifications\n• Revisiting their multi-omics data, the authors clarified that only a minority of upregulated inflammation, SASP, and STB genes are directly modified by m6A, and histone modifications play a more prominent regulatory role than m6A methylation in gene expression changes following METTL3 loss. They highlighted the interplay between epigenetic and epitranscriptomic mechanisms.\n• Rescue experiments with epigenetic inhibitors partially reversed the phenotype, indicating additional mechanisms exist. The authors also referenced other studies on METTL3's role in mitochondrial regulation and suggested this as prospective research directions.\nClarification of Methodological Details\n• The MeRIP-seq approach includes both mRNA and lncRNA, and the authors corrected counts and clarified annotation errors in the revised figures. Most m6A-modified RNAs are mRNAs.\n• They provided computational predictions for transcription factor regulation and described how METTL3 stabilizes TFs which then modulate gene networks in trophoblast cells.\nResponses to Timing and Histone Modification Questions\n• Earlier time point assessments revealed METTL3 protein persists initially after Dox withdrawal; significant changes in EP300/EZH2 and related histone marks occur by passage 2. They provided updated experiments and clarified which datasets and days their figures refer to, making corrections where needed.\n• Analysis showed no significant changes in global H3K4me3 upon METTL3 knockout, but suggested future studies could examine this at specific DEGs. The authors incorporated a discussion on H3K4me3 and H3K9me3 in the manuscript, as suggested.\nGeneral Corrections and Acknowledgements\n• The authors made corrections to figure references and labels as pointed out, improved precision in reporting, and acknowledged the complexity and the need for further studies on alternative pathways.\nThank you for your valuable feedback and advice.\nAdditional questions that could be addressed to provide a more precise understanding of the biological mechanisms and the translational relevance of the findings but are perhaps beyond the scope for the current manuscript.\n• Functional Clinical Validation: Can METTL3 expression or H3K27ac signatures be correlated with concrete clinical outcomes or functional PE phenotypes (e.g., abnormal placentation, maternal hypertension) rather than just transcriptomic or molecular markers?\nThis is an excellent suggestion that can be explored in the future study. Unfortunately, we are unable to provide at this moment direct experimental evidence linking defective METTL3 or H3K27ac to clinical outcomes or functional PE phenotypes. In this study, we elucidated the mechanism by which METTL3 regulates H3K27ac in hTSCs and validated their relationship in placental specimens from normal and PE pregnancies. Our cell model results revealed that METTL3 deficiency induced proliferation defects, premature senescence, and epigenomic alterations, which are the features observed in PE patients1-3. However, given that PE is a notoriously heterogeneous pregnancy complication and the limited number of samples we collected in this study, we cannot conclude that METTL3 deficiency or H3K27ac overexpression directly cause PE. Instead, our data, along with other published studies1,2, suggest that epitranscriptomic and epigenetic abnormalities are believed to play a significant role in the occurrence and development of PE. Furthermore, most existing studies on PE1,3,4 primarily focus on transcriptomic or molecular markers, making it challenging to establish a causal link between the potential factors and clinical PE phenotypes such as maternal hypertension.\n• Rescue Experiment Complexity: What additional cellular or molecular pathways are implicated in the partial rescue that occurs with epigenetic inhibitors, and can direct mitochondrial functional assays clarify the role of METTL3 in trophoblast bioenergetics or metabolism?\nThank you for the suggestion. Through re-analysis, we found that treatment with A-485 primarily downregulated the expression of STB genes, while other pathways- such as the inflammatory response, TNFα signaling via NF-κB, and SASP- remained unaffected (as shown below, left panel), suggesting that some defects caused by METTL3 loss cannot be rescued by A-485 treatment. These results indicate that there exist METTL3 functions independent of H3K27ac modification in hTSCs.\nFor the mitochondrial functional assays, we initially conducted relevant experiments that are not included in this manuscript. As shown below (right panel), we observed that METTL3 depletion decreased ATP production rate in mitochondria of hTSCs, as well as hESCs and hEPSCs, indicating that METTL3 plays a role in trophoblast bioenergetics and metabolism. However, whether A-485 can rescue the mitochondrial dysfunction caused by METTL3 loss remains to be determined and requires further investigation.\n• Epigenetic Interactions: Is the role of other histone marks (such as H3K4me3 and H3K9me3) in METTL3-deficient cells mapped at genomic loci corresponding to key differentially expressed genes. Would further CUT&Tag/ChIP-seq studies resolve patterns at single-cell or region-specific resolution?\nWe totally agree with the assessment. Conducting additional CUT&Tag assays for H3K4me3 and H3K9me3 would further clarify how these histone marks influence gene expression through METTL3 in hTSCs.\n• Longitudinal Analysis: What is the chronology of changes following METTL3 depletion, especially at intermediate time points (prior to passage 2), and do these correspond to initial drivers of cellular phenotype or later downstream effects?\nThank you for your insightful comment. In our current inducible knockout system, we observed residual METTL3 expression after DOX withdrawal at passage 1 (P1), which may mask immediate effects due to compensatory mechanisms. Consequently, we have focused on analyzing phenotypes at passages 2 (P2) and 3 (P3) to better understand the functions and underlying mechanisms of METTL3 in human trophoblast lineage development. It is possible that initial drivers of the cellular phenotype remain undetected because of the limitations of the current knockout strategy. Future studies can utilize the auxin-inducible degron technology to achieve rapid METTL3 depletion if it works well, enabling the observation of immediate knockout effects and better identification of these initial drivers.\n• Translational Potential: Are there therapeutic implications for targeting METTL3/epigenetic regulation in PE, and what are the safety and efficacy considerations in relevant clinical or animal models?\nThank you for the valuable comments. Indeed, targeted therapy addressing epitranscriptomic and epigenetic abnormalities in PE is a promising direction for future research. For example, endogenous retroviruses overexpression (ERV3-MLT1) induced by epigenetic alterations may serve as a potential biomarker detectable in maternal plasma for PE diagnosis4. However, before METTL3 (RNA methylation) can be established as a clinical therapeutic target for PE, many steps need to be taken, such as increasing sample sizes and validating its safety and efficacy in animal models. Since the treatment would be administered to pregnant women, safety considerations are crucial-particularly ensuring that the intervention does not cause side effects. To this end, we might utilize existing mouse models of PE5 to investigate the roles of METTL3 and H3K27ac, with the goal of targeting these factors to alleviate PE symptoms. Safety assessments could involve using nanomaterials for direct delivery of therapeutics to placental CTBs. Different groups could receive varying drug concentrations and treatment durations, with monitoring physiological indicators such as maternal blood pressure and offspring birth rates to comprehensively evaluate safety and efficacy. If METTL3 proves to be a viable therapeutic target, employing METTL3 activators6 for direct and specific delivery to placental CTBs would be a feasible and promising approach. We also included this consideration in the end of the Discussion section.\nReviewer #3 (Remarks to the Author):\nThe authors have done a thorough job addressing all my points of concern so I am happy to recommend that the manuscript be published.\nThank you for the advice and suggestions.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you for your insightful comments and advice."}]

Decision letter