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Cellular Biogenetic Law and Its Distortion by Protein Interactions: A Possible Unified Framework for Cancer Biology and Regenerative Medicine

Authors:
Alexander Vinogradov at Institute of Cytology
Alexander Vinogradov
  • Institute of Cytology
Olga Vladimirovna Anatskaya at Russian Academy of Sciences
Olga Vladimirovna Anatskaya
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Abstract and Figures

The biogenetic law (recapitulation law) states that ontogenesis recapitulates phylogenesis. However, this law can be distorted by the modification of development. We showed the recapitulation of phylogenesis during the differentiation of various cell types, using a meta-analysis of human single-cell transcriptomes, with the control for cell cycle activity and the improved phylostratigraphy (gene dating). The multipotent progenitors, differentiated from pluripotent embryonic stem cells (ESC), showed the downregulation of unicellular (UC) genes and the upregulation of multicellular (MC) genes, but only in the case of those originating up to the Euteleostomi (bony vertebrates). This picture strikingly resembles the evolutionary profile of regulatory gene expansion due to gene duplication in the human genome. The recapitulation of phylogenesis in the induced pluripotent stem cells (iPSC) during their differentiation resembles the ESC pattern. The unipotent erythroblasts differentiating into erythrocytes showed the downregulation of UC genes and the upregulation of MC genes originating after the Euteleostomi. The MC interactome neighborhood of a protein encoded by a UC gene reverses the gene expression pattern. The functional analysis showed that the evolved environment of the UC proteins is typical for protein modifiers and signaling-related proteins. Besides a fundamental aspect, this approach can provide a unified framework for cancer biology and regenerative/rejuvenation medicine because oncogenesis can be defined as an atavistic reversal to a UC state, while regeneration and rejuvenation require an ontogenetic reversal.
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Int.J.Mol.Sci.2022,23,11486.https://doi.org/10.3390/ijms231911486www.mdpi.com/journal/ijms
Article
CellularBiogeneticLawandItsDistortionbyProtein
Interactions:APossibleUnifiedFrameworkforCancerBiology
andRegenerativeMedicine
AlexanderE.Vinogradov*andOlgaV.Anatskaya
InstituteofCytology,RussianAcademyofSciences,194064St.Petersburg,Russia
*Correspondence:aevin@incras.ru
Abstract:Thebiogeneticlaw(recapitulationlaw)statesthatontogenesisrecapitulatesphylogenesis.
However,thislawcanbedistortedbythemodificationofdevelopment.Weshowedtherecapitula‐
tionofphylogenesisduringthedifferentiationofvariouscelltypes,usingameta‐analysisofhuman
single‐celltranscriptomes,withthecontrolforcellcycleactivityandtheimprovedphylostratigra‐
phy(genedating).Themultipotentprogenitors,differentiatedfrompluripotentembryonicstem
cells(ESC),showedthedownregulationofunicellular(UC)genesandtheupregulationofmulticel‐
lular(MC)genes,butonlyinthecaseofthoseoriginatinguptotheEuteleostomi(bonyvertebrates).
Thispicturestrikinglyresemblestheevolutionaryprofileofregulatorygeneexpansionduetogene
duplicationinthehumangenome.Therecapitulationofphylogenesisintheinducedpluripotent
stemcells(iPSC)duringtheirdifferentiationresemblestheESCpattern.Theunipotenterythroblasts
differentiatingintoerythrocytesshowedthedownregulationofUCgenesandtheupregulationof
MCgenesoriginatingaftertheEuteleostomi.TheMCinteractomeneighborhoodofaproteinen‐
codedbyaUCgenereversesthegeneexpressionpattern.Thefunctionalanalysisshowedthatthe
evolvedenvironmentoftheUCproteinsistypicalforproteinmodifiersandsignaling‐relatedpro‐
teins.Besidesafundamentalaspect,thisapproachcanprovideaunifiedframeworkforcancerbi‐
ologyandregenerative/rejuvenationmedicinebecauseoncogenesiscanbedefinedasanatavistic
reversaltoaUCstate,whileregenerationandrejuvenationrequireanontogeneticreversal.
Keywords:celldifferentiation;genephylostratigraphy;geneexpression;interactome;embryonic
stemcells;inducedpluripotentstemcells;recapitulationlaw;Heckel’slaw;humans;wholegenome
duplication;evolutionarymedicine
1.Introduction
Thebiogeneticlaw(recapitulationlaw,vonBaer’slaw,Heckel’slaw)statesthaton‐
togenesisrecapitulatesphylogenesis[1–3].Thislawassumesa‘terminaladdition’when
recentlyevolvedfeaturesareaddedatthelaststagesofdevelopment,nearingtheadult
state[4].However,recapitulationcanbedistortedbyevolutionarymodificationsappear‐
ingatanydevelopmentalstage,especiallybyembryonicadaptations[1,5].Foralong
time,thishasbeenadebatedtopic;however,recently,theconceptofontogeneticrecapit‐
ulationhasacquirednewsupportfrommolecularandanatomicalstudies[1,3,4].Cur‐
rently,thebiogeneticlawisbecomingespeciallyimportantbecauseoftheatavistictheory
ofoncogenesis,whichsuggeststhatcancerisanevolutionaryreversaltoaunicellular
state[6–10].
Thegenesofunicellular(UC)originareoverexpressedincancertissues,whereasthe
genesappearinginthemulticellular(MC)evolutionarystagesaredownregulated[11–
13].Thehumaninteractome(globalproteininteractionnetwork)containsgiantclusters,
oneofwhichisstronglyenrichedwiththegenesofUCoriginandcorresponding
Citation:Vinogradov,A.E.;
Anatskaya,O.V.CellularBiogenetic
LawandItsDistortionbyProtein
Interactions:APossibleUnified
FrameworkforCancerBiologyand
RegenerativeMedicine.Int.J.Mol.
Sci.2022,23,11486.https://doi.org/
10.3390/ijms231911486
AcademicEditor:CristoforoComi,
BenoitGauthier,DimitriosH.
RoukosandAlfredoFusco
Received:29August2022
Accepted:26September2022
Published:29September2022
Publisher’sNote:MDPIstaysneu‐
tralwithregardtojurisdictional
claimsinpublishedmapsandinstitu‐
tionalaffiliations.
Copyright:©2022bytheauthors.Li‐
censeeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsandcon‐
ditionsoftheCreativeCommonsAt‐
tribution(CCBY)license(https://cre‐
ativecommons.org/licenses/by/4.0/).
Int.J.Mol.Sci.2022,23,114862of18
functions,whiletheothersareenrichedwiththegenesofMCoriginandtheirfunctions,
whichsuggeststheexistenceofanMC/UCcontrastincellularnetworks[14].Thegenes
downregulatedwithhumanagingareenrichedintheUCcluster,whereastheupregu‐
latedgenesareoverrepresentedintheMCcluster.Theclustersshowdenserinteractions
withinthemthanbetweenthem;therefore,theycanserveasattractors(stablestatesof
dynamicsystems)forcellularprograms.Importantly,theUCclusterhasahigherin‐
side/outsideconnectionratiocomparedwiththeMCclusters(i.e.,itisdenser),whichsug‐
gestsastrongerattractoreffectandmayexplainwhythecellsofMCorganismsareprone
tooncogenesis(reversaltotheUCstate)[14].
TheUCclusterisupregulatedinhumancancers,whichwasshowninthecaseofthe
single‐celltranscriptomesofvariouscancertypeswiththecontrolofthecellcycleactivity
[15].Theexpressionofgenesinvolvedinthecellcycleiscorrelatedwiththeexpressionof
UCgenes,eveniftheoverlappedgenesareremoved;therefore,thecontrolofthecellcycle
activityisnecessaryforthedemonstrationofevolutionaryreversalincancercells.These
datasuggestthatoncogenesisisnotjustthealterationofafewgenesbuttheswitchto
ancientunicellularprograms.Therefore,thecomparisonofcancercellswiththeorgan‐
ismsbelongingtotheUCevolutionarystagemayhelpustoelucidatetheetiologyofdis‐
easesandagingandeventosuggestpossibleremedies.Forinstance,certainunicellular‐
specificdrugscanbeappliedforthetreatmentofcancer[16,17].Certainotherdiseases
canalsobeunderstoodasaresultofevolutionaryreversal[4].Thegeneexpressionshift
towardsearlierevolutionarystageswasalsoobservedinthepolyploidizationofsomatic
cells,whichcanbeconsideredastheactivationofthecellemergencyreserveunderstress‐
fulconditions[18].
Thebiogeneticprinciplemayalsobeimportantforregenerative/rejuvenationmedi‐
cine,whichisintrinsicallyintertwinedwithcancerbiology.Themaincontradictionof
multicellularity(MCM)isthatbetweenthecellularandorganismallevels[14].Thecell
pluripotencyandproliferativepotentialarevitalforthehealthydevelopmentandlongev‐
ityofMCorganismsifheldincheck.Inthiscase,theactivityoftheUClevelpromotesthe
organism’svitality.Incontrast,uncheckedunicellularityresultsinoncogenesiswhenthe
cellstendtobehaveasindependentevolutionaryunits[8,16,19].Inthiscase,theactivity
oftheUClevelunderminestheorganism’svitality.Themainproblemfortheapplication
ofstemcelltechnologyinregenerativemedicineisthequestionofhowtoavoidoncogen‐
esis[20,21].Thesetwooppositeforces—promotionvs.suppressionoftheMCorganism’s
vitalitybytheactivityoftheUClevel—areencapsulatedbytheterm‘MCM’.Asanexam‐
pleoftheUC/MCcontrastincellularnetworks,thetotalpluripotencysignature(PluriNet)
isenrichedintheUCgiantcluster,whereasthegenescontrollingpluripotency(theKEGG
pathway)areenrichedintheMCcluster[14].
Theatavistictheoryofcancerentailsthatthestudyofthebiogeneticlawatthecellu‐
larlevelisespeciallyimportant.Beforethestudyofapathology,itisnecessarytoknow
thebasicsofthenormaldevelopment,i.e.,howtheevolutionisrecapitulatedincelldif‐
ferentiation,whichconstitutesanessentialpartofontogenesis(andwhosereversalisan
essentialpartofoncogenesis).Thisknowledgecanalsobehelpfulforregenerativemedi‐
cineandrejuvenation(orprolongationofthehealthylifespan)becausethereversalofde‐
velopmentmaybeassociatedwiththereversalofexpressiontomoreancientgenesand
cellularprograms.Thisprocessmaybesimilartooncogenesisbutshouldincludediffer‐
ences,ensuringsafereversal.Thus,thecellular‐levelstudyofrecapitulationcanextend
theimportanceofthebiogeneticlawfromapurelyacademicfieldtothepracticaldimen‐
sionandhelpresearcherstobuildaunifiedframeworkforcancerbiologyandregenera‐
tive/rejuvenationmedicine.
Recently,theappearanceofsingle‐celltranscriptomeshasmadeitpossibleforre‐
searcherstoinvestigatethebiogeneticlawatthecellularlevel.Here,westudythecellular
recapitulationofphylogenesiswithanemphasisontheUC–MCevolutionarytransition.
Thisworkpresentsameta‐analysisofhumansingle‐celltranscriptomesinthepluripotent
embryonicstemcells(ESC),moredifferentiatedcells(multipotentprogenitorsand
Int.J.Mol.Sci.2022,23,114863of18
unipotenterythroblasts),embryoniccellsduringzygoticcleavage,andinducedpluripo‐
tentstemcells(iPSC).Weestimatedtherelativeeffectsofontogeneticrecapitulationand
developmentmodernization,assessingtheimpactoftheevolutionaryoriginoftested
genesandthegenesencodingforinteractomesoftheproteinsencodedbythetestedgenes
ontheexpressionofthetestedgenesduringcelldifferentiation.
Ourapproachisbasedontheconceptthatthemodernizationofdevelopmentcanbe
performedbytheinteractionoftheproteinsencodedbyoldergeneswithmorerecent
ones.Touncoverthepurerecapitulationeffects,wecontrolledforthecellcycleactivity.
Thiswasnecessarybecausetheearlierembryoniccellshaveahighercellcycleactivity
comparedwithmoredifferentiatedcells,andthehighercellcycleactivityisassociated
withtheupregulationofUCgenes[15].Thisconnectioncoulddistortthepurerecapitu‐
lationeffectsifstudiedwithoutthecorrectionforthecellcycleactivity.
2.Results
2.1.TheProofofConcept
Weanalyzedthetranscriptlevels(henceforthcalled“expression”forthesakeof
brevity)ofthegenesoriginatingatdifferentevolutionarystages(phylostrata)inthesin‐
gle‐celltranscriptomesofhumancells,whichdifferinthestateofcelldifferentiation.In
thefirstexample,thepluripotentembryonicstemcells(ESC)werecomparedwiththe
moredifferentiatedmultipotentprogenitors(MP).Asthecontrolforthecellcycleactivity,
weusedtheregressionlinesoftheexpressionofthetestedgenesontheexpressionofthe
cellcyclegenes,aspreviouslydescribed[15].ThegenesoriginatinginUCphylostrata
showedalowerregressionlineintheMPascomparedwiththeESC,whereasthegenes
fromtheMCphylostratashowedahigherline(Figure1;SupplementaryFiguresS1–S17).
2nd phylostratum (Eukaryota)
Cell cycle
A
ESC
12 3456 78
1.8
2.2
2.6
3
3.4
3.8
4.2
4.6
MP
Int.J.Mol.Sci.2022,23,114864of18
Figure1.Regressionlinesofthegenesbelongingtodifferentphylostrataonthecellcyclesignature
inthesingle‐celltranscriptomesofmultipotentprogenitors(MP)(blue)andpluripotentembryonic
stemcells(ESC)(red).Themeanexpressionofthegenesbelongingtoaphylostratumisplotted
versusthemeanexpressionofcellcyclesignaturegenes(withindividualcellsasseparatepoints).
(A)The2ndphylostratum(unicellularEukaryota);(B)the5thphylostratum(Eumetazoa).Forthe
differencebetweenintercepts,(A)p<10−103and(B)p<10−41.ThetranscriptomesarefromGSE75748
(‘celltype’dataset).
Importantly,inbothcelltypes,theexpressionofUC‐origingenescorrelateswiththe
expressionofcellcyclegenes(Figure1).IntheMCphylostrata,thiscorrelationsharply
decreases,whileinthepost‐Bilateriaphylostrata,itbecomesnegative(Figure2),butit
alsorequirescorrection.Thenegativecorrelationofthegenesfromthelaterphylostrata
isunderstandablebecausethesegenesaremostlyinvolvedindifferentiationandtissue‐
specificfunctions(whiletheUC‐origingenesareinvolvedinhousekeepingandthecell
cyclefunctions),whichareusuallyassociatedwiththesuppressionofthecellcycleactiv‐
ity.
Figure2.Evolutionaryprofileoftheslopeoftheregressionlinesoftheexpressionofgenesbelong‐
ingtodifferentphylostrataonthecellcyclesignatureinthesingle‐celltranscriptomes,with
5th phylostratum (Eumetazoa)
Cell cycle
B
ESC
MP
12 3456 78
0.9
1
1.1
1.2
1.3
1.4
1.5
Int.J.Mol.Sci.2022,23,114865of18
confidenceintervals(p=0.05).TheregressionlinesareshowninFigure1andSupplementaryFig‐
uresS1–S17.ThetranscriptomesarefromGSE75748(‘celltype’dataset).Phylostrata:1—cellular
organisms(Prokaryota);2—Eukaryota;3—Opisthokonta;4—Metazoa;5—Eumetazoa;6—Bilateria;
7—Chordata;8—Vertebrata;9—Euteleostomi;10—Tetrapoda;11—Amniota;12—Mammalia;13—
Theria;14—Eutheria;15—Boreoeutheria;16—Primates;17—Hominidae.Thepicturesatthetop
showrecentorganismscorrespondingtothephyleticbranchingusedforhumangenedating.
Moreover,theESCshowahigherexpressionofcellcyclegenesascomparedwith
theMP.Thesefactsjustifythecorrectionforthecellcycleactivity.Otherwise,theeffectof
theevolutionarygeneoriginontheESC–MPdifferenceinthegeneexpressionmaybe
distortedbythehighercellcycleactivityintheESC.Forthiscorrection,weusedthedif‐
ferenceintheinterceptsbetweentheregressionlinesfortheMPandESCatequalslopes
(seeMaterialsandMethods).Byextrapolation,thiscanbeinterpretedasthedifferencein
theexpressionbetweentheMPandESCatzerocellcycleactivity.
Forthewholepictureacrosstotalphylogenesis,weplottedtheMP–ESCdifferences
intheinterceptsforallthephylostrata(Figure3A).Therearethreephasesintheevolu‐
tionaryprofileofESC‐to‐MPdifferentiation.ThegenesthatoriginatedintheUCevolu‐
tionarystage(thefirsttwophylostrata)aredownregulatedintheMPascomparedwith
theESC.Then,atthethirdphylostratum,thereisasharptransitiontothesecondphase.
Thedifferenceintheinterceptschangessign,indicatingtheupregulationofgenesorigi‐
natinginthethird(andlater)phylostrataintheMPascomparedwiththeESC.Thethird
phylostratumisOpisthokonta(representedbytherecentcolonialChoanoflagellata),
whichcanbeconsideredasthelastunicellularsorfirstmulticellulars,dependingonthe
viewpoint.Thesecondphaseoftheevolutionaryprofile(theupregulationintheMP)con‐
tinuesuptothe9thphylostratum(Euteleostomi,bonyvertebrates).Beginningfromthe
10thphylostratum(Tetrapoda:amphibians,reptiles,birds,andmammals),anydifference
disappeared,whichindicatedthethirdphase(theabsenceofrecapitulation).
Int.J.Mol.Sci.2022,23,114866of18
Figure3.Evolutionaryprofiles:thedifferencesintheinterceptsbetweentheregressionlinesofthe
expressionofgenesbelongingtodifferentphylostrataonthecellcyclesignatureinthesingle‐cell
transcriptomes(regressionlinesasinFigure1)forthedifferentcelltypes,withconfidenceintervals
(p=0.05).(A)Multipotentprogenitors(differentiatedfromESC)vs.ESC(GSE75748,‘celltype’da‐
taset).(B)Erythrocytes(differentiatedfromerythroblasts)vs.erythroblasts(GSE123899).(C)
Hepatocyte‐likecells(differentiatedfromiPSC)vs.iPSC(GSE90749).Phylostrata:1—cellularorgan‐
isms(Prokaryota);2—Eukaryota;3—Opisthokonta;4—Metazoa;5—Eumetazoa;6—Bilateria;7—
Chordata;8—Vertebrata;9—Euteleostomi;10—Tetrapoda;11—Amniota;12—Mammalia;13—The‐
ria;14—Eutheria;15—Boreoeutheria;16—Primates;17—Hominidae.Thepicturesatthetopshow
recentorganismscorrespondingtothephyleticbranchingusedforhumangenedating.
Thus,theMP–ESCcomparisondemonstratesthatontogenesis,atthecellularlevel
(reflectedintheESC‐to‐MPcelldifferentiation),recapitulatesphylogenesisinaphase‐like
manner,withasharpUC/MCcontrast,butonlyuptotheEuteleostomi.Asimilarthree‐
phasepicture,withasharpUC/MCcontrastattheOpisthokontaandtheterminationof
therecapitulationaftertheEuteleostomi,canbeseenduringthe4daysoftheESCcultur‐
ing,demonstratingtheprocessofdifferentiation(SupplementaryFiguresS18andS19).
TheESCwererepresentedbytwocelllines(H1,H9)behavingsimilarly,whereasthe
MPwererepresentedbyfivecelllines,anditistheconsolidatedpicturethatisshownin
Figure3A.Takenseparately,theMPcelllinesshowacertainvariation,butthethree‐phase
patterngenerallyremains(SupplementaryFiguresS20andS21).Theonlydifferencein
thepatternoftheUC–MCtransitionwasobservedintheneuralprogenitors(NPC)(Sup‐
plementaryFigureS20A).IntheNPC,thegenesoriginatinginthethirdphylostratum
Intecept difference
B
1234567891011121314151617
Phylostrata
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
Phylostrata
Intercept difference
1234567891011121314151617
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
C
Int.J.Mol.Sci.2022,23,114867of18
(Opisthokonta)showalowerexpressionintheMPcomparedwiththeESC,andtheUC–
MCtransitionisthusdelayedtothefourthphylostratum(Metazoa,recentsponges).This
differencecanarisebecausethenervoussystemisofalaterevolutionaryorigin[22].After
the9thphylostratum(Euteleostomi),thereisalsosomelimitedvariation.TheNPCand
theendothelialcells(EC)showaslightlyhigher(butconsistentintheadjacentphy‐
lostrata)expressioncomparedwiththeESC,i.e.,acontinuedrecapitulationofphylogen‐
esis(SupplementaryFigureS20A,B).Atthesametime,theforeskinfibroblasts(HFF),
trophoblast‐likecells(TB)andendodermderivatives(DEC)showaslightly(butconsist‐
ently)lowerexpression,whichcanbeinterpretedasasmalldistortionoftherecapitula‐
tion(SupplementaryFigureS21A–C).
Themultipotentprogenitors(MP)arenotcompletelydifferentiatedcells.Forthelater
stages,westudiedthedifferentiationoftheunipotenterythroblaststhatareprecursorsof
erythrocytes(Figure3B).Theerythrocytesareprobablyoneofthemoststronglydifferen‐
tiatedcelltypes,whichultimatelylosetheirabilityforreplicationandeventranscription.
Inthedifferentiatingerythroblasts,thefirstphasetransitionisthesame(UC–MC),but
withamorecomplicatedpictureafterthatstage(Figure3B).Importantly,incontrastto
theESC–MPdifferentiation,thedifferentiatingerythroblastsshowapronouncedrecapit‐
ulationinthegenesoriginatingaftertheEuteleostomi,withthestrongesteffectinthelast
phylostratum(Hominidae).Thus,therecapitulationduringcelldifferentiationwasob‐
servedforthewholeevolutionaryrangefromtheunicellularstohominids(albeit,forthe
laterevolutionarystages,onlyintheterminallydifferentiatedcells).
2.2.ArtificialOntogeneticReversal
Theinducedpluripotentstemcells(iPSC)aretheresultofartificialontogeneticre‐
versal[20].Theevolutionaryprofileoftheirdifferentiationisqualitativelysimilartothe
differentiationoftheESC(Figure3C).However,intherangeof10–12phylostrata,there
isaconsistentdownregulationinthedifferentiatedcellsascomparedwiththeinitialiPSC.
Thisobservationindicatesadistortionofrecapitulation.ThetwootheriPSCexamples
showasimilarviolationinthisphylostraticarea,albeitlesspronounced(Supplementary
FigureS22A,B).However,asimilardistortionwasobservedinHFF,TB,andDEC,differ‐
ingfromESC(SupplementaryFigureS21A–C).Therefore,thisdistortionmaysimplyin‐
dicateavariationwithinthegeneralrecapitulationpatternduringthedifferentiationof
pluripotentcells.
2.3.AbOvo
Torevealtheearliestappearanceofcellularontogeneticrecapitulation,westudied
thezygoticcleavage.Atfirstglance,itmaybeexpectedthatthestrongestexpressionof
theUCgeneswilltakeplaceintheUContogeneticstage,i.e.,intheoocyteorzygote.But
thisisnotso.ThehighestupregulationoftheUCgeneswasobservedinthehatching
blastocystonthe6thdayafterfertilization(Figure4).ItisknownthattheESC existinthe
innercellmassofthehumanblastocyst from4thto7thdayafterfertilization,andthey
disappearafterthe7thday[23].Thus,theESCseemtobeveryclosetothestrongestreca‐
pitulationoftheUCstage,albeitthattheupregulationofUCgenesisslightlylowerinthe
culturedESCascomparedwiththe6‐dayblastocyst(Figure4A).
Int.J.Mol.Sci.2022,23,114868of18
Figure4.Ontogeneticprofile:thedifferenceintheinterceptsbetweentheregressionlinesofthe
expressionofunicellular‐origingenes(1–2phylostrata)onthecellcyclesignatureinthesingle‐cell
transcriptomesfromearlyembryonicdevelopment(withconfidenceintervals,p=0.05).(A)Embry‐
oniccellsvs.ESC(GSE36552).(B)Embryoniccellsvs.embryoniccellsatday6(E‐MTAB‐3929).
2.4.RegulatoryGeneGroups
TheESC‐to‐MPdifferentiationwaschosenforthefunctionalanalysis(asitprovides
theclearestrecapitulationpatternoftheUC–MCevolutionarytransition).Controllingfor
thecellcycleactivity,westudiedtheexpressionofregulatorygenegroups,whoseexpan‐
sioninthehumangenomewasstudiedpreviouslyusingthesamephylostratigraphicda‐
ting[24].Thechaperones,epigeneticfactors,andcofactorsofthetranscriptionfactors(TF)
aredownregulatedinMP(comparedwithESC),whereastheproteinmodifiers,TF,biva‐
lentgenes,andsignalingreceptorsareupregulatedinMP(Figure5A).
Developmental stage
Intercept difference
ESC
A
Oocyte Zygote 2-cell 4-cell 8-cell Morulae Blastocyst
4 h 19 h 27 h 2 day 3 day 4 day 6 day
1 2 3 4 5 6 7
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
Intecept difference
Developmental stage
B
3 day 4 day 5 day 6 day 7 day
3 4 5 6 7
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
Int.J.Mol.Sci.2022,23,114869of18
Figure5.Thedifferenceintheinterceptsbetweentheregressionlinesoftheexpressionofdifferent
genegroupsonthecellcyclesignatureinthesingle‐celltranscriptomesofMPvs.ESC(GSE75748,
‘celltype’dataset),withconfidenceintervals(p=0.05).(A)Differentregulatorygenegroups.(B)UC
genes,MCgenes,andUCgiantclustergenes.(C)UCandMCgeneswithdifferentfractionsofMC
orUCproteinsintheone‐stepinteractomeneighborhoodoftheirproteins(e.g.,‘0.25MC’means
0.25oralesserfractionoftheMCproteinsintheneighborhoodofaUCprotein).Thebluecircles
showtheinterceptvalues,theredtrianglesshowtheirconfidenceintervals.
Chaperones Protein Epigenetic TF cofactors TF Signaling
modifiers factors receptors
Intercept difference
Bivalent
genes
1234567
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
A
UC genes MC genes UC cluster UC genes UC genes MC genes
in UC clu ster in UC clu sterout UC cluster
Intercept difference
B
1 2 3 4 5 6
-1.6
-1.3
-1
-0.7
-0.4
-0.1
0.2
Fraction of interactants
UC genes
0.25 MC 0.5 MC 0.75 MC 1.0 MC
MC genes
0.25 UC 0.5 UC
Intercept difference
0.75 UC 1.0 UC
C
1 2 3 4 5 6 7 8
-3
-2.6
-2.2
-1.8
-1.4
-1
-0.6
-0.2
0.2
Int.J.Mol.Sci.2022,23,1148610of18
2.5.TheStrengthofOldandNewTies
Inlightofthesuggestionthatthemodernizationofdevelopment,whichdistortsre‐
capitulation,canbefulfilledbytheinteractionofproteinsencodedbyoldergeneswith
morerecentgenes,westudiedthedependenceofthegeneexpressionontheevolutionary
ageofgenesencodingfortheinteractantsofproteinsencodedbythetestedgenes.The
effectoftheinteractomeprovedtobeconsiderable.Thus,albeitthatthegenesofMC
originareupregulatedintheMP(comparedwithESC),theMCgenesinsidetheUCgiant
interactomeclusteraredownregulated(Figure5B).FortheUCgenes,thiseffectiseven
morestriking.TheUCgenesinsidetheUCclusteraremuchmoredownregulatedinMP
(comparedwithESC)thanthetotalUCgenes,whereastheUCgenesoutsidetheUCclus‐
terbecomeevenupregulatedinMP(insteadofbeingdownregulated),thusbehavingsim‐
ilarlytothetotalMCgenes(Figure5B).
Atthelevelofdirect(one‐step)interactions,westudiedtheeffectofthegradualin‐
creaseintheMCfractionintheneighborhoodofproteinsencodedbythetestedgenes.
WiththeincreaseintheMCfractionintheone‐stepneighborhoodofaUCprotein,the
encodingUCgeneshowedagradualtransitionfromdownregulationtoupregulationin
MP(comparedwithESC)(Figure5C).WiththedecreaseintheMCfractionintheone‐
stepneighborhoodofanMCprotein,theencodingMCgeneshowedagradualtransition
fromupregulationtodownregulationinMP,albeitthatthiseffectofsignchangingwas
weakerthanitwasinthecaseofUCgenesinthehigh‐MCenvironment(Figure5C).
2.6.FunctionalAnalysisoftheProteinsinDifferentOne‐StepInteractomeNeighborhoods
WestudiedthefunctionsoftheUCandMCproteinsdifferingintermsoftheMC
fractionintheirone‐stepinteractomeneighborhoods.FortheUCproteins,theconserva‐
tiveUCenvironment(i.e.,alowfractionofMCproteinsintheneighborhood)ismain‐
tainedfortheproteinsinvolvedincellmetabolism,translation,ribonucleoproteincom‐
plexes,andpluripotencysignatures(Figure6A;SupplementaryTablesS1–S8).The
evolvedenvironmentofUCproteins(highfractionofMCproteinsintheneighborhood)
isobservedmostlyinthemembraneandincludesfunctionsrelatedtosignaling(Figure
6A;SupplementaryTablesS9–S16).Thesameoutcomeisobservedforproteinmodifiers
(Figure6A).Importantly,theevolvedMCenvironmentisalsofoundinthenetworkof
cancerproteins(Figure6A).
Observed/expected gene number ratio
0.25
0.05
e-05
0.9
e-58
e-15
0.04
e-4
e-10
e-4
0.5 0.4
e-136
e-94
e-13
0.9
A
e-138
0.06
e-11
e-6
e-4
e-17
e-3
0.8
e-9
0.02
e-30.9
e-8 e-21
0.9
0.9
0.9
0.9
e-10
e-10
UC genes
-1
0
1
2
3
4
5
6
7
Cellular metabolic process
Ribonucleopr otein co mplex
Plasma membrane
G protein recepto r pathway
Mitotic cell cycle
Protein modification process
Netw ork of ca ncer g enes
ESC signature
PluriNet
0.25 MC
0.5 MC
0.75 MC
1.0 MC
Int.J.Mol.Sci.2022,23,1148611of18
Figure6.ThefunctionalenrichmentoftheUCandMCproteinswithdifferentfractionsofMCor
UCproteinsintheirone‐stepinteractomeneighborhood.(A)UCproteinsintheMCenvironment.
(B)MCproteinsintheUCenvironment.Thesignificanceiseitherforenrichment(ifobserved/ex‐
pected>1)orforunderrepresentation(ifO/E<1).
FortheMCproteins,theneighborhoodwiththehighUCfractionisobservedforthe
proteinsrelatedtoRNAprocessing(Figure6B;SupplementaryTablesS17–S24).Theen‐
vironmentwithahighMClowUCfractionisobservedfortheproteinsrelatedtodevel‐
opment,celldifferentiation,cellcommunication,theregulationoftranscription,andtran‐
scriptionfactors(Figure5B6B;SupplementaryTablesS25–S32).Thebivalentgenes,ohno‐
logs,tumorsuppressors,and‘cosmic’genes(whosemutationsarefoundincancercells)
alsoshowastepwiseenrichmentwiththeincreaseintheMCfractionintheinteractome
oftheirproteins(Figure6B;SupplementaryTablesS20,S24,S28,andS32).
3.Discussion
3.1.CellularBiogeneticLaw
Wedemonstratedtheontogeneticrecapitulationofphylogenesisatthecellularlevel.
ThehighestupregulationofUCgeneswasobservednotinthesingle‐celloocyteorzygote
butinthehatchingblastocyst(aboutthe6thdayafterfertilization).Thismayappeartobe
adistortionofthebiogeneticlaw,butitonlysupportsitbecausethisobservationcanbe
explainedbythematernalmRNAsinthezygote.BecauseofthematernalmRNAs,the
oocyteorzygotedoesnotcorrespondtotheUCevolutionarystagebutpresentsaproduct
oftheMCorganism.Probably,onlyinthehatchingblastocystdoesthematernal‐to‐zy‐
gotictransition(MZT)causethecompletedecayofmaternalmRNAs[25],andtheblasto‐
cysttranscriptomebecomesofapurelyzygoticorigin.Thisontogeneticstage(containing
abouttencells)isthestrongestrecapitulationoftheUCevolutionarystage.Theupregu‐
lationofUCgenesinthehatchingblastocystisonlyslightlyhigherthaninthecultured
embryonicstemcells(ESC).Notably,theculturedESCwereinitiallytakenfromonlythe
hatchingblastocyst[23].
DuringthedifferentiationofthepluripotentESCintomultipotentprogenitors(MP),
thedownregulationofUCgenesandtheupregulationofMCgenestakeplace,albeitonly
thoseMCgenesthatoriginateuptotheEuteleostomi(bonyvertebrates).Thispicture
strikinglyresemblestheevolutionaryprofileofregulatorygeneexpansionduetogene
Observed/Expected gene number ratio
e-34
e-15
0.6
e-23
e-181
e-17
e-7
e-5
e-52 e-65
e-9
0.9
B
e-128
e-16
e-7e-2
e-48
e-14
e-2
e-2
e-137
e-2
e-6
e-6
e-56
e-250.1
e-3
e-79
e-7
0.8
0.3
e-15 e-20
0.6
0.9
e-10 e-11
0.6
0.3
MC genes
-1
0
1
2
3
4
5
6
RNA processing
System development
Regulation of transcription
Transcription factors
Cell differentiation
Cell communication
Bivalent genes
Tumor suppressors
Cosmic
Ohnologs
0.25 UC
0.5 UC
0.75 UC
1.0 UC
Int.J.Mol.Sci.2022,23,1148612of18
duplicationinthehumangenome,whichshowsasimilardecayaftertheEuteleostomi
[24].Theupregulationoftheregulatorygenegroupsalsoresemblestheevolutionarypro‐
fileofthesegroups’expansions.Thechaperones,epigeneticfactors,andcofactorsoftran‐
scriptionfactors(TF)areupregulatedintheESC,whereastheproteinmodifiers,TF,biva‐
lentgenes,andsignalingreceptorsareupregulatedintheMP.
Theonlyexceptionistheproteinmodifiers.Inthehumangenome,thechaperones,
epigeneticfactors,TFcofactor,andproteinmodifiersexpandedattheUCevolutionary
stage,whereastheTF,bivalentgenes,andsignalingreceptorsmostlyexpandedattheMC
stages[24].Theexceptionoftheproteinmodifiersisprobablyrelatedtothefactthatthey
wereadoptedfortheMCregulationinthecourseofevolution..Therefore,theybecame
upregulatedinthemoredifferentiatedcells(MPvs.ESC),wheretheMCgenesaregener‐
allyupregulated.Similarly,theproteinmodifiers,whichfirstlyexpandedinthegenomes
ofprokaryotes,asthemainprokaryoticregulatorylevel,wereadoptedintheUCeukary‐
otestoplaytheroleofepigeneticfactors,therebyanticipatingantecedentingtheexpan‐
sionofTF[24].Forinstance,histonemodifiers,HATsandHDACs,acetylateanddeacety‐
latethousandsofotherproteinsbesideshistones[26].Thus,therecapitulationpatternof
theexpressionofregulatorygenegroupsinthecourseofESC‐to‐MPdifferentiation,in
general,coincideswiththeevolutionarycourseoftheexpansionofthesegenegroupsin
thehumangenomeduetogeneduplication(exceptforproteinmodifiers),providingad‐
ditionalsupportforthecellularbiogeneticlaw.
TheEuteleostomievolutionarystage,inwhichtherecapitulationduringESC–MP
differentiationiscompleted,isclosetothecladewherethevertebratephylotypicstageis
mostpronounced[5,27].Aphylotypicstageisadevelopmentalstage,wheretheembryos
ofdifferentspeciesbelongingtoaclademoststronglyresembleeachother[1,28].The
similarityintheearlierontogeneticstagesisdistortedbyembryonicadaptations,inthe
laterstages ‐‐byterminaladditionsinthecourseofcladediversification[5,28].Inthe
ontogenesis,thephylotypicstageisclosetotheonsetoforganogenesis,andthedifferen‐
tiationofMPfromESCisnecessaryfororganogenesis[29–31].Therecapitulationofthe
laterevolutionarystagescanbeobservedduringthedifferentiationoftheunipotent
erythroblasts,wherethegenesoriginatingatthemorerecentphylostrata(uptothe
Hominidae)wereupregulated.Thisdifferentiationcorrespondstothemaintenanceofde‐
finitivetissues.
3.2.ModificationofDevelopment
Themodificationofdevelopmentdistortstherecapitulationlaw.Thisprocessisman‐
ifestedin(andprobablycausedby)theinteractomeofproteinsencodedbythegenesun‐
derconsideration.ThemoststrikingeffectfortheMCenvironmentisthatontheexpres‐
sionofUCgenes.ThereisastepwisereductioninthedownregulationofUCgenesinMP
(comparedwithESC)dependingontheMCfractionintheone‐stepinteractomeofthe
UCproteins.Moreover,intheenvironmentwithafractionofMCproteinsofabout3/4or
higher,eventheupregulationofUCgenestakesplace.Similarly,theMCgenesencoding
forproteinsintheenvironmentwithaUCfractionabove3/4aredownregulatedinstead
ofbeingupregulated.Genesworkintheformofproteins,whichinturnactasparticipants
ofproteininteractionnetworks.Itisreasonabletosuggestthat,aftertheproteininterac‐
tionswererewired,theexpressionoftheencodinggenesbecomeadaptedtothenewcon‐
ditions,inwhichtheencodedproteinsfoundthemselvesintherewiredinteractome.This
meansthatanevolutionarychangemaybeginwithachangeintheproteinsequence
(causingchangesintheproteininteractions)followedbytheadjustmentofthecoding
geneexpression.
TheevolvedenvironmentoftheUCproteins(i.e.,ahighfractionofMCproteinsin
theinteractomeofaUCprotein)includesfunctionsrelatedtosignaling,whicharemostly
performedbyproteinmodifiers.Thisfactcanexplainwhyproteinmodifiersareupregu‐
latedinthemoredifferentiatedcells(MPvs.ESC),albeitthattheirexpansioninthehu‐
mangenometookplaceattheUCevolutionarystage[24].Thesignalingisinvolvedin
Int.J.Mol.Sci.2022,23,1148613of18
intercellularcommunications,whoseroledrasticallyincreasesinthemulticellulars.The
signalingshouldbeperformedswiftly,andthiscanbebetterachievedbyproteinmodifi‐
cationascomparedwithchangesinthetranscription.TheevolvedMCenvironmentis
alsofoundinthenetworkofcancerproteins,whichindicatesthatthecontrolofoncogen‐
esisistheprerogativeoftheMClevel.
FortheMCproteins,theenvironmentwithahighUCfractionwasobservedinthe
proteinsrelatedtoRNAprocessing.TheenvironmentwithalowUCfractionwasob‐
servedintheproteinsrelatedtodevelopment,celldifferentiation,cellcommunication,
andregulationoftranscription.Thebivalentgenes,whichenablerapidswitchingbetween
cellularprograms[32],alsoshowastepwiseenrichmentwiththedecreaseintheUCfrac‐
tionintheinteractomesoftheirproteins.Asimilarpicturewasobservedforthetumor
suppressorand‘cosmic’genes(whosemutationswerefoundincancercells).Notably,
ohnologs(genesretainedinduplicatesafterwholegenomeduplication)alsoshowastep‐
wisegrowthwiththedecreaseintheUCfractionintheirinteractomeenvironment.Ohno‐
logsaremoststronglyinvolvedinboththeregulatorylevelsofMCorganisms,thenucle‐
omeandthenervoussystem[33].
3.3.AUnifiedFrameworkforCancerBiologyandRegenerativeMedicine
Besidestheirimportanceforevolutionaryanddevelopmentalbiology,studiesofthe
cellularbiogeneticlawcanprovideaunifiedframeworkforcancerbiologyandregenera‐
tive/rejuvenationmedicine.TheCancerGenomeProjectrevealedamultitudeandgreat
diversityofsomaticmutationsincancercells[34].Inaddition,alargenumberofepige‐
nomicalterationswereuncovered[35,36].Theseunexpectedresultsraisedconcernswith
respecttotheclassic‘somaticmutationtheory’ofoncogenesis,whichassumesthatcancer
iscausedbythealterationofafewoncogenes,andstimulatedinterestinthemoresys‐
temicexplanations[34,37,38].Oneofthemostprominentsystemicconceptsistheatavistic
theory,suggestingthatcancerarisesbecauseofMCcellreversaltoaUCstate[6–10].Sim‐
ilarly,theregeneration/rejuvenationrequiresareversaltoayoungerorganismstate,
which,inaccordancewiththerecapitulationlaw,mayresembleearlierevolutionary
stages.
Regenerationisverystronglyandparadoxicallyintertwinedwithbothphylogeny
andoncogenesis.Theregenerativeabilityishigherinsimplerorganisms[39–41].Moreo‐
ver,inhighlyregenerativeanimals(suchassalamandersandfrogs),regenerativepro‐
cessescanrevertmalignantcellsbacktoaphysiologicalstate[39].Inhumans,theregen‐
erativeabilityisstrongerinearlierdevelopment,whenitcanbeassociatedwithanticancer
activity.Thus,themicroenvironmentofhumanembryonicstemcellswasreportedtosup‐
pressthetumorigenicphenotypeofaggressivecancercells[42].Atthesametime,the
applicationofstemcelltechnologyforthepurposeofregenerationishinderedbytheon‐
cogenicpotentialofstemcells[20,21].Thecellularbiogeneticlawanditsnormal(evolu‐
tion‐acquired)distortionbythemodificationofdevelopmentmayofferasystemicframe‐
workfordisentanglingthisknotofintertwinedandcontroversialphenomena.
Thegenesworknotseparatelybutaspartsofcellularprograms,andtheseprograms
wereformedinthecourseofevolution.Probably,theywerecreatedbytheadditionof
extralayerstocellularnetworks,becausethehumaninteractomeshowsacore‐to‐periph‐
eryevolutionarygrowth[14],whichwasaccompaniedbynetworkrewiring,mixingnovel
andancientgenesandcausingthedistortionofthebiogeneticlaw.Beforethestudyofa
pathology,itisnecessarytoobtainaclearpictureofnormalrecapitulation(accompanied
bytheevolution‐acquiredmodificationofdevelopment).Thedeviationfromthenormal
recapitulationcanelucidatetheetiologyofpathologicalconditions.
Becausetheregenerativeabilityishigheramongsimplerorganisms,thecontrolled
activationofearlierevolutionaryprogramsinhumansmayfacilitateinjuryhealingand
rejuvenation.‘Controlled’isakeywordhere,becausethedangerofoncogenesisisthe
mainproblemconcerningstemcellusageforregeneration.Probably,healthyregeneration
shouldinvolvetheontogeneticreversaltoayoungerstatewithoutthephylogenetic
Int.J.Mol.Sci.2022,23,1148614of18
reversaltoaunicellularstate.Thesearchforcriticaldifferencesbetweenhealthyontoge‐
neticreversalandpathologicalphylogeneticreversalcouldbenefitfromaphylostrati‐
graphicframeworkrepresentingthehistoryofcellularnetworkbuilding.“Everythingis
thewayitisbecauseitgotthatway”[43](i.e.,everythingisexplainedbyitshistory).The
biogeneticlawlinkingdevelopmentandevolutionmightofferacentralconceptforsys‐
temicanalyses.
Theevolutionaryapproachisalsoimportantbecausemanybiomedicalproblemsare
studiedusingthemodelorganisms(e.g.,rodents,zebrafish,fruitflies,andnematodes).
Notably,cancerappearedintheevolutionasearlyasthebasaleumetazoans(itwasfound
inhydraandcorrals)[19].Ourunderstandingofthedifferentevolutionarytrajectoriesof
modelorganismscoupledwiththeirrecapitulationinontogenesisisnecessaryforthecor‐
recttranslationofobtainedresultstohumans.
4.MaterialsandMethods
Thehumansingle‐celltranscriptomeswereacquiredfromGeneExpressionOmnibus
[44]andBioStudies[45].ThedatabaseswereGSE75748(twodatasets:‘celltype’and‘time
course’)[46],GSE123899[47],GSE90749(twodatasets:‘hepatocyte‐like’and‘whiteadi‐
pocytes’)(unpublished),GSE36552[48],E‐MTAB‐3929[49],andGSE81252[50].Thecell
typesareindicatedinthefigurelegends(withdatasetidentifiers).
Thecontrolforcellcycleactivitywasconductedaspreviouslydescribed[15].Briefly,
thedatawerenormalizedusingthe‘limma’softwareimplementedintheRpackageusing
the‘quantile’normalizationmethod[51].Thenormalizedtranscriptlevelsofthegenes
belongingtoatestedgenegroup(e.g.,thegenesfromaphylostratum)wereaveragedfor
eachgenegroupineachcelltranscriptome.Thelimmaprovideslogtransformation.After
genegroupaveraging,themeanswereback‐transformed.Weanalyzedtheregressionof
themeanofatestedgenegrouponthemeanofthecellcyclesignature(thegenesfrom
theGOcategoryGO:0000278,‘mitoticcellcycle’),withthetranscriptomesofindividual
cellstakenasseparatepoints.Inthetext,thetranscriptleveliscalled“expression”forthe
sakeofbrevity.Tocomparethetworegressionlines(e.g.,MPvs.ESC),weusedthedif‐
ferenceintheinterceptsbetweentheseregressionlines(atequalslopes),withthecorre‐
spondingstatisticalsignificance.TheanalyseswereperformedusingtheStatgraphics
CenturionXVIIIpackage.
Asafirstapproximation,weusedthelinearmodelbecauseitenablesthestrictcom‐
parisonoftheregressionlines(withthedeterminationofthestatisticalsignificanceofthe
interceptdifferencebetweenthelines).Thecomparisonofinterceptsfornonlinearcurves
ispointless.Moreover,thelinearmodelgraspstheoverwhelmingpartofthevarianceof
thedependentvariableexplainedbythenonlinearmodel(>90%).Forinstance,thelinear
modelfortheESCinFigure1Aexplained33.6%ofthevariance(r‐squaredcoefficient),
whilethe2‐orderpolynomialmodelexplained35.9%.(Thehigher‐orderpolynomial
membersarenotsignificant.)Inotherwords,linearmodelrepresents94%ofthenonlinear
model.FortheMPinFigure1A,ther‐squaredvaluesare34.7%and35.5%,respectively.
Here,thelinearmodelrepresents98%ofthenonlinearmodel.FortheESCinFigure1B,
ther‐squaredvaluesare6.5%and6.6%,respectively.Here,linearmodelrepresents98%
ofthenonlinearmodel.FortheMPinFigure1B,ther‐squaredvaluesare18.9%and19.7%.
Here,thelinearmodelrepresents96%ofthenonlinearmodel.
Theevolutionarystratificationofhumangenes(phylostratigraphy,orgenedating)
wasacquiredfrom[24],wheretheproblemsofdifferentgenedatingresultsweredis‐
cussed.Here,weusedshallowphylostratigraphy,whichisbasedonthestrictgeneorthol‐
ogyobtainedusingthebestreciprocalhitswiththeaccurateSmith–Watermanalgorithm.
(Incontrast,deepphylostratigraphyincludesin‐paralogs,thusprovidingthedatingof
wholegenefamilies.)
ThehumanproteininteractionswereacquiredfromtheSTRINGdatabase[52].We
selectedtheinteractionswithatop‐halfconfidence(>0.5),whichisslightlyhigherthan
thedefaultconfidenceusedbytheSTRINGserver(>0.4).
Int.J.Mol.Sci.2022,23,1148615of18
ThegenesencodingfortheproteinsbelongingtotheUCandMCgiantclustersof
thehumaninteractome(usedinFigure5B)wereacquiredfrom[14].Forthedetermination
ofthefractionsofUC‐andMC‐originproteinsintheone‐stepinteractomeneighborhood
ofaprotein(usedinFigures5Cand6),theinteractantsofthisproteinweretakenfrom
theSTRING.Phylostraticgenedatingwasappliedtothegenesencodingfortheseinter‐
actants.Then,thefractionsoftheUC‐andMC‐origingeneswerecalculatedforthisgene
set.
Thefunctionalover‐andunder‐representationanalysiswasperformedaspreviously
described[53].Foreachgeneontology(GO)category,wecollectedallitssubcategories
usingGOdirectedacyclicgraphs(DAG),andagenewasregardedasbelongingtoagiven
categoryifitwasmappedtoanyofitssubcategories.Thisisnecessarybecause,forin‐
stance,onlyonegeneismappedtothe‘proteinmodificationprocess’(GO:0036211)di‐
rectly,whereas2500+genescanbemappedtothisprocessusingtheGODAG(because
proteinmodifiersaredistributedamongspecificproteinmodificationprocesses).Themo‐
lecularpathwayswereacquiredfromtheNCBIBioSystems.Aredundancyofthisre‐
source,whichconstitutesamostcompletecompendiumofthepathwaysfromdifferent
databases,wasremovedbyunitingtheentrieswithidenticalgenesets.
Tothispathwayscompendium,weaddedthefollowinggenesignatures:theMolec‐
ularSignaturesDatabase(MSigDB)[54],tumorsuppressorgenesfromtheTSGdatabase
[55],genesfromtheCatalogueofSomaticMutationsinCancer(COSMIC)[56],human
transcriptionfactorsfrom[57]andAnimalTFDB[58],bivalentgenesfrom[32],and
genesfromtheOHNOLOGSdatabase[59].Asthepluripotencysignatures,weused
PluriNetfromMSigDBandthesetofgenesupregulatedintheESCvs.differentiatedcells
observedinatleastthreeindependentstudies[60].
Thehypergeometricdistributionofprobability(implementedintheRenvironment)
wasusedforthedeterminationofthestatisticalsignificanceoftheratioofobservedto
expectednumbersofgenesbelongingtoaGOcategory/pathwayinatestedgenesample.
Theexpectednumberwascalculatedonthebasisofthenumberofcategory/pathway
genesinthetotalgenedataset(assumingarandomgenedistributionacrosscatego‐
ries/pathways).Afterthedeterminationoftheenrichedcategories/pathways,thestatisti‐
calsignificanceoftheenrichmentwascorrectedformultipletests,accordingto[61].
SupplementaryMaterials:Thefollowingsupportinginformationcanbedownloadedat:
https://www.mdpi.com/article/10.3390/ijms231911486/s1,FiguresS1–S22,TablesS1–S32.
AuthorContributions:A.E.V.designedthestudy,performedtheanalyses,andwrotethepaper.
O.V.A.analyzedthedataandwrotethepaper.Allauthorshavereadandagreedtothepublished
versionofthemanuscript.
Funding:ThisworkwasfundedbytheMinistryofScienceandHigherEducationoftheRussian
Federation(AgreementNo.075‐15‐2021‐1075,signed28.09.2021).
InstitutionalReviewBoardStatement:Notapplicable.
InformedConsentStatement:Notapplicable.
DataAvailabilityStatement:Thedataunderlyingthisarticleareavailableinthearticleandits
onlineSupplementaryMaterials.
Acknowledgments:Wethankthethreeanonymousreviewersfortheirvaluablecomments.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
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... We assume that these random walks can simulate random alterations in the interactome caused by mutations in encoding genes or disturbances in gene expression or protein configuration. It was shown previously that alterations in protein interactions are associated with changes in gene expression [57]. ...
... Thus, there can be a synergism between the high gene expression in the UC center and the UC interactome attractor that is triggered by intensive cell stress, especially during cell proliferation. As a result, the Waddington epigenetic landscape of ontogenesis [70,71], which, in accordance with the biogenetic law, roughly recapitulates phylogenesis [57,72], can turn over. (The biogenetic law was validated on the cellular level [57].) ...
... As a result, the Waddington epigenetic landscape of ontogenesis [70,71], which, in accordance with the biogenetic law, roughly recapitulates phylogenesis [57,72], can turn over. (The biogenetic law was validated on the cellular level [57].) In normal cells, this landscape is slanted towards cell differentiation, yet under stressful conditions it can be counteracted by the activity of UC attractor, causing landscape turnover and cell dedifferentiation ( Figure 11). ...
Systemic Alterations of Cancer Cells and Their Boost by Polyploidization: Unicellular Attractor (UCA) Model
Article
Full-text available
  • Mar 2023
  • INT J MOL SCI
Using meta-analyses, we introduce a unicellular attractor (UCA) model integrating essential features of the ‘atavistic reversal’, ‘cancer attractor’, ‘somatic mutation’, ‘genome chaos’, and ‘tissue organization field’ theories. The ‘atavistic reversal’ theory is taken as a keystone. We propose a possible mechanism of this reversal, its refinement called ‘gradual atavism’, and evidence for the ‘serial atavism’ model. We showed the gradual core-to-periphery evolutionary growth of the human interactome resulting in the higher protein interaction density and global interactome centrality in the UC center. In addition, we revealed that UC genes are more actively expressed even in normal cells. The modeling of random walk along protein interaction trajectories demonstrated that random alterations in cellular networks, caused by genetic and epigenetic changes, can result in a further gradual activation of the UC center. These changes can be induced and accelerated by cellular stress that additionally activates UC genes (especially during cell proliferation), because the genes involved in cellular stress response and cell cycle are mostly of UC origin. The functional enrichment analysis showed that cancer cells demonstrate the hyperactivation of energetics and the suppression of multicellular genes involved in communication with the extracellular environment (especially immune surveillance). Collectively, these events can unleash selfish cell behavior aimed at survival at all means. All these changes are boosted by polyploidization. The UCA model may facilitate an understanding of oncogenesis and promote the development of therapeutic strategies.
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... Among these features are the response to DNA and telomere damage and the impairment of circadian entrainment [73,[132][133][134]. Our data also uncovered ploidy-related manifestations of the fetal phenotype in the signaling pathways and metabolism [88,[135][136][137]. For example, the associations between polyploidy and the activation of pathways of meiosis, female gametogenesis, programs of unicellularity, and signaling of multipotency (TGFbeta, JAK-STAT, c-KIT) were well documented in heart diseases, cancer, and normal tissues [79,89,134,[138][139][140][141][142][143][144]. ...
Long-Term Transcriptomic Changes and Cardiomyocyte Hyperpolyploidy after Lactose Intolerance in Neonatal Rats
Article
Full-text available
  • Apr 2023
  • INT J MOL SCI
Many cardiovascular diseases originate from growth retardation, inflammation, and malnutrition during early postnatal development. The nature of this phenomenon is not completely understood. Here we aimed to verify the hypothesis that systemic inflammation triggered by neonatal lactose intolerance (NLI) may exert long-term pathologic effects on cardiac developmental programs and cardiomyocyte transcriptome regulation. Using the rat model of NLI triggered by lactase overloading with lactose and the methods of cytophotometry, image analysis, and mRNA-seq, we evaluated cardiomyocyte ploidy, signs of DNA damage, and NLI-associated long-term transcriptomic changes of genes and gene modules that differed qualitatively (i.e., were switched on or switched off) in the experiment vs. the control. Our data indicated that NLI triggers the long-term animal growth retardation, cardiomyocyte hyperpolyploidy, and extensive transcriptomic rearrangements. Many of these rearrangements are known as manifestations of heart pathologies, including DNA and telomere instability, inflammation, fibrosis, and reactivation of fetal gene program. Moreover, bioinformatic analysis identified possible causes of these pathologic traits, including the impaired signaling via thyroid hormone, calcium, and glutathione. We also found transcriptomic manifestations of increased cardiomyocyte polyploidy, such as the induction of gene modules related to open chromatin, e.g., “negative regulation of chromosome organization”, “transcription” and “ribosome biogenesis”. These findings suggest that ploidy-related epigenetic alterations acquired in the neonatal period permanently rewire gene regulatory networks and alter cardiomyocyte transcriptome. Here we provided first evidence indicating that NLI can be an important trigger of developmental programming of adult cardiovascular disease. The obtained results can help to develop preventive strategies for reducing the NLI-associated adverse effects of inflammation on the developing cardiovascular system.
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Gradistics: An underappreciated dimension in evolutionary space
Article
  • Feb 2023
  • BIOSYSTEMS
The growth of complexity is an unsolved and underappreciated problem. We consider possible causes of this growth, hypotheses testing, molecular mechanisms, complexity measures, cases of simplification, and significance for biomedicine. We focus on a general ability of regulation, which is based on the growing information storage and processing capacities, as the main proxy of complexity. Natural selection is indifferent to complexity. However, complexification can be inferred from the same first principle, on which natural selection is founded. Natural selection depends on potentially unlimited reproduction under limited environmental conditions. Because of the demographic pressure, the simple ecological niches become fulfilled and diversified (due to species splitting and divergence). Diversification increases complexity of biocenoses. After the filling and diversification of simple niches, the more complex niches can arise. This is the 'atomic orbitals' (AO) model. Complexity has many shortcomings but it has an advantage. This advantage is ability to regulatory adaptation, including behavioral, formed in the evolution by means of genetic adaptation. Regulatory adaptation is much faster than genetic one because it is based on the information previously accumulated via genetic adaptation and learning. Regulatory adaptation further increases complexity of biocenoses. This is the 'regulatory advantage' (RA) model. The comparison of both models allows testable predictions. We focus on the animal evolution because of the appearance of higher regulatory level (nervous system), which is absent in other lineages, and relevance to humans (including biomedical aspects).
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Polyploidy as a Fundamental Phenomenon in Evolution, Development, Adaptation and Diseases
Article
Full-text available
  • Mar 2022
  • INT J MOL SCI
DNA replication during cell proliferation is ‘vertical’ copying, which reproduces an initial amount of genetic information. Polyploidy, which results from whole-genome duplication, is a fundamental complement to vertical copying. Both organismal and cell polyploidy can emerge via premature cell cycle exit or via cell-cell fusion, the latter giving rise to polyploid hybrid organisms and epigenetic hybrids of somatic cells. Polyploidy-related increase in biological plasticity, adaptation, and stress resistance manifests in evolution, development, regeneration, aging, oncogenesis, and cardiovascular diseases. Despite the prevalence in nature and importance for medicine, agri- and aquaculture, biological processes and epigenetic mechanisms underlying these fundamental features largely remain unknown. The evolutionarily conserved features of polyploidy include activation of transcription, response to stress, DNA damage and hypoxia, and induction of programs of morphogenesis, unicellularity, and longevity, suggesting that these common features confer adaptive plasticity, viability, and stress resistance to polyploid cells and organisms. By increasing cell viability, polyploidization can provide survival under stressful conditions where diploid cells cannot survive. However, in somatic cells it occurs at the expense of specific function, thus promoting developmental programming of adult cardiovascular diseases and increasing the risk of cancer. Notably, genes arising via evolutionary polyploidization are heavily involved in cancer and other diseases. Ploidy-related changes of gene expression presumably originate from chromatin modifications and the derepression of bivalent genes. The provided evidence elucidates the role of polyploidy in evolution, development, aging, and carcinogenesis, and may contribute to the development of new strategies for promoting regeneration and preventing cardiovascular diseases and cancer.
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Database resources of the national center for biotechnology information
Article
Full-text available
  • Dec 2021
The National Center for Biotechnology Information (NCBI) produces a variety of online information resources for biology, including the GenBank® nucleic acid sequence database and the PubMed® database of citations and abstracts published in life science journals. NCBI provides search and retrieval operations for most of these data from 35 distinct databases. The E-utilities serve as the programming interface for the most of these databases. Resources receiving significant updates in the past year include PubMed, PMC, Bookshelf, RefSeq, SRA, Virus, dbSNP, dbVar, ClinicalTrials.gov, MMDB, iCn3D and PubChem. These resources can be accessed through the NCBI home page at https://www.ncbi.nlm.nih.gov.
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Time to synchronize our clocks: Connecting developmental mechanisms and evolutionary consequences of heterochrony
Article
Full-text available
  • Nov 2021
Heterochrony, defined as a change in the timing of developmental events altering the course of evolution, was first recognized by Ernst Haeckel in 1866. Haeckel's original definition was meant to explain the observed parallels between ontogeny and phylogeny, but the interpretation of his work became a source of controversy over time. Heterochrony took its modern meaning following the now classical work in the 1970–80s by Steven J. Gould, Pere Alberch, and co-workers. Predicted and described heterochronic scenarios emphasize the many ways in which developmental changes can influence evolution. However, while important examples of heterochrony detected with comparative morphological methods have multiplied, the more mechanistic understanding of this phenomenon lagged conspicuously behind. Considering the rapid progress in imaging and molecular tools available now for developmental biologists, this review aims to stress the need to take heterochrony research to the next level. It is time to synchronize the different levels of heterochrony research into a single analysis flow: from studies on organismal-level morphology to cells to molecules and genes, using complementary techniques. To illustrate how to achieve a more comprehensive understanding of phyletic morphological diversification associated with heterochrony, we discuss several recent case studies at various phylogenetic scales that combine morphological, cellular, and molecular analyses. Such a synergistic approach offers to more fully integrate phylogenetic and ontogenetic dimensions of the fascinating evolutionary phenomenon of heterochrony. Highlights • Heterochrony research focuses on comparative morphology but lags on mechanistic understanding. • We propose integrating the different levels of study into a single analysis flow: from morphology to cells to molecules and genes, using modern techniques.
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Growth of Biological Complexity from Prokaryotes to Hominids Reflected in the Human Genome
Article
Full-text available
  • Oct 2021
  • INT J MOL SCI
The growth of complexity in evolution is a most intriguing phenomenon. Using gene phylostratigraphy, we showed this growth (as reflected in regulatory mechanisms) in the human genome, tracing the path from prokaryotes to hominids. Generally, the different regulatory gene families expanded at different times, yet only up to the Euteleostomi (bony vertebrates). The only exception was the expansion of transcription factors (TF) in placentals; however, we argue that this was not related to increase in general complexity. Surprisingly, although TF originated in the Prokaryota while chromatin appeared only in the Eukaryota, the expansion of epigenetic factors predated the expansion of TF. Signaling receptors, tumor suppressors, oncogenes, and aging- and disease-associated genes (indicating vulnerabilities in terms of complex organization and strongly enrichment in regulatory genes) also expanded only up to the Euteleostomi. The complexity-related gene properties (protein size, number of alternative splicing mRNA, length of untranslated mRNA, number of biological processes per gene, number of disordered regions in a protein, and density of TF-TF interactions) rose in multicellular organisms and declined after the Euteleostomi, and possibly earlier. At the same time, the speed of protein sequence evolution sharply increased in the genes that originated after the Euteleostomi. Thus, several lines of evidence indicate that molecular mechanisms of complexity growth were changing with time, and in the phyletic lineage leading to humans, the most salient shift occurred after the basic vertebrate body plan was fixed with bony skeleton. The obtained results can be useful for evolutionary medicine.
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Quantitative lineage analysis identifies a hepato-pancreato-biliary progenitor niche
Article
Full-text available
  • Sep 2021
  • NATURE
Studies based on single cells have revealed vast cellular heterogeneity in stem cell and progenitor compartments, suggesting continuous differentiation trajectories with intermixing of cells at various states of lineage commitment and notable degrees of plasticity during organogenesis1,2,3,4,5. The hepato-pancreato-biliary organ system relies on a small endoderm progenitor compartment that gives rise to a variety of different adult tissues, including the liver, pancreas, gall bladder and extra-hepatic bile ducts6,7. Experimental manipulation of various developmental signals in the mouse embryo has underscored important cellular plasticity in this embryonic territory⁶. This is reflected in the existence of human genetic syndromes as well as congenital malformations featuring multi-organ phenotypes in liver, pancreas and gall bladder⁶. Nevertheless, the precise lineage hierarchy and succession of events leading to the segregation of an endoderm progenitor compartment into hepatic, biliary and pancreatic structures have not yet been established. Here we combine computational modelling approaches with genetic lineage tracing to accurately reconstruct the hepato-pancreato-biliary lineage tree. We show that a multipotent progenitor subpopulation persists in the pancreato-biliary organ rudiment, contributing cells not only to the pancreas and gall bladder but also to the liver. Moreover, using single-cell RNA sequencing and functional experiments we define a specialized niche that supports this subpopulation in a multipotent state for an extended time during development. Together these findings indicate sustained plasticity underlying hepato-pancreato-biliary development that might also explain the rapid expansion of the liver while attenuating pancreato-biliary growth.
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Genetic, Epigenetic, and Post-Transcriptional Basis of Divergent Tissue Regenerative Capacities Among Vertebrates
Article
Full-text available
  • May 2021
Regeneration is widespread across the animal kingdom but varies vastly across phylogeny and even ontogeny. Adult mammalian regeneration in most organs and appendages is limited, while vertebrates such as zebrafish and salamanders are able to regenerate various organs and body parts. Here, we focus on the regeneration of appendages, spinal cord, and heart—organs and body parts that are highly regenerative among fish and amphibian species but limited in adult mammals. We then describe potential genetic, epigenetic, and post‐transcriptional similarities among these different forms of regeneration across vertebrates and discuss several theories for diminished regenerative capacity throughout evolution.
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Whole-Genome Duplications in Evolution, Ontogeny, and Pathology: Complexity and Emergency Reserves
Article
  • Nov 2021
Whole-genome duplication (WGD), or polyploidy, increases the amount of genetic information in the cell. WGDs of whole organisms are found in all branches of eukaryotes and act as a driving force of speciation, complication, and adaptations. Somatic-cell WGDs are observed in all types of tissues and can result from normal or altered ontogenetic programs, regeneration, pathological conditions, aging, malignancy, and metastasis. Despite the versatility of WGDs, their functional significance, general properties, and causes of their higher adaptive potential are unclear. Comparisons of whole-transcriptome data and information from various fields of molecular biology, genomics, and molecular medicine showed several common features for polyploidy of organisms and somatic and cancer cells, making it possible to understand what WGD properties lead to the emergence of an adaptive phenotype. The adaptation potential of WGDs may be associated with an increase in the complexity of the regulation of networks and signaling systems; a higher resistance to stress; and activation of ancient evolutionary programs of unicellularity and pathways of morphogenesis, survival, and life extension. A balance between the cell and organismal levels in controlling gene regulation may shift in stress towards the priority of cell survival, and the shift can lead to cardiovascular diseases and carcinogenesis. The presented information helps to understand how polyploidy creates new phenotypes and why it acts as a driving force of evolution and an important regulator of biological processes in somatic cells during ontogeny, pathogenesis, regeneration, and transformation.
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Reverting to single-cell biology: The predictions of the atavism theory of cancer
Article
  • Aug 2021
Cancer or cancer-like phenomena pervade multicellular life, implying deep evolutionary roots. Many of the hallmarks of cancer recapitulate unicellular modalities, suggesting that cancer initiation and progression represent a systematic reversion to simpler ancestral phenotypes in response to a stress or insult. This so-called atavism theory may be tested using phylostratigraphy, which can be used to assign ages to genes. Several research groups have confirmed that cancer cells tend to over-express evolutionary older genes, and rewire the architecture linking unicellular and multicellular gene networks. In addition, some of the elevated mutation rate - a well-known hallmark of cancer - is actually self-inflicted, driven by genes found to be homologs of the ancient SOS genes activated in stressed bacteria, and employed to evolve biological workarounds. These findings have obvious implications for therapy.
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