www.sciencemag.org/content/352/6293/1576/suppl/DC1

Supplementary Materials for

Identification of an NKX3.1-G9a-UTY transcriptional regulatory network that controls prostate differentiation

Aditya Dutta, Clémentine Le Magnen, Antonina Mitrofanova, Xuesong Ouyang, Andrea Califano, Cory Abate-Shen*

*Corresponding author. Email: cabateshen@columbia.edu

Published 24 June 2016, Science 352, 1576 (2016) DOI: 10.1126/science.aad9512

This PDF file includes:

Materials and Methods Supplementary Text Figs. S1 to S11 Tables S1 to S6 References

Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/352/6293/1576/DC1)

Databases S1 to S4

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Materials and Methods

DescriptionoflentiviralvectorsAllproceduresusing lentivirusesweredoneaccording toapprovedproceduresas

specifiedbytheOfficeofEnvironmentalHealthandSafetyatColumbiaUniversityMedicalCenter. For expression of exogenous genes,we used the pTRIPZ vector (Dharmacon, GEHealthcareLifeSciences),whichwasengineeredtointroduceeitherIRES-RFPorIRES-GFPbetweenuniqueEcoRIandMluIsites(hereafterreferredtoas“modifiedpTRIPZ”).cDNAsequencesencodinggenesofinterest(i.e.,NKX3.1andothers)wereclonedintouniqueAgeIandEcoRIsitesinthemodifiedpTRIPZplasmid;theserestrictionsiteswereintroducedinthecDNAsbyPCRamplificationusingprimersthatalsocontainedsequencestointroduceFLAG and/or HA epitope tags at the 5-prime end. The pTRIPZ plasmid also contains atetracycline response element (TRE) such that gene expression can be regulated in adoxycycline-dependent manner. Gene knock-down studies were done using pGIPZlentiviruses (Dharmacon, GE Healthcare Life Sciences), which constitutively express therelevantshRNAmir(mircoRNA-adaptedshRNA,hereafterreferredtoasshRNA)aslistedinTableS5.AminimumoftwoindependentshRNAwereusedforeachgene;dataareshownforoneof them.Lentivirusesweregeneratedusingsecondgenerationpackagingvectors,psPAX2 and pMD2.G (Addgene), in HEK-293T cells (ATCC), and concentrated using theLenti-XConcentratorreagent(Clonetech)accordingtothemanufacturer.

AnalysesofNkx3.1germlinemutantmiceAllexperimentsusinganimalswereperformedaccordingtoprotocolsapprovedby

theInstitutionalAnimalCareandUseCommittee(IACUC)atColumbiaUniversityMedicalCenter.TheNkx3.1germlinemutantmicehavebeendescribedpreviously(3).Expressionprofilingof anteriorprostate and seminal vesicle from15monthNkx3.1mutantorwild-typemice was done on an Affymetrix platform (Mu74AV2) as described previously (6).Geneexpressionprofileswereanalyzedasin(29).DifferentiallyexpressedgenesfromthemouseseminalvesiclearelistedinDatasetS2.

Forexpressionprofilingofanteriorprostatefrom4-monthmice,RNAwaspreparedusing MagMAX-96 Total RNA Isolation Kit (Life Technologies, Grand Island, NY). RNAsequencinganalysiswasdoneat the JPSulzbergerColumbiaGenomeCenteratColumbiaUniversity Medical Center. A TruSeq RNA Sample Prep Kit v2 (Illumina) was used forlibrarypreparation followedby sequencing (30million reads, singleend)onan IlluminaHiSeq 2500. RNAseq data raw counts were normalized and the variance was stabilizedusingDESeqpackage(Bioconductor) inR-systemv3.1.1(TheRFoundation forStatisticalComputing,ISBN3-900051-07-0).TherawandnormalizeddatafilesaredepositedinGeneExpression Omnibus (GEO) (GSE81440). Differentially expressed genes are listed inDatasetS1.

For histological and immunofluorescence analyses, anterior prostatewas fixed in10%formalinandembedded inparaffin. Immunofluorescencestainingwasdoneusing3µmparaffinsectionsandanalyzedusingaLeicaTCSSP5confocalmicroscopeasdescribedpreviously(4,30).DetectionofmouseNkx3.1wasenhancedusingtyramideamplification(Perkin Elmer) with horseradish peroxidase (HRP)-conjugated secondary antibody(Invitrogen), followed by incubation with tyramide 488 as described (4). Details of all

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primaryandsecondaryantibodiesusedinthisstudyareprovidedinTableS6.Allantiserawerepurchased commerciallywith the exceptionof the antisera against Svp2,whichweproducedinrabbitsagainstabacterially-expressedrecombinantprotein.Quantificationofstainingwas done using 5-10 independent sections (as indicated) from4-5 independentmice as done previously (4). Quantitative Real-time PCR (qRT-PCR) analysis wasperformed using total RNA isolated from anterior prostate using the QuantiTect SYBRGreenPCRkit(Qiagen)asdescribedpreviously(30).ThesequencesofallprimersusedinthisstudyareprovidedinTableS5.

AnalysesofRWPE1humanprostatecellsRWPE1cells(31)wereobtainedfromATCCandgrowninKeratinocyte-serumfree

media (K-SFM; Gibco, Life technologies). Sub-confluent cells were infected with pTRIPZlentiviruses expressing exogenous genes (or control) for 2 consecutive days at amultiplicity of infection (MOI) of approximately5, followedby selectionwithpuromycin(0.5μg/ml))forupto4days.Whereindicated,cellsweresubsequentlyinfectedwithpGIPZlentiviruses expressing the relevant shRNA (or control) using a similar procedure.Exogenous gene expressionwas inducedwith doxycycline (0.5 μg/ml).Where indicated,infectedcellswereenrichedbyFACSsortingonaBD-FACSAriacellsorter(BDBiosciences)to isolate those expressing RFP (561nm excitation, 582nm emission) and/or GFP (488excitation, 530nm emission). For expression profiling, RNA was prepared from infectedcellsandRNA-sequencinganalysiswasperformedasdescribedabove.Rawandnormalizeddata files are deposited in Gene Expression Omnibus (GEO) (GSE81440). DifferentiallyexpressedgenesarelistedinDatasetS4.

Forwesternblotanalysis, totalproteinextractswerepreparedby lysisof infectedcellswith1Xradioimmunoprecipitationassay(RIPA)buffer(0.1%SDS,1.0%deoxycolate-sodium salt, 1.0% Triton X-100, 0.15 M NaCl, 10 μmol/L Tris-HCl (pH 7.5), 1 mmol/LEDTA)with fresh 1% protease inhibitor (#1697498; Roche Basel) and 1% phosphataseinhibitor(#P2850;Sigma-Aldrich).Proteinlysates(20µgperlane)wereresolvedbySDSpage,followedbyimmunoblottingwiththeappropriateprimaryandsecondaryantibodies,andvisualizedusinganECLPlusWesternBlottingDetectionKit(GEHealthcare/AmershamBiosciences).DetailsofprimaryandsecondaryantibodiesusedinthisstudyareprovidedinTableS6.

Nuclearextractswerepreparedbylysisofcellsinhypotonicbuffer(20mMHEPES(pH 7.4), 5 mM NaCl, 1 mM EDTA (pH 8.0), 10 mM MgCl2, 1 mM DTT with fresh 1%protease inhibitor (#1697498; Roche Basel) and 1% phosphatase inhibitor (#P2850;Sigma-Aldrich). Nuclei were collected by centrifugation and proteins extracted withnuclearextractionbuffer(20mMHEPES(pH7.4),0.45MNaCl,0.2mMEDTA(pH8.0),25%glycerol,0.5mMDTT,0.5mMPMSFwith fresh1%protease inhibitor(#1697498;RocheBasel)and1%phosphataseinhibitor(#P2850;Sigma-Aldrich))followedbycentrifugation.Gel shift assays were done using nuclear extracts (5 μg) as described (32) with a DNAprobe corresponding to a previously describedNKX3.1 binding site containing themotif(TAAGTA)(33,34).

For identification of NKX3.1-interacting proteins by mass spectrometry, nuclearextractsfromcellsexpressingtheFlag-HA-taggedNKX3.1weresubjectedtotwosuccessiveroundsofimmunoprecipitation,firstwithAnti-Flag(M2)agarose(Sigma-Aldrich,A-2220)

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and eluted with a Flag peptide (Sigma-Aldrich, F3290), and then with Anti-HA (HA-7)agarose (Sigma-Aldrich, A2095) and eluted with HA peptide (Sigma-Aldrich, I2149).Immunoprecipitated protein complexes were resolved by SDS PAGE gel electrophoresisand visualized by silver staining (for detection of protein bands) or Coomassie blue (formassspectrometricanalysis).

Mass spectrometric analysiswas done at the Proteomics Shared Resource at theHerbert IrvingComprehensiveCancerCenter,ColumbiaUniversityMedicalCenter. In-geldigestionwascarriedoutonrehydrateddriedgelpiecesusingTrypsinGold(Promega)in50mMammoniumbicarbonateovernightat37°C.Peptideswereextractedwithamixtureofacetonitrile(ACN)andformicacid(FA)andLC-MS/MSanalysiswasperformedusingaThermoFusionTribridmassspectrometerequippedwithanEASY-Spraysource(ThermoScientific).Massspectrometer-scanningfunctionsandHPLCgradientswerecontrolledbyan Xcalibur data system (Thermo Finnigan, San Jose, CA). Tandem mass spectra weresearched against a Bsubilits168 human protein database using the ProteomeDiscoverer1.4(ThermoFinnigan,SanJose,CA),whichusesaprobability-basedscoringsystemtoratethe relevance of the best matches found by the SEQUEST algorithm. For validation ofinteracting proteins, RWPE1 cells expressing Flag-HA-taggedNKX3.1were lysed inRIPAbuffer (asabove),pre-clearedusingProteinA/G, followedby immunoprecipitationusingAnti-Flag (M2) agarose (Sigma, A-2220). Eluted proteins were subject to western blotanalyses.

TissueandcellrecombinationassaysTissue recombination assays were done using adult mouse epithelium and rat

embryonic urogenitalmesenchyme as previously described (3,4,35). Briefly, embryonicurogenital sinus mesenchyme was isolated from embryonic day 18.0 rat embryos, anddissociated cell suspensions were obtained following manual dissection and a series ofenzymatic digestions as described (4, 35). For isolation of the mouse epithelial cells,prostate (all lobes combined) or seminal vesicle was isolated from 8-10week oldmalemiceandepithelialcellswereisolatedfollowingdissociationbymanualdissectionfollowedbyaseriesofenzymaticdigestionsasdescribed(4,35).Thedissociatedmouseepithelialcellswereinfectedusingthespininfectionmethod.Briefly,500,000cellsin1mlofmedia(DMEM+10%FBS+1%PenStrep)supplementedwith8μg/mlpolybrenewereinfectedwith lentiviruses (MOIof15) inacentrifugepreheated to32°Cat1500g for60minutes.Cells were resuspended by pipetting, transferred to a 1.5 ml tube and recentrifuged asabove. For recombinantsmadewithhumanRWPE1 cells as a source of epithelium, cellswere infected with indicated lentiviruses followed by FACS sorting to enrich for cellsexpressingtherelevantlentivirusesasdescribedabove.

Tissuerecombinantsweremadebymixingthedissociatedmesenchymalcellswiththeepithelialcells(mouseorhuman;250,000totalcells)inaratioof3:1(mesenchymaltoepithelial cells) in rat collagen(BDBiosciences) insettingsolution (10XEarle’sBalancedSalt Solution (Life Technologies), 0.2MNaHCO3, 50mMNaOH). The recombinantswereculturedovernight inDMEMmediaplus10%FBS(formouserecombinants)or inK-SFMmedia (for RWPE1 recombinants) supplementedwith 10-7M dihydrotestosterone (DHT,Sigma-Aldrich) and 1x Rock Inhibitor (Stem Cell Technologies). On the following day,recombinants were grafted under the kidney capsule of 6-8 week old male nude mice

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(Taconic); the mice were implanted with a testosterone pellet (90 day release, 12.5mg/pellet, Innovative Research of America). The grafts were grown for 90 days duringwhich time the nude host mice were fed doxycycline in drinking water to maintainexogenous gene expression. At the conclusion, host mice were sacrificed and the renalgrafts collected for histological and immunochemical analyses, as described above. Forexpressionprofiling,RNAwasprepared from the recovered tissue recombinants (mouseonly) and RNA-sequencing analysis was performed as described above. Raw andnormalized data files are deposited in Gene Expression Omnibus (GEO) (GSE81440).DifferentiallyexpressedgenesarelistedinDatasetS3.

AnalysisofNKX3.1targetgenesThe NKX3.1 target genes studied herein were selected from those reported

previouslyformouseprostate(36)thatwerealsofoundtobetargetgenesforNKX3.1inhumancells(RWPE1).GeneswereprioritizedbyfocusingonGOannotationsforprocessesassociated with development or differentiation, namely “embryonic organ development”,“chromatinmodification”,“multicellularorganismaldevelopment”,“Wntsignalingpathway”,“endothelial cell proliferation”, “post-embryonic development”, “anterior/posterior patternspecification”, “determination of left/right symmetry”, and “pattern specification process”.ConfirmationofNKX3.1bindingtothesesiteswasdonebychromatinimmunoprecipitationfollowedbyrealtimePCR(ChIP-qPCRanalysis)asdescribed(37).Theexpressionlevelsoftarget genes in RWPE1 cells or NKX3.1-expressing RWPE1 cells was performed asdescribed(30).ChIPdataareexpressedasfoldenrichmentofNKX3.1orG9Abinding,andnormalized to input; IgG controls were used for all pull down experiments. PrimersequencesandantibodiesaredescribedinTablesS5andS6,respectively.

StatisticalanalysisStatistical analyses were conducted using a 2-tailed t-test, χ2 test, or Fisher's exact test as

appropriate. Differential gene expression was estimated with a 2-tailed student t-test. Gene set enrichment analysis (GSEA) (38) was performed as described previously (30); p-value<0.05 was considered significant. GraphPad Prism software (Version 5.0) and R-system v3.1.1 (The R Foundation for Statistical Computing, ISBN 3-900051-07-0) were used for all statistical analyses and data visualization.

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Supplementary Text

Loss-of-function of Nkx3.1 in aged and young mice leads to impaired prostatedifferentiation

ToinvestigatemolecularchangesfromprostatesofNkx3.1wild-type(Nkx3.1+/+)andNkx3.1mutant(Nkx3.1—/—)mice,wecomparedexpressionprofilesfromyoungadults(i.e.,~2-4months of age), which display differentiation defects, with agedmice (i.e.,~12-15monthsofage),whichdisplayaprominentPINphenotype(3,29)(Figs.1,S1,andS2). Inparticular, in prostates from both young and aged Nkx3.1—/— versus Nkx3.1+/+ mice,differentially-expressed genes include down-regulation of those associatedwith prostatedifferentiation and luminal epithelial cell differentiation, and up-regulation of thoseassociated with basal cells (Figs. 1A,B, S1B, S2A,B; Dataset S1 and (6)). In addition, weobserved up-regulation of genes that are not normally expressed in prostate, includingthoseexpressed in theseminalvesicle(Figs.1A,B,S1B,S2A,B;DatasetsS1andS2, (7,39,40).AmonggenesthatdistinguishprostateandseminalvesicleinbothmiceandhumansisFOXA1, which has been shown to play a role in prostate differentiation and cancerpredisposition(41).

Expressionofaprostate-specificmarker,probasin,wassignificantlyreducedinyoungandagedNkx3.1—/—prostatesatboththeproteinandmRNAlevels;conversely,expressionof the seminal vesicle marker, Svp2, was significantly up-regulated in the Nkx3.1—/—

prostate, while it was rare/negligible in Nkx3.1+/+prostate (Figs. S1C,D, S2C; Table S1).Although Svp2-postive cells were present in both young and agedNkx3.1—/—prostates,theywereapproximately4-timesmoreprevalentintheagedmice,wheretheyrepresented8% (N=683/8727)of the total epithelial cells (Figs. S1E;Tables S2,3).Notably, all of theSvp2-positivecellsexaminedinboththeyoungandagedprostates(N=782total)expresscytokeratin8 (Ck8), consistentwith their relationship toNkx3.1-expressing luminal cells(Fig. S1E; Tables S2,3). These Svp2-positive cells retained the ability to proliferate,particularly in the young mice in which approximately 13% of the Svp2-positive cells(N=27/213) express Ki67, a marker of cellular proliferation (Fig. S1E; Tables S2,3).Interestingly,theseSvp2-positivecells,particularlyintheagedmice,frequentlyco-expresscytoplasmic p21 (43% of the Svp2-positive cells; N=277/648) (Fig. S1E; Tables S2,3),which has oncogenic functions (42), suggesting that these Svp2-positive cells mayrepresentpre-neoplasticcells.

InductionofprostatedifferentiationbyNKX3.1ismediatedbyUTY(KDM6c)WeinvestigatedbonafidetargetgenesofNKX3.1,whichwedefineasthosethatare

bothdirectlyboundbyNKX3.1at theirpromoter/enhancerregions inprostatecells,andwhoseexpressionisregulatedbyNKX3.1.Amongpotentialcandidates(36),wefocusedonthose that have functions predicted for differentiation, and that are conserved betweenmice and humans (Fig. S8). Although NKX3.1 has been reported to function as atranscriptional repressor in transient transfection assays (43), its bona fidetarget genesinclude those that are both activated and repressed by NKX3.1 in mouse and humanprostate (Fig. S8B,C). Someof these targetgenes, includingbothactivatedand repressedones,werealsoboundbyG9a(Fig.4A,S9).Notably,althoughbindingbyG9atothesegeneswasdependentonNKX3.1,bindingofNKX3.1wasnotdependentonG9a(Fig.4A,S9).

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Supplementary Figures Figure S1

Fig. S1. Loss-of-function of Nkx3.1 leads to impaired prostate differentiation in aged mice (A) Diagram of the male urogenital system in adult mice highlighting the embryological origins of the tissues. Tissues in dark grey (i.e., prostate and others) are derived from the urogenital sinus, which is of endodermal origin, whereas those in light grey (i.e., seminal vesicle and others) are derived from the Wolffian duct, which is of mesodermal origin. Reprinted from (2, 3). (B) Heat map representations of differentially expressed genes comparing mouse (top; 15 months of age) or human (bottom) normal (i.e., wild-type) prostate versus seminal vesicle. The mouse heatmap (top) represents a subset of differentially expressed genes listed in Dataset S2 (p < 0.0001), and the human heatmap (bottom) represents a subset of differentially expressed genes published in (44). (C) Histological analyses of anterior prostate from Nkx3.1+/+ or Nkx3.1-/- mice at 12 months of age. Representative H&E stained sections or confocal images showing co-staining of Probasin or Svp2 with E-cadherin and DAPI as indicated. Scale bars represent 50 µm

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in the H&E images and 20 µm in the immunofluorescence images. (D) (Left) Real-time PCR showing relative mRNA levels for probasin or Svp2(Svs4) in anterior prostate. (Right) Quantification of Svp2-positive cells in anterior prostate. (E) (Left) Confocal images from representative sections of anterior prostate from Nkx3.1-/- mice at 12 months of age showing co-staining of Svp2-positive cells with Ck8, a marker of luminal cells, Ki67, a marker of cellular proliferation, and cytoplasmic p21, a marker of neoplasia. (Right) Quantification of co-expression of these markers with Svp2. The graphs in D and E show quantification of the data provided in Table S1-3.

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Figure S2

Fig. S2. Loss-of-function of Nkx3.1 leads to impaired prostate epithelial differentiation in young mice

(A)HeatmaprepresentationsofdifferentiallyexpressedgenesfromtheanteriorprostateofNkx3.1+/+versusNkx3.1-/-miceat4monthsofage.TheheatmaprepresentsasubsetofdifferentiallyexpressedgeneslistedinDatasetS1(p<0.0001).(B)GSEAperformedusingas a query gene set the differentially-expressed genes from the mouse seminal vesicleversusprostate(wild-type)(fromDatasetS2(p<0.0001))comparedwithareferencegenesignatureofNkx3.1-/-versusNkx3.1+/+mouseprostate(4monthsofage)(fromDatasetS1).(C)HistologicalanalysesofanteriorprostatefromNkx3.1+/+orNkx3.1-/-miceat4monthsof age. Shown are representative H&E stained sections or immunofluorescence imagesshowingco-stainingofProbasinorSvp2withE-cadherinandDAPI,asindicated. Scalebarsrepresent 50 µm in the H&E images and 20 µm in the immunofluorescence images.QuantificationoftheseimmunofluorescenceisprovidedinTablesS1-3.

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Figure S3

Fig.S3.Controlexperimentsfortissuerecombinationstudies(A) Whole mount images showing low and high power views of control recombinantsgenerated using only rat urogenital mesenchyme (left) or mouse prostate epithelium(right).Therulershowscmscale.Summaryofdata isprovided inTableS4. (B)Confocalimages using DAPI staining of tissue recombinants to distinguish ratmesenchymal cellsandmouseepithelialcells,asshown.

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Figure S4

Fig. S4. Analyses of expression profiling of mouse tissue recombinants

These analyses compare tissue recombinants made from mouse prostate epithelium (PE) or mouse seminal vesicle epithelium (SVE), which were infected with a control lentivirus or with a lentivirus expressing a mouse Nkx3.1 (+Nkx3.1). The epithelial tissues were recombined in vitro with mesenchyme from rat embryonic urogenital sinus and grown in host mice for 3 months. Following sacrifice, RNA was made from the tissue recombinants and the samples were

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subjected to RNASeq analysis (see materials and methods). A complete list of differentially expressed genes is provided in Dataset S3. (A) Real-time PCR showing relative mRNA levels for the indicated genes in the indicated tissue recombinants. Experiments were done using 4 samples per group and performed in triplicate; values were normalized to GAPDH. The p-values were calculated using a 2-tailed t-test. (B) Principal component analysis showing a comparison of the gene expression profiles from the PE, SVE, and SVE+Nkx3.1 tissue recombinants. (C) GSEA performed using as a query gene set the differentially-expressed genes from the mouse seminal vesicle versus prostate (wild-type) (from Dataset S2) compared with a reference gene signature of SVE and SVE+Nkx3.1 (left) or SVE and PE (right) (from Dataset S3).

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Figure S5

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Fig.S5.PhenotypeofhumanRWPE1cellsShown are wholemounts and H&E images of the tissue recombinant phenotypesmadefrom human RPWE1 cells, which had been infected with a control lentivirus or with alentivirusexpressinghumanNKX3.1withorwithouttheindicatedshRNA.Epithelialcellswere recombined in vitro with mesenchyme from rat embryonic urogenital sinus andgrowninhostmicefor3months(asinmaterialsandmethods).Representativeimagesareshown;asummaryofalltissuerecombinantsisprovidedinTableS4.Scalebarsrepresent500µm,100µm,and50µm,intheupper,middle,andlowerpanels,respectively.

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Figure S6

Fig.S6.Massspectrometryanalysis(A)DiagramoftheexperimentalstrategyforFigs.3A,B,S6andS7.HumanRWPE1prostateepithelialcellswereinfectedwitha lentivirusexpressinghumanNKX3.1taggedattheN-terminus with Flag and HA epitopes (Flag-HA-NKX3.1), or with a series of truncatedNKX3.1 proteins, or the control vector (see Fig. S7). Nuclear extracts from the Flag-HA-NKX3.1-expressingRWPE1 cells (or the control)were subjected to immunoprecipitationfollowed by mass spectrometry to identify the associated proteins (Fig. 3A, S6B,C).Alternatively, extracts were analyzed by immunoprecipitation or gel retardation assays(Fig.3B,S7).(B)SilverstainshowingtheresultsofimmunoprecitationofFlag-HA-NKX3.1to identify endogenous interacting proteins. *NS, non-specific protein bands. Relativepeptide counts are indicated for NKX3.1 (bait) and other interacting partners. (C) Tabledescribingtheproteinsidentifiedfrommassspectrometryanalysis.

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Figure S7

Fig.S7:NKX3.1interactswithG9ahistonemethyltransferaseviathehomeodomain(A)Diagramshowingrelevantproteindomainspresent in theNKX3.1truncatedproteinsas well as a summary of their DNA binding properties and protein interactions. (+++)indicates strong interaction, (-) indicates no detectable interaction. (B) Western blotanalysis following immunoprecipitation of Flag-HA-NKX3.1, or the indicated truncatedproteins, fromRWPE1cellsconfirmingassociationwithG9aand itspartnerGLP,butnotwithtwounrelatedhistonemethyltransferases,EZH2andSUV39H1.Inputshows5%ofthetotal inputprotein, and immunoprecipitation (IP) showsproteins recovered following IPusinganantibodytoFlag.Actinisacontrolforproteinloading.(C)GelretardationanalysisdoneusingnuclearextractsfromRWPE1cellsexpressingthecontrolvector,NKX3.1,orthevarious truncated proteins as shown. The arrow indicates the free DNA probe. Allimmunoprecipitation and gel shift experiments were performed with 3 independentbiologicalreplicates;representativedataareshown.

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Figure S8

Fig. S8: Gene expression and chromatin binding analyses of NKX3.1 target genes

(A) Table describing NKX3.1 target genes investigated in this study. These represent asubsetof thegenesdescribed in (36). (B)Real timePCRshowingexpressionanalysesofNKX3.1targetgenesinmouseprostate(left;comparingNkx3.1wild-typeandmutantmice)andhumanRWPE1prostatecells(right;comparingcellsexpressingexogenousNKX3.1orthe control vector). (C) ChIP-qPCR analyses showing the relative levels of NKX3.1 atNKX3.1binding sites at target genes inRWPE1 cells expressing exogenousNKX3.1. ChIPdataisexpressedasenrichmentofNKX3.1bindingrelativetoinput.Realtimeexpressionanalysiswasperformedwith4independentbiologicalreplicates.ChIP-qPCRanalysiswasperformedwith3 independentbiologicalreplicates.Statisticalanalyseswasdoneusinga2-tailedt-test;dataareindicatedasmean+/-SD.

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Figure S9

Fig.S9.BindingofG9atoNKX3.1targetgenes(A) Diagram of the experimental strategy for Figs. 4A and S9. Human RWPE1 prostateepithelialcellswereinfectedwithalentivirusexpressinghumanNKX3.1(withRFP)orthecorrespondingcontrolvector, followedbyinfectionwithanshRNAforG9a(withGFP)orthe corresponding control vector. The cells were FACS sorted to enrich for those co-infectedwithbothplasmids(i.e.,expressingbothRFPandGFP)andanalyzedbyreal-timePCRorChIPanalysis.(B,C)ChIP-qPCRanalysesshowingtherelativelevelsofNKX3.1(B)orG9a(C)atNKX3.1bindingsitesinRWPE1cellswithcontrolvector(control),expressingexogenous NKX3.1, exogenous G9a, and/or with shG9a, as indicated. ChIP data areexpressed as enrichment ofNKX3.1orG9abinding relative to input. ChIP-qPCRanalysiswasperformedwith3independentbiologicalreplicates.Statisticalanalysiswasdoneusinga2-tailedt-test;dataareindicatedasmean+/-SD.

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Figure S10

Fig. S10. Analyses of expression profiling of mouse tissue recombinants

These analyses compare expression profiles of tissue recombinants made from mouse prostate epithelium (PE) or seminal vesicle epithelium (SVE) infected with a control lentivirus or with a lentivirus expressing a mouse Nkx3.1 (+Nkx3.1) (as in Fig. S4) compared with those generated using SVE expressing an shUty (SVE+shUty) or with SVE expressing an shUty as well as exogenous Nkx3.1 (SVE+Nkx3.1+shUty). A complete list of differentially expressed genes is provided in Dataset S3. (A) Principal component analysis showing a comparison of PE, SVE, and SVE+Nkx3.1 with SVE+shUTY and SVE+Nkx3.1+shUTYtissue recombinants. (B) GSEA performed using as a query gene set differentially expressed genes from the mouse seminal vesicle versus prostate (wild-type) (from Dataset S2) compared with a reference gene signature of SVE+Nkx3.1+shUty and SVE+Nkx3.1.

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Figure S11

Fig.S11.Model(A)DiagramdepictingtheabilityforexogenousNkx3.1tore-specifymouseseminalvesicleepithelium to form prostate in vivo, which is abrogated following knock-down of Uty(shUty). In human RPWE1 cells, exogenous NKX3.1 promotes prostate growth in vivo,which isabrogated followingknock-downofG9a(shG9a)orUTY(shUTY). (B)Schematicdiagramshowing theexpressionofNKX3.1 in threescenariosand itsrelationship toG9aandUTY.Innon-prostatictissues,NKX3.1isnotexpressedandthereforedoesnotrecruitG9a to target genes, such asUTY. Scenario 2 shows normal prostate,whereinNKX3.1 isexpressed and recruits G9a to target genes such as UTY to promote their expression.Scenario 3 shows abnormal prostate,wherein reduced levels of NKX3.1 lead to reducedactivation of target genes such at UTY, which is likely associated with cancerpredisposition.

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Table S1: Quantification of SVP2-positive cells in mouse anterior prostate

Mouse # Age (months)

Total epithelial cells

Total SVP2+ cells

Percentage of SVP2+cells

Nkx3.1-/- 1 12 2869 178 6.2% 2 12 2847 324 11.8% 3 12 3011 181 6.0% Total 8727 683 8.0% +/- 3.3 4 4 3353 65 1.9% 5 4 2626 32 1.2% Total 5979 97 1.6%+/- 0.5 Nkx3.1+/+ 1 12 2104 0 0% 2 12 1942 0 0% Total 4046 0 0% 3 4 2478 0 0% 4 4 1565 0 0% Total 4043 0 0%

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Table S2: Cellular characteristics of SVP2 positive cells in the Nkx3.1—/— anterior prostate

Cytokeratin Staining Mouse # Age

(mths) Total

SVP2+ Total

SVP2+ CK8+

Percent SVP2+ CK8+

Total SVP2+ CK5+

Percent SVP2+ CK5+

1 12 194 193 99.5 1 0.5 2 12 219 219 100 0 0 3 12 141 141 100 1 0.7 Total 554 553 99.8 +/- 0.3 2 0.4 +/- 0.4 4 4 97 97 100 0 0 5 4 131 131 100 0 0 Total 228 228 100 +/- 0 0 0 p21 staining Mouse # Age

(mths) Total SVP+

Total SVP2+ cyto 21+

Percent SVP2+ cyto 21+

1 12 270 96 35.6 2 12 102 45 44.1 3 12 276 136 49.2 Total 648 277 43.0 +/- 6.9 4 4 54 12 22.2 5 4 135 41 30.4 Total 189 53 26.3 +/- 5.8 Ki67 (proliferation) Mouse # Age

(mths) Total

SVP2+ Total SVP2+

Ki67+ Percent SVP2+

Ki67+ 1 12 185 6 3.2 2 12 109 2 1.8 Total 294 8 2.5 +/- 1.0 3 4 35 7 20.0 4 4 116 11 9.5 5 4 62 9 14.5 Total 213 27 14.7 +/- 5.3

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Table S3: Cellular characteristics of SVP2 positive cells in Nkx3.1+/+ anterior prostate

Cytokeratin Staining Mouse # Age

(mths) Total

SVP2+ Total

SVP2+ CK8+

Percent SVP2+ CK8+

Total SVP2+ CK5+

Percent SVP2+ CK5+

1 12 0 0 0 0 0 2 12 0 0 0 0 0 Total 0 0 0 0 0 3 4 0 0 0 0 0 4 4 0 0 0 0 0 Total 0 0 0 0 0 p21 staining Mouse # Age

(mths) Total SVP+

Total SVP2+ cyto 21+

Percent SVP2+ cyto 21+

1 12 0 0 0 2 12 0 0 0 Total 0 0 0 3 4 0 0 0 4 4 0 0 0 Total 0 0 0 Ki67 (proliferation) Mouse # Age

(mths) Total

SVP2+ Total SVP2+ plus Ki67+

Percent SVP2+ plus Ki67+

1 12 0 0 0 2 12 0 0 0 Total 0 0 0 3 4 0 0 0 4 4 0 0 0 Total 0 0 0

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Table S4: Summary of cell and tissue recombination assays

Description Phenotypec # Analyzed

Control grafts

UGMa without epithelium No tissue growth 4

Mouse prostate epithelium (PE) without UGM No tissue growth 2

Mouse seminal vesicle epithelium (SVE) without UGM No tissue growth 2

Human RWPE1 cells alone without UGM No tissue growth 2

Grafts made with mouse epitheliumb

PE infected with control vector + UGM (Positive control for prostate)

Prostate phenotype 19

SVE infected with control vector + UGM (Positive control for seminal vesicle)

Seminal vesicle phenotype 20

SVE infected with Nkx3.1 + UGM Prostate phenotype 26

SVE infected with NKX3.1 (human) + UGM Prostate phenotype 4

SVE infected with Msx1 + UGM Seminal vesicle phenotype 3

SVE infected with shUty + UGM Seminal vesicle phenotype 8

SVE infected with Nkx3.1 + shUty + UGM Seminal vesicle phenotype 8

Grafts made with human epithelium (RWPE1 cells)

RWPE1 infected with control vector + UGM No tissue growth 12

RWPE1 infected with NKX3.1 + UGM Prostate phenotype 15

RWPE1 infected with NKX3.1(T164A) + UGM No tissue growth 7

RWPE1 infected with shG9A + UGM No tissue growth 9

RWPE1 infected with NKX3.1 plus shG9A + UGM No tissue growth 9

RWPE1 infected with shSUV39H1 + UGM No tissue growth 7

RWPE1 infected with NKX3.1 plus shSUV39H1 + UGM Prostate phenotype 9

RWPE1 infected with shUTY + UGM No tissue growth 8

RWPE1 infected with NKX3.1 plus shUTY + UGM No tissue growth 8

RWPE1 infected with shEDEM2 + UGM No tissue growth 4

RWPE1 infected with NKX3.1 plus shEDEM2 + UGM Prostate phenotype 4

a) UGMstandsforurogenitalsinusmesenchymeobtainedfromratembryosb) Mouseepitheliumobtainedfrom2-4montholdadultmice(wild-type)c) Prostate phenotype was assessed by the following criteria: (1) morphological appearance of

prostaticducts;(2)histologicalappearanceofprostate-likemorphology;(3)expressionofprostatemarkersincludingprobasinandFOXA1.

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Table S5: Description of primers and shRNA used in this study

Real Time qPCR Forward Reverse

Human genes

ASNS GGAAGACAGCCCCGATTTACT AGCACGAACTGTTGTAATGTCA

HDLBP GAGAGCGACCCTCCAACCTA CTCCAGGGGTACATGGAACAC

PRDX6 GTGACAGCTCGTGTGGTGTT CTGGGGTGGCAACCCTTTT

CTSB AGAGTTATGTTTACCGAGGACCT GATGCAGATCCGGTCAGAGA

NEDD4L GGACAATTTAGGCCGAACTTACT CTCCAAGTCTTCGCTGATGTG

UTRN CCATCCGTGAGGTAGAGACAC CTCGGGGACTCTCATGCTC

EDEM2 CTGGTCGGCAACCACATTGAT CATCGAAGCGGGTGTAGTTCC

UTY TTAGCCTGACAGTCGAGGAAA GTAGGGTCTTCGTTCTGGCG

IGF1R TCGACATCCGCAACGACTATC CCAGGGCGTAGTTGTAGAAGAG

HK2 TGCCACCAGACTAAACTAGACG CCCGTGCCCACAATGAGAC

ANGPT2 AACTTTCGGAAGAGCATGGAC CGAGTCATCGTATTCGAGCGG

GAPDH ACAACTTTGGTATCGTGGAAGG GCCATCACGCCACAGTTTC

Mouse genes

Asns GCAGTGTCTGAGTGCGATGAA TCTTATCGGCTGCATTCCAAAC

Hdlbp GGAAAGAGCGGAGCCAACAT CCCTCAATTCGGATCAGGTTAC

Prdx6 CGCCAGAGTTTGCCAAGAG TCCGTGGGTGTTTCACCATTG

Ctsb TCCTTGATCCTTCTTTCTTGCC ACAGTGCCACACAGCTTCTTC

Nedd4l CACGGGTGGTGAGGAATCC GCCGAGTCCAAGTTGTGGT

Utrn GTATGGGGACCTTGAAGCCAG ATCGAGCGTTTATCCATTTGGT

Edem2 ATGCCTTTCCGGCTACTCATC CCTTGACTCGCTCCCTGTAGT

Uty GAGGTTTTGTGGCATGGGAG TGCAGAAGATAACGAAGGAGCTA

Igf1r GTGGGGGCTCGTGTTTCTC GATCACCGTGCAGTTTTCCA

Hk2 TGATCGCCTGCTTATTCACGG AACCGCCTAGAAATCTCCAGA

Angpt2 CCTCGACTACGACGACTCAGT TCTGCACCACATTCTGTTGGA

Gapdh AGGTCGGTGTGAACGGATTTG TGTAGACCATGTAGTTGAGGTCA

Probasin AAGGCTCACCATTGAGAACCT CAGTTGGCACTTAGTCCCTTTC

Svp2 GTTCTGAGCCAGAGGTATTTGTT TCGAGTGCATCTTGAGAAAACC

Exogenous Nkx3.1 GATTACAAGGATGACGACGATAAGGGT GAAGGAGGTGAGCCGCTTG

Total Nkx3.1 ATGCTTAGGGTAGCGGAGC TGCGGATTGCCTGAGTGTC

Sva TGAACCCCGTAACTACACACT GGCATTGCTTCACGTTGTTTT

FoxA1 ATGAGAGCAACGACTGGAACA TCATGGAGTTCATAGAGCCCA

ChIP Forward Reverse

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ASNS CTGATGTAATTAGTAAAATACGATTTAAGA CCCCATCTCTAAAACAATAAAATC

HDLBP ATTCCGTTCCGCCCAGT ATTCCTTATGTCCCTCACTTTTATAAACTA

PRDX6 TCGCCATGCCCGGAGGTCTGCTTC GGCTGAGCGGCCGGGAGG

CTSB GAAGGTGGCGGGAGGGGG GAGAGCCTGCAGCCCAGCC

NEDD4L CCAAGTAGGCACTTTGACGTTTCCTTTT CACCCAGCTACCGTTTCTCTAGC

UTRN CTCGGGGCCTCAGTTTAGCTCCC ATCACTGGCCCAGCGCCC

EDEM2 CTTCCTTCTTTAACCCGGT ATGTTTTAATAATAACAGCTAGCATGTATT

UTY CGAGCTGTCCCTCTTACC AACACCACTTTTTTCTTACTTTCTCAATTG

IGF1R GCAAGGCAGTTTTTCATCTAA TGTCATTAAATTAATATTTGGGTTTGAATC

HK2 CTGGGCAACATATCGAGTCCCTGC AAAGGTTCAGGGAAGGAAGATGAAGGTCTG

ANGPT2 AGGAACTTTCATTGTACTTCAACATTAAAG GAATGCCTGCCCTCCAAC

Gel Shift Top strand Bottom strand

NKX3.1 GATCCATATATTAAGTATATATAGGATC GATCCTATATATACTTAATATATGGAT

shRNA Clone ID Mature Antisense

shG9a #1 V3LHS_364364 TGAGTTGAAGCGCAAACCA

shG9a #2 V2LHS_44409 TAAATTCCTGGAGCAATCG

shSUV39H1 #1 V2LHS_153686 TGTAATCAAAGGTGAGCTC

shSUV39H1 #2 V3LHS_403672 AGAGCAGGTAGGAGCAGGT

shUTY #1 V3LHS_380228 TAAACTAGACTTGTAGTCT

shUTY #2 V3LHS_380229 TCATTGTTAGCTTCTACCA

shEDEM2 #1 V2LHS_156353 TATCAAAGTCCACGCTGTC

shEDEM2 #2 V2LHS_156355 TATACTCTAGGAACATGGC

shUty #1 V3LMM_491456 ATCTGAACATGTTGATTCA

shUty #2 V2LMM_50864 TTATTAAGGCTGGAAATCG

shUty #3 V2LMM_49926 TAGAGAAAGTGTTTCTGTG

Scramble RHS4743 N/A

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Table S6: List of antibodies used in this study

Antigen Company Catalog # Type Use and dilution

IF Western ChIP IP AR Abcam ab133273 Rabbit mAb 1:100 1:1000

β-actin Cell Signaling 4970 Rabbit mAb 1:2000

Cytokeratin 5 Covance SIG-3475 Chicken pAb 1:1000

Covance PRB-160P Rabbit pAb 1:1000

Cytokeratin 8 Covance MMS-162P Mouse mAb 1:500 1:1000

E-cadherin BD Biosciences 610181 Mouse mAb 1:1000

Cell Signaling 3195 Rabbit mAb 1:500

Edem2 Sigma E1782 Rabbit pAb 1:1000

Ezh2 Millipore 07-689 Rabbit pAb 1:2000

Flag Sigma A2220 Mouse mAb 1:10 1:40

FoxA1 Abcam ab55178 Mouse mAb 1:100 1:1000

G9a MBL D141-3 Hamster mAb 1:1000

Abcam ab40542 Rabbit pAb 1:40

Glp MBL D220-3 Hamster mAb 1:1000

H3K9me2 Abcam ab1220 Mouse mAb 1:2000 1:40

H3K9me3 Abcam ab8898 Rabbit pAb 1:3000

H3K27me3 Millipore 07-449 Rabbit pAb 1:2000

HA Sigma A2095 Mouse mAb 1:40

HoxB13 Santa Cruz sc66923 Rabbit pAb 1:50 1:200

Ki67 eBiosciences 14-5698-82 Rat 1:500

NKX3.1 Abate-Shen Lab 67 Rabbit pAb 1:1000

Nkx3.1 Gift from C. Bieberich

0315 Rabbit pAb 1:1000

Abate-Shen Lab DE1 Rabbit pAb 1:2000 1:2000

p21 BD Biosciences 556431 Mouse mAb 1:10

P63 Santa Cruz sc-8431 Mouse mAb 1:500

Probasin Santa Cruz sc-17124 Goat pAb 1:500

PSA Dako M0750 Mouse mAb 1:500

Suv39h1 Millipore 07-550 Rabbit pAb 1:250

SVP2 Abate-Shen Lab -- Rabbit pAb 1:700

T TMPRSS2 Abcam ab92323 Rabbit mAb 1:1000

UTY Abcam ab85969 Rabbit pAb 1:500

Secondary antibodies used:

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Antibody Fluorochrome Company Catalog # Dilution

Goat anti-rabbit IgG Alexa Fluor® 488 Life Technologies A11008 1/1000

Goat anti-rabbit IgG Alexa Fluor® 555 Invitrogen A21428 1/1000

Goat anti-mouse IgG Alexa Fluor® 488 Life Technologies A11001 1/1000

Goat anti-mouse IgG Alexa Fluor® 555 Life Technologies A21424 1/1000

Goat anti-mouse IgG Alexa Fluor® 647 Life Technologies A21237 1/1000

Goat anti-chicken IgG Alexa Fluor® 488 Invitrogen A11039 1/1000

Donkey anti-goat IgG Alexa Fluor® 555 Invitrogen A21432 1/1000

Donkey anti-mouse IgG Alexa Fluor® 488 Invitrogen A21202 1/1000

Goat anti-rabbit IgG HRP None Invitrogen T30954 1/1000

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Additional Data table S1 (separate file)

DatasetS1:DifferentiallyexpressedgenescomparingNkx3.1wild-typeandmutantmiceat4months(p<0.0001)

DatasetS2:Differentiallyexpressedgenescomparingprostateandseminalvesicleat15months(p<0.001)

DatasetS3:Differentiallyexpressedgenescomparingmousetissuerecombinants(p<0.0001)

Dataset S4: Differentially expressed genes comparing control and NKX3.1-expressingRWPE1cells(p<0.0001)

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