Stem cell-specific activation of an ancestral myc protooncogene with conserved basic functions in

15 Pages·2010·1.6 MB·English

Stem cell-specific activation of an ancestral
myc protooncogene with conserved basic
functions in the early metazoan Hydra
Markus Hartla,1, Anna-Maria Mitterstillerb,1, Taras Valovkaa, Kathrin Breukerc, Bert Hobmayerb,2, and Klaus Bistera,2
InstitutesofaBiochemistry,bZoology,andcOrganicChemistry,CenterforMolecularBiosciences,UniversityofInnsbruck,Austria
EditedbyPeterK.Vogt,TheScrippsResearchInstitute,LaJolla,CA,andapprovedJanuary15,2010(receivedforreviewSeptember25,2009)
Thec-mycprotooncogeneencodesatranscriptionfactor(Myc)with Drosophila melanogaster, a triploblastic bilaterian model organ-
oncogenic potential. Myc and its dimerization partner Max are ism(9,10).DrosophilaMyc(dMyc)controlscellgrowthandcell
bHLH-Zip DNA binding proteins controlling fundamental cellular size(11)andregulatescellcompetitioninadose-dependentman-
processes.Deregulationofc-mycleadsto tumorigenesisandisa ner (12). dMyc and dMax bind to a large number of genomic
hallmarkofmanyhumancancers.Wehaveidentifiedandexten- E-boxes to regulate the expression of many genes including
sively characterized ancestral forms of myc and max genes from
key regulators of ribosome biogenesis (13, 14).
theearlydiploblasticcnidarianHydra,themostprimitivemetazoan
ThefreshwaterpolypHydraisaclassicdiploblasticmodelsys-
organismemployedsofarforthestructural,functional,andevolu-
tem to study pattern formation, regeneration, and stem cell dy-
tionaryanalysisofthesegenes.Hydramycisspecificallyactivated
namics in an evolutionary context (15–19), and belongs to the
inallstemcellsandnematoblastnestswhichrepresenttherapidly
animal phylum cnidaria that branched off almost 600 million
proliferating cell types of the interstitial stem cell system and in Y
R
proliferatingglandcells.Interminallydifferentiatednervecells,ne- yearsago.Itssimplebodyplaniscomposedofthreeindependent ST
matocytes,orepithelialcells,mycexpressionisnotdetectablebyin celllineages.Twolineagesofepithelialmusclecellsformunicel- EMI
H
situhybridization.Hydramaxexhibitsasimilarexpressionpattern lularsheets(ectoderm,endoderm),shapethebodyofthepolyp, OC
ininterstitialcellclusters.TheancestralHydraMycandMaxpro- andcarryitsmorphogeneticcapacities.Thethirdlineageisastem BI
teins display the principal design of their vertebrate derivatives, cellsystemdispersedintheinterstitialspacesbetweenepithelial
with the highest degree of sequence identities confined to the musclecells.Themultipotentstemcellsarelocatedintheecto-
bHLH-Zip domains. Furthermore, the 314-amino acid Hydra Myc dermofthegastricregion.Theygiverisetoectodermalanden-
proteincontainsbasicformsoftheessentialMycboxesIthrough dodermal differentiation products that are essential for the
III.ArecombinantHydraMyc/Maxcomplexbindstotheconsensus polyp’s behavior (nerve cells, nematocytes, and gland cells)
DNA sequence CACGTG with high affinity. Hybrid proteins com-
and sexual reproduction (gametes) (20). Ectodermal and endo-
posed of segments from the retroviral v-Myc oncoprotein and
dermalepithelialmusclecellsproliferatewithacellcyclelength
theHydraMycproteindisplayoncogenicpotentialincelltransfor-
of about 3.5 d, coincident with the polyp growth rate (21). The
mationassays.Ourresultssuggestthattheprincipalfunctionsof
interstitial stem cells proliferate much faster with a cell cycle
theMycmasterregulatoraroseveryearlyinmetazoanevolution,
allowingtheirdissectioninasimplemodelorganismshowingre- lengthofabout1d(22).However,only60%ofthedaughtercells
generativeabilitybutnosenescence. remainstemcells,whereas40%differentiateintonervecells,ne-
matocytes, or gland cells (23, 24).
cellproliferation∣cnidaria∣development∣transcriptionfactor Here, we describe the cloning and structural and functional
analysis of myc and max genes from Hydra, representing the
The myc oncogene was originally identified as a highly onco- oldestandmostprimitivemetazoanorganismusedsofarformo-
genicretroviralallele(v-myc),derivedfromthecellularc-myc lecularanalysesofthesegenesandtheirproteinproducts.Intri-
protooncogenebytransduction(1).Theproteinproduct(Myc)of guingly, the basic biochemical and biological functions defined
c-myc represents the central part of a transcriptional regulator
forvertebrateMycandMaxproteinsarealreadylaidoutintheir
network controlling the expression of up to 15% of all human
earlyancestralhomologs.Furthermore,thespecificexpressionof
genesandregulatingfundamentalcellularprocesseslikegrowth,
Hydra myc and max in all rapidly proliferating cell types of the
proliferation, differentiation, metabolism, and apoptosis (2, 3).
interstitialstemcellsystempointstoanearlyroleinfundamental
Deregulationofc-mycleadingtoelevatedlevelsofMycisafre-
cellular regulation, rendering Hydra an ideal model system for
quenteventintumorigenesis,occuringinabout30%ofallhuman
cancers(4,5).MycisabHLH-Zipproteincontainingproteindi- biochemical and genetic analyses of these important regulatory
merizationdomains(helix-loop-helix,leucinezipper)andaDNA genesandtheirpleiotropicimpactongrowthanddevelopment.
contactsurface(basicregion)(2).Mycformsheterodimerswith
the bHLH-Zip protein Max, binds to specific DNA sequence
elements(E-boxes),andispartofatranscriptionfactornetwork Authorcontributions:M.H.,B.H.,andK.Bisterdesignedresearch;M.H.,A.-M.M.,T.V.,
andK.Breukerperformedresearch;M.H.,K.Breuker,B.H.,andK.Bisteranalyzeddata;
includingadditionalbHLH-Zipproteins(2).Myc-Maxheterodi-
andM.H.,B.H.,andK.Bisterwrotethepaper.
mersareimplicatedintranscriptionalactivationofdistincttarget
Theauthorsdeclarenoconflictofinterest.
genes,butMychasalsobeenassociatedwithtranscriptionalre-
ThisarticleisaPNASDirectSubmission.
pression(2,3,6).ManyofthegenesactivatedbyMycarerelated
Data deposition: The sequences reported in this paper have been deposited in the
to processes of cell growth and metabolism, including protein
GenBankdatabase(accessionnos.GQ856263,GQ856264,GU199337).
synthesis, ribosomal biogenesis, glycolysis, mitochondrial func-
1M.H.andA.M.contributedequallytothiswork.
tion, and cell cycle progression (3, 7, 8). Most of the genes re-
2Towhomcorrespondenceshouldbeaddressed.E-mail:[email protected].
pressed by Myc are involved in cell cycle arrest, cell adhesion,
[email protected].
and cell-to-cell communication (3, 7, 8). Invertebrate orthologs
This article contains supporting information online at www.pnas.org/cgi/content/full/
of Myc and Max proteins have been identified in the fruit fly 0911060107/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.0911060107 PNAS∣March2,2010∣vol.107∣no.9∣4051–4056
Results chemically conservative substitutes (Fig. S1B). The specific se-
IdentificationandCloningofHydramycandmaxGenes.Startingfrom quence identities among the Hydra, chicken, and human Myc
theHydraEST(expressedsequencetags)databasecontainingen- and Max proteins are summarized in Fig. S1C. The location of
tries related to vertebrate myc and max genes, a bioinformatic myc1 and myc2 on the Hydra genome is shown in Fig. S1D.
search of the emerging Hydra magnipapillata genome database Intriguingly, myc2 maps directly adjacent to the CAD gene, a
ledtotheidentificationofopenreadingframesencodingputative bona fide Myc target in mammals (7, 8), encoding the multi-
HydraMycandMaxproteins.Arecentsearchusingthemostcur- functional protein carbamoyl-phosphate synthetase/aspartate
rentreleaseoftheHydragenomedatabaserevealedthepresence transcarbamoylase/dihydroorotase. Homology modeling of the
of additional myc-related sequences in the completed genome dimerization/DNA binding domain of Hydra Myc/Max, using
sequence. Two deduced protein sequences (termed Myc1 and the 3D structure available for the corresponding region of the
Myc2) display the principal topography of Myc proteins, one humanproteins(29)asatemplate,revealedspecificconservation
(Myc1) encoded by the gene initially cloned in this study and ofresiduesatthecontactsurfaces(Fig.S2).Molecularphyloge-
nowtermedmyc1.Comparisonofthededucedproteinsequences netic analyses revealed that the Hydra Myc and Max protein
with those of the human or chicken c-Myc and Max homologs sequences branch off from basal positions within the trees
revealed overall sequence identities of 31.5% or 27.4% for the (Fig.S3AandB).Also,theanalysessuggestthatthediversifica-
Myc1protein,and55.0%or54.4%fortheMaxprotein,respec- tionofC-/B-,L-,andN-/S-Mycsubfamiliesoccurredonlywithin
tively(Fig.1).Thehighestdegreeofsequenceidentitywasfound the vertebrate lineage (Fig. S3A).
in the bHLH-Zip regions of Myc1 (44.7% identity) and Max ThepredictedcodingregionsofHydramyc1(314aminoacids)
(81.0% identity), necessary for dimerization and sequence- and max (176 amino acids) (Fig. 1) were cloned by using total
specific DNA binding (Fig. 1). The amino-terminal Hydra mRNAs isolated from whole Hydra magnipapillata animals as a
Myc1domainexhibitslessoverallsequenceidentitytothehuman templateforcDNAsynthesisfollowedbyPCR.Sequenceanalysis
homolog, although Myc boxes I, II, IIIa, and IIIb, essential for ofthefull-length942-bpHydramyc1andthe528-bpHydramax
Myc-induced cell transformation and transcriptional regulation coding regions revealed that the max sequence was identical to
(25–28), display identities of 30.0%, 43.8%, 33.0%, and 41.7%, thesequencededucedfromtheHydragenome,whereastwonu-
resp. The structures of Hydra Myc1 determined here and of cleotide substitutions were found in the myc1 coding sequence
thededucedMyc2proteindisplaythesameprincipaltopography leading to one amino acid substitution (G188D) in comparison
and similar evolutionary relationship to the human protein with the predicted Hydra Myc1 protein from the database.
(Fig.S1A),butshareonly23.6%overallidentitiesamongthem- Furthermore, only a small fraction of the Hydra myc1-specific
selves.Notably,theexon-exonjunctionsofHydramyc1andmyc2, cDNAs encode the full-length 314-amino acid Myc1 protein,
andofhumanc-myc,shareverysimilarnucleotidesequences,and whereasthemajorityofcDNAsencodeashorter289-aminoacid
the amino acids around the junctions are either identical or version starting with the methionine at position 26 (Fig. 1).
Fig.1. AminoacidsequencesofHydraMyc1andMaxproteinsandalignmentwiththeirvertebratehomologs.(A)Alignmentofhuman(Hu)c-Myc,chicken
(Ck)c-Myc,andHydra(Hy)Myc1sequences(GenBankaccessionnos.:huc-Myc,NP_002458;ckc-Myc,NP_001026123;hyMyc1,GQ856263).(B)Alignmentof
human,chicken,andHydraMaxsequences(GenBankaccessionnos.:huMax,NP_002373;ckMax,P52162;hyMax,GQ856264).Identicalresiduesareshadedin
Blue,gapsareindicatedbyDashes.ThepositionsoftheMycboxes(MB)I–IVinthetransactivationdomainsofthevertebrateproteinsandofthebHLH-Zip
regionintheMycorMaxDNAbindingdomainsareindicated.ConservedheptadrepeatresiduesintheleucinezipperregionsaremarkedbyAsterisks.The
positionscorrespondingtoexon-exonjunctionsareindicatedbyArrowsabove(HU,CK)andbelow(HY)thealignments.Alignmentsweregeneratedbyusing
theClustalWalgorithmwithadditionalmanualadjustments.
4052 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0911060107 Hartletal.
In a Northern analysis using polyðAÞþ RNAs from whole preparations (Fig. 3A, C–F). Also, the myc1 gene was not tran-
polyps and Hydra myc1 and max cDNAs as probes, single tran- scriptionallyupregulatedduringmorphogeneticprocessessuchas
scripts with sizes of 1.20 kb and 1.25 kb, resp., were detected, asexual bud formation and regeneration. Taken together, myc1
whichiscompatiblewiththesizesofthepredictedopenreading wasspecificallyactivatedinallstemcellsandnematoblastnests
frames (Fig. 2A). Whereas the total expression level of myc1 in which represent the rapidly proliferating cell types of the inter-
thewholeanimalwasratherlow,maxwasabundantlyexpressed. stitialstemcellsystem,andinproliferatingglandcells(Fig.3G).
HybridizationofthesamefilterswithaHydraCADcDNAprobe InintactHydra,themaxgeneexhibitedanexpressionpattern
revealed thepresenceofasingle7.0-kbtranscript (Fig.2A).By similartothemyc1geneincellclustersoftheinterstitialstemcell
usingpolyclonalantibodiesgeneratedagainstrecombinantHydra lineage(Fig.3BandFig.S4B).Quantitativeanalysesofnestsizes
Myc1andMaxproteins,endogenousHydraMyc1andMaxpro- showed similar results for the myc1 and max genes (Fig. S4C),
teinsweredetectedbyimmunoprecipitationorimmunoblotana- withmyc1-andmax-positivenestscontaining4,8,or16cells.Pre-
lysis(Fig.2BandC).Forsizecomparison,invitrotranslatedfull- vious analyses of genes activated in post-mitotic, differentiating
length Hydra Myc1 (Mr 36,082) and Max (Mr 20,234) proteins, nematocytenestslikeHvJNK(31)andNowa(32)hadshownnest
and the shorter Myc1 protein (Mr 33,174) were included in the sizesbetween8and32cells.Inaddition,doubleinsituhybridiza-
analysis.BothinvitrotranslatedandendogenousHydraMaxpro- tion exhibited basically no overlap between myc1- and Nowa-
teinsdisplayanapparentMr of22,000.Mostoftheendogenous expressing nests (Fig. S4D–F). Hence, myc1 and max are likely
HydraMyc1proteinhasanapparentMr of35,000,similartothe coactivated in proliferating nematoblasts (Fig. 3G). At present,
invitrotranslatedshorter289-aminoacidform(p36),whereasin itisuncleartowhatextentmaxisupregulatedininterstitialstem
vitrotranslatedfull-lengthHydraMyc1displaysanapparentMr and gland cells. Whole mount preparations clearly showed max
of 39,000. expression also in epithelial cells throughout the entire polyp
(Fig. 3B and Fig. S4B). This is consistent with our results from
Hydramyc1andmaxGenesareActivatedintheInterstitialStemCell Northern hybridization (Fig. 2A) and with the more ubiquitous
Lineage.Inintactpolyps,insituhybridizationrevealedmyc1gene activation patterns observed for max in various bilaterians.
expressioninalargenumberofcellsandcellnestsoftheinter-
Y
stitial stem cell lineage (Fig. 3A and Fig. S4A). Expression was BiochemicalPropertiesofHydraMycandMaxProteins.Totestifthe TR
restricted to the gastric region; whereas head (hypostome and principal biochemical functions of Myc and Max proteins have MIS
tentacles) and foot (lower peduncle and basal disc) were free already emerged in the early metazoan Hydra, the coding se- HE
C
of myc1 expressing cells (Fig. 3A). To analyze cell type-specific quences of full-length Hydra Max and of the carboxyl-terminal BIO
expression in more detail, we performed in situ hybridization domainofHydraMyc1containingthebHLH-Zipdomain(amino
on macerated Hydra single cells spread onto microscope slides, acidresidues201–314)(Fig.1)wereinsertedintopETvectorsfor
permittingunambiguousclassificationofcelltypesbasedoncell recombinantproteinexpressioninEscherichiacoli.Asacontrol,
shapeandnuclearmorphology(30).Wefoundmyc1mRNAinall the chicken Max p14 protein containing the bHLH-Zip region
interstitial stem cells(Fig. 3CandE), inallproliferating nema- (amino acid residues 22–113) (33) was expressed analogously.
toblastnests(Fig.3DandF),andinamajorfractionofglandcells The proteins Hydra Max p22 (Mr 20,234), Hydra Myc1 p16
(Fig.3CandE).Thedifferentiatingandfullydifferentiatedcell (Mr 13,745), and chicken Max p14 (Mr 10,927) were efficiently
typesofthenervecellandnematocytepathwaysshowednomyc1 expressed,andthesolublefractionswerepurifiedtohomogeneity
expression. Ectodermal and endodermal epithelial cells also byusingliquidchromatographysystems(Fig.4A).Theidentities
showednomyc1expressioninbothwholemountandmaceration ofrecombinantHydraMyc1p16andMaxproteinswereverified
bymassspectrometry(Fig.S5),fragmentionmapping(Fig.S6),
andimmunoblotanalysis(Fig.4A).TotestifHydraMyc1p16and
Hydra Max p22 proteins show DNA binding activity, similar to
thatofchickenMaxp14(33),electrophoreticmobilityshiftana-
lyses (EMSA)were performed. The analysis showed that Hydra
Myc1p16bindstodouble-strandedDNAcontainingaconsensus
Myc binding site with high efficiency, whereas Hydra Max p22
displayed lower binding affinity (Fig. 4B). The apparent size of
theHydraMyc1p16-DNAcomplexishigherthanexpectedpos-
siblyduetobindingofhigherorderMycoligomersasproposed
previously (29, 34). In agreement with results obtained with
chicken proteins (33), the DNA affinity is enhanced when an
equimolar mixture of Hydra Myc1 p16 and Hydra Max is used,
allowing the formation of heterodimeric protein complexes
(Fig. 4B). The presence of both proteins in these complexes
wasverifiedbycoincubationwithantibodiesdirectedagainstHy-
draMyc1orMaxleadingtopartialsupershift(α-hyMax)orin-
Fig.2. ExpressionofHydramyc1andmaxgenesandtheirproteinproducts. hibition (α-hy Myc1) of DNA binding. To quantify the Hydra
(A)Northernanalysesusingaliquots(2.0μg)ofpolyðAÞþ-selectedRNAsfrom
Myc1orMaxDNAbindingaffinities,increasingamountsofpro-
wholeHydraanimalsandHydramyc1ormaxspecificcDNAprobes.Eachfil-
teinswereaddedtoconstantamountsofDNAinEMSAanalyses
terwasstrippedandrehybridizedwithaHydraCADspecificcDNAprobe.(B)
Immunoprecipitationanalysisusingaliquots(5×106cpm)ofboiledcellex- (Fig. S7A), the ratios of bound to total DNAwere determined,
tractsfrom½35S(cid:1)methionine-labeledHydraanimalsandapolyclonalantiser- and the dissociation constants (Kd) for the protein-DNA com-
umdirectedagainstHydraMyc1recombinantprotein(α-hyMyc1),ornormal plexes were calculated (Fig. S7B). The Kd values for protein-
rabbitserum(NRS).Forcomparison,½35S(cid:1)methionine-labeledHydraMyc1p39 DNAcomplexesformedbyHydraMyc1p16/HydraMaxp22,Hy-
andp36proteinswerealsoproducedbyinvitrotranslationofcorresponding dra Myc1 p16, and Hydra Max p22 were determined to
cDNAs cloned in Bluescript(BS) vectors andimmunoprecipitated.Proteins 1.7×10−8 M, 3.2×10−7 M, and 1.0×10−6 M, resp. Compared
w30e-rμegaanliaqluyozetdofbtyotSaDlSc/ePlAlpGrEot(e1i0n%sf,rowmt∕vwohl)o.le(CH)yImdrmaupnoolybplostanadnaolfysiinsvoiftroa with the Kd for the avian Myc/Max heterodimer binding to the
sameDNA(33),theDNAaffinityofHydraMyc1/Max isabout
translatedHydraMaxusingapolyclonalantiserumdirectedagainstHydra
Maxrecombinantprotein(α-hyMax).ProteinswereresolvedbySDS/PAGE 100-fold lower. Specific DNA binding activity could also be
(12.5%,wt∕vol). demonstrated for the nearly full-length Hydra Myc1 p36
Hartletal. PNAS ∣ March2,2010 ∣ vol.107 ∣ no.9 ∣ 4053
Fig.3. ExpressionpatternsofHydramyc1andmaxvisualizedbyinsituhybridization.(A,B)myc1andmaxareactivatedincellsbelongingtotheinterstitial
stemcelllineageinthegastricregionofintact,buddingpolyps.Inaddition,maxisexpressedatalowerlevelintheepitheliumthroughouttheentirebody
column.(C–F)myc1expressionininterstitialstemcells(CandE),proliferatingnematoblastnests(DandF),andaglandcell(InsertinCandE),visualizedbyin
situ hybridization in macerated single cell preparations. (C and D) bright field optics; (E and F) phase contrast optics. Ecto: ectodermal epithelial cell;
Endo:endodermalepithelialcell;andNV:nervecell.(G)Schemeofthedifferentiationpathwaysintheinterstitialstemcellsystemwithmyc1expressing
subpopulationsshadedinYellow.
recombinant protein, following solubilization from inclusion ment,tumorigenesis,orboth,isseverelycomplicatedbythelarge
bodies and renaturation (Fig. S7C). numberofdirectorindirectMyctargetsidentifiedbyexpression
profilingorgenomicDNAbinding(3).Furthermore,thepossible
Hydra-Viral Myc Hybrid Proteins Induce Cell Transformation. To ex- roleofMycasacoactivatororcorepressorofothertranscription
plore if some of the principal biological functions of vertebrate factors,andtherecentevidencethatmanybiologicalfunctionsof
Myc are already associated with their ancestral counterparts, MycareindependentofMaxorevenE-boxbindingamplifythe
the Hydra myc1 and max coding regions, and hybrids between range of possible Myc targets (3, 36).
Hydraandviralmycwereinsertedintothereplication-competent Identificationandanalysisofmycandmaxgenesandproteins
retroviral RCAS vector and tested for their potential to induce fromgeneticallytractableinvertebrateorganismslikeDrosophila
celltransformationinavianfibroblasts.Inthehybridconstructs, substantiallyenhancedtheanalysisoftheMyc/Maxnetwork(10,
the amino-terminal transcriptional regulation and carboxyl- 36). Hence,characterization ofthesegenes fromyetmuchsim-
terminal DNA binding domains had been mutually exchanged pler pre-bilaterianorganismsatthebaseofmetazoanevolution
(hy/v-myc, v/hy-myc) (Fig. 5A and Fig. S8A). The empty RCAS mayprovidefurtherinsightintothebasicbiochemistryandbiol-
vector and the RCAS-v-myc construct encoding the 416-amino ogyoftheseimportantcellularregulators.Usinginsilicogenomic
acidviralMyc(v-Myc)proteinwereusedascontrols.Theiden- analyses,thepresenceofmyc-likeormyc-relatedgeneshasbeen
tities of the DNA inserts were verified by sequencing and by in predicted for all sequenced genomes of metazoans with the ex-
vitro translation and immunoprecipitation of the protein pro- ceptionofthenematodeCaenorhabditiselegans,wherethelackof
ducts by using specific antibodies recognizing the Hydra Myc1 mycgenesisduetosecondaryloss(37,38).Representingthesis-
p39, Hydra Max p22, and avian v-Myc proteins (Fig. S8B). The tergrouptothemetazoanclade,thechoanoflagellateMonosiga
invitrotranslatedhybridproteinshy/vMyc(Mr 34,910)andv/hy- brevicollis is the most simple organism for which a protein dis-
Myc(Mr 47,210),andthev-Mycprotein(Mr 46,095)displayan tantlyrelatedtometazoanMycproteinshasbeenpredictedfrom
apparent Mr of 37,000, 53,000, or 52,000, resp. (Fig. S8B). The the genome sequence (37, 39). We have identified, cloned,
retroviralconstructsweretransfectedintoprimaryquailembryo andextensivelycharacterizedauthenticmycandmaxgenesfrom
fibroblasts(QEF),andcellswerepassagedseveraltimes.Expres- a diploblastic cnidarian organism, Hydra magnipapillata, that
sionofectopicviralandhybridMycproteinsandoftheendogen- branched off in metazoan evolution almost 600 million years
ous c-Myc protein comigrating with the v-Myc protein was ago. The Hydra Myc1 protein shares the principal topography
monitored by immunoprecipitation analysis (Fig. 5B). The c- withvertebrateMycproteins.Adirectcomparisonrevealsshort
Mycproteinisnotdetectableincellsthatcontainhighlevelsof conserved amino acid motifs in the N-terminal Myc boxes I, II,
v-Mycorv/hy-Myc,whichisduetonegativetranscriptionalreg- IIIa,andIIIb.Hence,thispartoftheproteinevolvedinthecom-
ulationofc-mycbyv-Myc(35).EctopicexpressionofHydraMax mon cnidarian-bilaterian ancestor. The C-terminal bHLH-Zip
didnotcauseanysignificantcellularalterations,andHydraMyc1 domain represents an ancient class of eukaryotic DNA binding
inducedonlyamarginalincreaseincellproliferation.Incontrast, domains, with high sequence identity and structural homology
expressionofbothhybridproteins,hy/v-Mycandv/hy-Myc,ledto between Hydra Myc1 and vertebrate Myc proteins (Fig. 1 and
cell transformation manifested by focus formation, enhanced Fig. S2). Notably, the position of the exon-exon junction that
proliferation, and anchorage-independent growth (Fig. 5C and isfixedacrossbilaterianmycgenes(10)isalsoconservedinthe
D).Transformationbybothhybridproteinswaslessefficientthan pre-bilaterian Hydramyc1gene (Fig.S1).TheHydraMyc1pro-
thatinducedbytheauthenticv-Myconcoprotein.However,these teindimerizeswithHydraMaxandbindstoE-boxDNAwithhigh
resultsshowthatboththeamino-terminalandthecarboxyl-term- affinity,indicatingaveryearlyoriginofthesebasicbiochemical
inaldomainofHydraMyc1areprincipallycapabletosubstitute properties. Intriguingly, oncogenic activity of truncated mutant
for the correspondingregions inthe highly oncogenicretroviral versions of the retroviral v-Myc protein can, at least partially,
v-Myc protein. be rescued bythe corresponding HydraMyc1 protein segments.
Hence,evenbasicbiologicalfunctionsofvertebrateMycproteins
Discussion emerged very early in their ancestral homologs.
Conversetranscriptionalregulationofgrowthpromotingorsup- Recentdatabaseupdatesrevealfourmyc-relatedgenesinthe
pressingtargetsisimplicatedinMyc-inducedoncogenesis(3,6– Hydragenome.Twoofthesegenes,myc1isolatedhere,andmyc2,
8).Theidentificationofsuchgenesrelevantfornormaldevelop- areclearmycorthologsdisplayingstructuralconservationintheir
4054 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0911060107 Hartletal.
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Fig.5. CelltransformingactivitiesofHydraandviralMychybridproteins.
(A)Schematicdiagramofthecodingregionsofv-myc(Blue),andofhy/v-myc
Fig.4. HydraMyc1andMaxrecombinantproteinsandtheirbiochemical and v/hy-myc hybrids (Hydra sequences shown in Yellow). The constructs
activity.(A)SDS/PAGE(5.0–17.5%gradient,wt∕vol)of2-μg(Coomassiebril- wereinsertedintotheuniqueClaIsiteofthereplication-competentretroviral
liantbluestaining)or50-ng(immunoblotting)aliquotsofpurifiedrecombi- pRCASvectorusedforDNAtransfectionintoquailembryofibroblasts(QEF).
nantHydraMyc1p16(aminoacids201–314),HydraMax,a1∶1mixtureof (B) Immunoprecipitation of endogenous c-Myc and ectopic Myc proteins
bothproteins,andchickenMaxp14(aminoacids22–113).Specificantibodies using 1.0×107-cpm aliquots of lysates from ½35S(cid:1)methionine-labeled QEFs
are indicated below the Blots. (B) EMSA using the recombinant proteins transfectedwiththepRCASconstructsshowninA,andantibodiesdirected
shown in A and 0.3-ng (25,000 cpm) aliquots of a [32P]-labeled double- againstamino-terminal(N)orcarboxyl-terminal(C)segmentsofv-Myc,or
stranded18-merdeoxyoligonucleotide33containingtheMyc/Max-binding normal rabbit serum (NRS). Proteins were resolved by SDS/PAGE (10%,
motif5′-CACGTG-3′.Antibodieswereaddedtothebindingreactionsasin- wt∕vol).(C)ProliferationratesofQEFstransfectedwiththepRCASconstructs
dicated.Finalproteinconcentrationsareindicatedbelow. showninAandpassagedseveraltimes.Equalnumbers(7.5×105)ofcells
were seeded onto 60-mm dishes, and cell numbers were determined at
theindicatedtimepoints.(D)Top:QEFson60-mmdishesweretransfected
bHLH-ZipdomainsandMyc-boxesItoIII(Fig.1andFig.S1). with4-μgaliquotsofDNAfromthepRCASconstructsshowninA,keptunder
Two other predicted genes encode proteins with Myc-related agaroverlayfor2wk,andthenstainedwitheosinmethyleneblue.Numbers
bHLH-Zip domains but highly divergent N-terminal parts. Our offociperdishareindicated.Middle:QEFsweretransfectedwiththepRCAS
molecular phylogenetic analysis suggests that the diversification constructsshowninAandpassagedseveraltimes.Thedoublingtimes(Td)of
of vertebrate myc genes into C/B-, N/S-, and L-myc subfamilies the cell populations are indicated below the phase-contrast micrographs.
Bottom:Equalnumbers(1.0×105)oftransfectedandpassagedcellswere
doesnotoriginatefromadiversificationinlowermetazoans,but
seeded in soft agar and incubated for 2 wk. Numbers of colonies per
occured later, presumably at the basis of vertebrate evolu-
1,000cellsseededareshownbelowthebright-fieldmicrographs.
tion (Fig. S3).
The in situ hybridization shows that myc1 is transcriptionally
activated in interstitial stem cells, nematoblasts, and gland cells notypesofmyc1-expressingcells,weproposetwofunctions.First,
(Fig.3).Thesecelltypesrepresenttheproliferatingfractionsof interstitialstemcellsandnematoblastsarefastcyclingcellswith
theHydrainterstitialstemcellsystemthatevolvedatthebasisof cell cycle lengths three to four times shorter than other Hydra
animal evolution (20). Our results corroborate the general re- cells, particularly epithelial muscle cells. Gland cells comprise
quirement of Myc transcription factors in stem and progenitor a population of differentiated stem cell products with retained
cells to regulate proliferation and self-renewal and to perturb capacity for proliferation. Hence, also in Hydra high levels of
orinhibitterminaldifferentiation,asproposedinhighermetazo- Myc1 may act to trancriptionally activate genes involved in cell
ans like Drosophila or vertebrates (3). The detailed action of cycleprogressionorconverselytodown-regulategenesinvolved
Myc1 and Max in Hydra is yet unknown, but based on the phe- in cell cycle arrest (3). Secondly, Myc1 may directly act in
Hartletal. PNAS ∣ March2,2010 ∣ vol.107 ∣ no.9 ∣ 4055
ribosome biogenesis. Under phase contrast optics (Fig. 3E and InsituHydridization.Wholemountinsituhybridizationwithdigoxigenin-la-
F), myc1-expressing interstitial stem cells, nematoblasts, and beledRNAprobeswasdoneasdescribed(41).Forinsituhybridizationinsin-
glecellpreparations,Hydraweremaceratedfor20min(30).Cellsuspensions
gland cells show a characteristic, dark cytoplasm based on the
were fixed in 4% paraformaldehyde for 30 min, spread onto microscope
presenceofverylargenumbersofribosomes(30).Furthermore,
slides,anddriedbriefly.Hybridizationwasasdescribed(42).
a recent in silico analysis of metazoan and non-metazoan gen-
omes has shown that Myc- and Max-specific bHLH-Zip DNA CellsandRetroviruses.Cellculture,DNAtransfection,andtransformationas-
binding domains coevolved with the appearance of E-box sites saysofquailembryofibroblasts(QEF)wereperformedasdescribed(6,40,
in the core promoters of nearly all genes involved in ribosome 43). The construction of retroviral and Bluescript vectors carrying Hydra
mycandmaxgenesorHydra-viralhybridgenesisdescribedinSIText.
biogenesis(38).Theprincipalstructuralandfunctionalsimilari-
ties between Hydra Myc and Max proteins and their vertebrate
Protein Chemistry. In vitro translation, immunoprecipitation of
derivativesprovidethebasisforusingthissimpleorganismnear L-½35S(cid:1)methionine-labeled proteins, SDS/PAGE, and immunoblotting were
the base of metazoan evolution as a model system for cellular doneasdescribed(6,40).Expressionandpurificationofrecombinantpro-
regulation by these proteins. teins was performed essentially as described previously (33). Construction
ofpETexpressionvectorscarryingHydramycandmaxgenesequences,mass
Materials and Methods spectrometryandsequencingofrecombinantproteins,generationofpoly-
clonalantiseradirectedagainstHydraMycandMaxrecombinantproteins,
Animals.HydravulgarisstrainBaselandHydramagnipapillatawildtypestrain
andmetaboliclabelingofHydraproteinsaredescribedinSIText.
105wereusedinthisstudy.Masscultureswerekeptat18°Candfeddaily
withfreshlyhatchedArtemianauplii.Experimentalanimalswerecollected
EMSA. EMSA analysis was performed as described (33, 35). DNA binding
24hafterthelastfeeding(19).
reactions(20μL)wereperformedat25°Cfor45mininabuffercontaining
10mMTrisHClpH7.5,0.5mMEDTA,65mMKCl,5mMMgCl2,1mMDTT,
DNACloningandNucleicAcidAnalysis.Molecularcloning,DNAsequencing, 100μg∕mL BSA, 10% (vol∕vol) glycerol. For supershift analysis, polyclonal
and Northern analysis have been described (6, 40). For RNA isolation, antisera(0.5μL)wereaddedafter15min.Protein-DNAcomplexeswerere-
∼1;500polypsofHydramagnipapillatawerelysedin50mLofabuffercon- solvedbynative6%(wt∕vol)PAGE,andradioactivesignalswerequantified
taining 4 M guanidine thiocyanate, 25 mM sodium acetate pH 6.0, and byusingaPhosphorImager(MolecularDynamics).
0.835%(vol∕vol)2-mercaptoethanol.TotalRNAwasisolatedbyCsCldensity
centrifugation,andmRNAwasenrichedbypolyðAÞþ-RNAselectionasde-
ACKNOWLEDGMENTS.WethankJ.FuchsandK.Liedlfortemplate-basedmod-
scribed (40). Cloning of the coding regions of the predicted Hydra myc eling. This work was supported by Austrian Science Fund (FWF) Grants
andmaxgenesbycDNAsynthesisandPCRisdescribedinSIText. P17041,P18148,P20734,andY372
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Supporting Information
Hartl et al. 10.1073/pnas.0911060107
SI Text passingthefull-lengthHydramaxcodingsequence(hymax),the
DNACloning.ForconstructionoftheadaptorplasmidpA-v-myc, Hydra myc codons 26–314 (p36hymyc) or 201–314 (p16hymyc)
thecodingregionofthev-mycalleleofavianacuteleukemiavirus were inserted into the pET11d vector (Novagen). In the case
MC29 (1,2)wasamplified byPCRbyusingthe v-myc-insert of of the p16hymyc construct, an additional initiating methionine
pRc-Myc (3) as a template and 5′-CCGCTCAGCGCCAGCC- codonwasderivedfromthevector.ThepET3d-p14maxconstruct
TC-3′ and 5′-TCTAGAGGATCCCTATGCACGAGAGTTCCT- encoding chicken Max has been described previously (5).
TA-3′ oligonucleotides as primers. After digestion with BamHI, Fortheexpressionoffull-lengthHydraMaxprotein(176ami-
the PCR product was inserted into the adaptor plasmid pA- no acids), the plasmid pET11d-hymax was transformed into
CLA12NCO (4). To clone the coding regions of the predicted E.colistrainBL21(DE3)CodonPlus-RIL(Stratagene).Anover-
Hydra myc and max genes, cDNA synthesis was performed as nightcultureinLBbrothsupplementedwith2 mg∕mL glucose,
described (4), followed by PCR using the primer pairs 5′- 50μg∕mL ampicillin, and 25μg∕mL chloramphenicol was di-
TATTTTGAGAAGACATTTAATACAG-3′/5′-AAGCTTGGA- luted 1∶50 to yield a 1-l culture. Bacteria were grown at 37°C
TCCTTAAAAGCGAAACTTCATTTCTTCTTGACG-3′(hymyc) toanopticaldensityof0.7at600nm,recombinantproteinsynth-
and 5′-AGTGACGAAGACAAAGAAG-3′/5′-CAGCTCGGATC- esiswasinducedbytheadditionofisopropyl-β-D-thiogalactoside
CTAATGTTCTGTTTTCACTCG-3′ (hy max). The primer se- (IPTG)toafinalconcentrationof1mM,andincubationwascon-
quenceswerededucedfromtheHydramagnipapillatagenome(hy- tinuedfor4h.Thebacterialpelletwasresuspendedin30mLice-
drazome.metazome.net) that was searched for sequence coldbufferB(20mMTris-HClpH7.5,1MNaCl, 1mMDTT,
contigs(38932and37686)relatedtovertebratemycandmaxgenes, 1mMPMSF)andlysedat1,300psiusingaFrenchPress.Tore-
respectively.ThePCRproductsweredigestedwithBamHIandin- duceviscosity,thecellextractwassupplementedwith1.7 μg∕mL
sertedintotheNcoI(blunt-ended)/BamHIsitesofpA-CLA12NCO, DNaseIand0.3mMMgCl2andincubatedfor35minonice.The
yielding pA-hymyc and pA-hymax. To construct the plasmids pA- sample was then centrifuged at 18;000 × g for 20 min at 4°C.
HM-VM and pA-VM-HM encoding hybrid proteins of Hydra Theclarifiedsupernatantwasadjustedto30%ammoniumsulfate
(hy) Myc (HM) and v-Myc (VM), the corresponding hy Myc and saturationbyaddingasaturatedsolutionofthesalt,andthemix-
v-Myc-specificsegmentscontainingoverlappingsequencesweream- turewasstirredonicefor30min.Theprecipitatedproteinswere
plified in four different PCRs using the primer pairs 5′-CACTT- pelletedbycentrifugationat11;000 × gfor30minat4°C.The
CAAGATCTTGCAG-3′/5′-GTGCGTTCGCC- supernatantwasadjustedto40%ammoniumsulfatesaturation,
TCTTGTCACTTAACGAACCGTTTGA-3′ (HM1), 5′-TCAAA- andincubatedoniceasabove.Theprecipitatewascollectedby
CGGTTCGTTAAGTGACAAGAGGCGAACGCAC-3′/5′- centrifugationasaboveanddissolvedin6mLofbufferA(20mM
TCTAGAGGATCCCTATGCACGAGAGTTCCTTA-3′ (VM2), Tris-HClpH7.5,80mMNaCl,1mMEDTA,1mMDTT).The
5′-CCGCTCAGCGCCAGCCTC-3′/5′-GTTTTCGAGAGGTC- solutionwasdialyzedagainst1lofthesamebufferfor12hat4°C
GAGGGTTCTCCTCTGAGTCTAA-3′ (VM1), 5′-TTAGACT- and loaded onto a Resource™ Q anion exchange column (GE
CAGAGGAGAACCCTCGACCTCTCGAAAAC-3′/5′-TCT- Healthcare) equilibrated in buffer A. Chromatography on an
AGAGGATCCTTAAAACC-3′(HM2).IntwosubsequentPCRs, ÄKTAPurifier(GEHealthcare)wascarriedoutbyinitialelution
dilutedpoolsfrompairsofthefirstPCRswereemployedastem- withbufferA,followedbyalineargradientfrom0to0.5MNaCl
plates (HM1þVM2 or VM1þHM2) using the primer pairs inthesamebufferataflowrateof1 mL∕min.HydraMaxcon-
5′-CACTTCAAGATCTTGCAG-3′/5′-TCTAGAGGATCCCTA- taining fractions were concentrated by using an Amicon Ultra
TGCACGAGAGTTCCTTA-3′ (HM-VM) and 5′-CCGCT- centrifugalfilterdevice(MWCO10,000)(Millipore)andloaded
CAGCGCCAGCCTC-3′/5′-TCTAGAGGATCCTTAAAACC-3′ ontoaSuperdex-75gelfiltrationcolumn(GEHealthcare)equi-
(VM-HM).TheresultingPCRproductswerecleavedwithBglII/ librated with buffer C (50 mM NaH2PO4∕Na2HPO4 pH 7.2,
BamHIorBamHIandinsertedintotheBglII/BamHI-sitesofpA- 140mMNaCl,1mMEDTA,1mMDTT)andthenelutedwith
hymycortheNcoI(blunt-ended)/BamHIsitesofpA-CLA12NCO. the same buffer. Hydra Max containing fractions were pooled,
TheinsertsofpA-hymyc,pA-hymax,pA-HM-VM,pA-VM-HM, concentratedandstoredinliquidnitrogen.Thefinalyieldofpur-
andpA-v-mycwerereleasedbypartialdigestionwithClaIandin- ifiedHydraMaxproteinwasapproximately3mgperliterofbac-
sertedintotheretroviralRCAS-BPvector,orintotheBluescript teria culture.
pBSSKIIþvectorforinvitrotranslationasdescribed(4).Toclone FortheexpressionofHydraMycp16(115aminoacids)from
thecodingregionofthepredictedHydramyc2gene,cDNAsynth- the pET11d-p16hymyc construct, bacterial culture, and protein
esis was performed as above, followed by PCR using the expressionwereperformedasabove.Thebacterialpelletwasre-
primer pair 5′-ACTGGTTCAAATTGGTGTACACACG-3′/5′- suspended in 30 mL of buffer D (50 mM NaH2PO4∕Na2HPO4
AGTCAGTCGACGAATTTCAAAAATAAAAAAAATTATT- pH 7.2, 1 M NaCl, 1 mM EDTA, 1 mM DTT) and lysed as
TAAAAAGTAT-3′.ThePCRproductwasdigestedwithSalIand describedabove.AfterDNaseItreatment,thelysatewasclarified
insertedintotheNcoI(blunt-ended)/SalIsitesofpA-CLA12NCO, by centrifugation and the recombinant protein was precipitated
yieldingpA-hymyc2.Toclonepartofthecodingregionofthepre- at 30% ammonium sulfate saturation. The precipitate was
dictedHydraCADgene,PCRwasperformedusingtheprimerpair collected by centrifugation, dissolved in buffer E (50 mM
5′-TCGGAATTCGCGGCATTATGTGGTGGAG-3′/5′-TCGG- NaH2PO4∕Na2HPO4 pH 7.2, 80 mM NaCl, 1 mM EDTA,
AATTCGTGGTTTCACTTTTGCTTGCTG-3′. The purified 1 mM DTT), and dialyzed against the same buffer overnight
534-bpPCRproductwasdirectlyusedforhybridization,andalso at4°C.Thesamplewasclarifiedbycentrifugationandthesuper-
insertedintotheEcoRIsiteofpUC19. natantwasloadedontoaMonoScationexchangecolumn(GE
Healthcare)equilibratedwithbufferE.Proteinswereelutedwith
Recombinant Protein Expression and Purification. To generate the alineargradientfrom0–0.5MNaClinthesamebufferataflow
prokaryotic expression vectors pET11d-hymax, pET11d-p16hy- rateof0.5 mL∕min.FractionscontainingtheHydraMycp16pro-
myc, and pET11d-p36hymyc, Hydra myc or max-specific coding teinwerepooledandconcentratedbyusinganAmiconUltracen-
regionswereamplifiedfromcDNAs.ThePCRsegmentsencom- trifugalfilterdevice(MWCO3,000)(Millipore).Theproteinwas
Hartletal. www.pnas.org/cgi/doi/10.1073/pnas.0911060107 1of9
then purified on a Superdex-75 gel filtration column (GE dra Myc p16 protein, CAD, and ECD gave b, c, y, and z-type
Healthcare) and stored as described above. The final yield of fragmentionsfromcleavageat113outof114interresiduesites
purifiedHydraMycp16wasapproximately1mgperliterofbac- (Fig.S6A),correspondingtoasequencecoverageof99.1%.For
teria culture. theHydraMaxprotein,fragmentionsfromcleavageat139outof
Chicken Max p14 protein was purified as described previ- 174 interresidue sites (Fig. S6B) gave a sequence coverage
ously (5) with minor modifications. Protein expression from of 79.9%.
the pET3d-p14max construct in the E. coli strain BL21 (DE3)
CodonPlus-RIL (Stratagene) was induced with 1 mM IPTG, Protein Analysis. The recombinant full-length 176-amino acid
and the bacterial culture was incubated for 5 h at 37°C. After HydraMaxprotein,the289-aminoacidHydraMycp36protein,
cell lysis, chicken Max p14 protein was precipitated by ammo- anda161-aminoacidpolypeptidecontainingtheamino-terminal
niumsulfateprecipitationintwostepsasdescribedaboveforHy- 140 residues from v-Myc and additional 21 residues from the
dra Max. The recombinant protein precipitating between 50% Gag-MycborderintheMC29Gag-Mychybridprotein(2)were
and60%ammoniumsulfatesaturationwaspurifiedbycationex- purifiedandusedtogeneratetherabbitpolyclonalantiseraα-hy
changechromatographyonaMonoScolumn(GEHealthcare), Max,α-hyMyc,andα-Myc-N,resp.,asdescribedpreviously(8).
followedbygelfiltrationchromatographyonaSuperdex-75col- Theantiserumα-Myc-Cdirectedagainsta105-aminoacidrecom-
umn (GE Healthcare). The final yield of purified chicken Max binant protein encompassing the carboxyl-terminal 103 amino
p14proteinwasapproximately1mgperliterofbacteriaculture. acidresiduesofv-MycandtwoadditionalresiduesðM;VÞatthe
aminoterminushasbeendescribed(8).Invitrotranslation,im-
MassSpectrometry(MS)ofHydraMycandMaxRecombinantProteins. munoprecipitationofL-½35S(cid:2)methionine-labeledproteins,immu-
Proteins were desalted by using Vivaspin 500 PES centrifugal noblotting, and SDS/PAGE were done as described (3, 4). For
concentrators (MWCO 5,000) (Sartorius). Centrifugal concen- metaboliclabelingofHydraproteins,20animalswereincubated
tration and addition of 100 mM ammonium acetate in H2O for3hat18°Cin1mLofHydrasolution(1mMCaCl2,1mM
(18MΩ)tothesupernatantwasrepeatedfivetimes,followedby TrisHClpH7.5,0.1mMMgCl2,0.1mMKCl,1mMNaHCO3)
sixcyclesofconcentrationanddilutionwithH2O(18MΩ).Final supplemented with 20 μM reduced glutathione, and 200 μCi of
protein concentration of the electrospray ionization (ESI) solu- L-½35S(cid:2)methionine (1;000Ci∕mmol). The polyps were washed
tion (H2O∶MeOH 1∶1, 1% vol∕vol acetic acid) was 1 μM. ESI three times in Hydra solution. Lysis was performed in 0.3 mL
MS using a 7 Tesla Fourier transform ion cyclotron resonance of a buffer containing 10 mM sodium phosphate pH 7.2, 0.5%
(FT-ICR) instrument (Bruker) gave mass values for the most (wt∕vol) SDS, 2μg∕mL aprotinin, and the lysate was boiled
abundant isotopic peak of 13;613.364(cid:1)0.016Da for Hydra for5min.Thecooledextractwasdilutedwith1.2mLofabuffer
Myc p16 (theoretical mass without initiating methionine: containing 10 mM sodium phosphate pH 7.2, 187.5 mM NaCl,
13,613.357Da),and20;102.074(cid:1)0.010DaforHydraMax(the- 1.25%(vol∕vol)IgepalCA630,1.25%(wt∕vol)sodiumdeoxycho-
oreticalmasswithoutinitiatingmethionine:20,102.071Da)with late,2 μg∕mLaprotinin,andthesolutionwasclarifiedbycentri-
internal calibration (error<1 ppm) using polyethylene glycol fugationat20;000 × gfor1hat4°C.Thesupernatantwasused
1000ascalibrant.TheESImassspectraofproteinwithoutinter- forimmunoprecipiationanalysisasdescribed(3).Cellextractsfor
nal calibrant (Fig. S5) show high protein purity; ions of lower immunoblotting were prepared from 30 animals by lysis for
abundance correspond to protein adducts from residual phos- 20 min on ice in 1.2 mL of a buffer containing 20 mM Tris
phate buffer. HCl pH 8.0, 200 mM LiCl, 0.5% (vol∕vol) Igepal CA-630, 1
HydraMycandMaxproteinsequenceswereanalyzedintop- mM EDTA, 2 μg∕mL aprotinin, 1 μg∕mL leupeptin, and 1 μg/
downMSexperiments(6,7)usingcollisionallyactivateddissocia- mLpepstatinA.Thelysatewasclarifiedasabove,andthesuper-
tion(CAD)andelectroncapturedissociation(ECD).FortheHy- natant was used for immunoblot analysis.
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toproteinswithmassesgreaterthan200kilodaltons.Science314:109–112. 18:691–699.
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Fig.S1. StructuralrelationshipofHydraMyc1andMyc2proteins.(A)Alignmentofaminoacidsequencesofhuman(hu)c-MycandHydra(hy)Myc1andMyc2
proteins(GenBankaccessionnos.:huc-Myc,NP_002458;hyMyc1,GQ856263;hyMyc2,GU199337),usingtheClustalWalgorithmwithadditionalmanual
adjustments. Identical and BLOSUM62 matrix-defined similar residues are shaded in Blue, gaps are indicated by Dashes. Annotations of Myc boxes,
bHLH-Zipregions,andpositionscorrespondingtoexon-exonjunctionsareasinFig.1.ConservedphosphorylationsitesinMBI(9)andtheprimarynuclear
localizationsignal(10)arehighlightedbyGreenandOrangeBoxes,resp.(B)Nucleotideandaminoacidsequencesaroundtheexon-exonjunctions.(C)Overall
andsegment-specificsequenceidentitiesderivedfrompairwisealignmentsoftheproteinsindicated.(D)TopographyofHydramagnipapillatagenomicloci
(accession nos. NW_002158519, NW_002159544) containing the Hydra myc1 and myc2 genes. Adjacent genes encoding Hydra proteins similar to CAD
(carbamoyl-phosphatesynthetase/aspartatetranscarbamoylase/dihydroorotase),E2F-7(E2Ftranscriptionfactor7),andCHDH(cholinedehydrogenase)are
alsoshown.GeneorientationsareindicatedbyArrows.
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Fig.S2. Template-basedmodelingofthebHLH-ZipregionsofHydraMyc1andMax.(A)HomologymodelofaheterodimerformedbyMyc1(Red,residues
P220–Q307)andMax(Blue,residuesD30–A112)incomplexwiththedouble-strandeddeoxyoligonucleotide5′-CGAGTAGCACGTGCTACTC-3′(E-boxunder-
lined).Residueschangedincomparisontothehumanproteinsequencesareshowninbrightercolors.(B)HomologymodeloftheHydraMyc1/Maxhetero-
dimerasinA,withresiduescoloredaccordingtotheirBLOSUM62score(11):conservedandevolutionarysimilarresiduesareshowninGreen,andincreasing
evolutionarydissimilarityisindicatedbyspectralchangesviaYellowtoRed.ThemodelswerebuiltinMOE(MolecularOperatingEnvironment2008.10;Che-
micalComputingGroup,Inc.)usingthestructure(PDBcode1NKP)ofthehumanMyc/Max/DNAcomplex(12)asthetemplatewiththehighestsequence
identity(Fig.S1C).ThemodelwasbuiltinpresenceoftheDNAtorefinecontacts.ThestructurewasoptimizedbyusingtheAMBER99-forcefield(13)with
reactionfieldsolvationmodel(14)yieldingafinalstructurewitharmsgradientbelow1.0kcal∕ðmolÅÞ.ThermsdforthematchedCαatomsoftargetand
templateis0.255Å.PicturesweregeneratedusingPyMOL(15).
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