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DOI: 10.1038/ncb2798

c

0 2 4 6Transcription factor activity

Pattern specification processTranscription regulator activity

Embryonic morphogenesis

Embryonic organ developmentDNA dependent regulation of transcription

Regulation of RNA metabolic processNeuron differentiation

DNA bindingChordate embryonic development

Embryonic development ending in birth*tchingtRNA metabolic process

Transcription factor complex

Sequence specific DNA bindingEmbryonic organ morphogenesis

-log(p-value)

Repressed Genes

0 2 4 6Extracellular matrix

Extracellular region partProteinaceous extracellular matrix

Extracellular regionPositive regulation of development

Calcium ion bindingRegulation of cell motion

Neuron developmentCell motion

Positive regulation of cell differentiationCell projection organization

Neuron differentiationRegulation of neurogenesis

Exocrine system developmentRegulation of nervous system development

-log(p-value)

Induced Genes

Figure-S1 (Surani)

100 101 102 103 1040

20

40

60

80

100

100 101 102 103 1040

20

40

60

80

100

LO

HI

Positive

EGFPBLIMP1-EGFP

24h 48h a b

24h LO 48h 24h LO 48hEGFP BLIMP1-EGFP

Prdm1Rhox9Gata2Igf2CompSpo11Gata6Snai3Wnt6Krt8Krt18Nanos3Olig1KitGadd45bCol18a1Dnmt3lIfitm1Rhox6Jmjd1aGli2Evx1Setdb2Chd1Jmjd5Hoxa2Fgf5Dnmt3bNanogWnt5bMycPolr1bChek2Etv1Taf7Chek1HopxHoxa5EomesEn1Wnt8aTcfap2aEn2Eras

−1 0 1Row Z−Score

InducedRepressed

Figure S1 Expression profiling of P19ECs with ectopic expression of BLIMP1. (a). Flow cytometric plot showing the fluorescence intensity of EGFP and BLIMP1-EGFP transfected cells. Gates employed for cell sorting are indicated. (b) Heat map showing differentially regulated genes

in P19ECs upon BLIMP1 expression. Each column represents a time-point assayed in triplicate. The colours indicate the z-score for differential expression. (c). Gene ontology analysis of the top 10% of genes repressed and induced upon BLIMP1 expression in P19ECs.

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0 1 2 3 4 5 6 7 8

lysosomeextracellular matrix

GO:0031982~vesicleGO:0001568~blood vessel development

GO:0001942~hair follicle developmentGO:0000904~cell morphogenesis …

GO:0051270~regula�on of cell mo�onGO:0006643~membrane lipid metabolic …

GO:0060284~regula�on of cell …GO:0042325~regula�on of …

GO:0048667~cell morphogenesis …GO:0016477~cell migra�on

GO:0001501~skeletal system developmentGO:0050770~regula�on of axonogenesis

GO:0043433~nega�ve regula�on of …GO:0007276~gamete genera�on

GO:0007272~ensheathment of neuronsGO:0008406~gonad development

GO:0014031~mesenchymal cell …GO:0050804~regula�on of synap�c …

GO:0016310~phosphoryla�onGO:0050771~nega�ve regula�on of …

GO:0007276~gamete genera�onGO:0001701~in utero embryonic …

48h UP 0 5 10 15 20

nuclear lumen

tRNA processing

ribosome biogenesis

nucleo�de-binding

amino acid biosynthesis

mitochondrial envelope

RNA methyltransferase ac�vity

atp-binding

dna-directed rna polymerase

GO:0022411~cellular component …

GO:0000278~mito�c cell cycle

gtp-binding

GO:0006220~pyrimidine nucleo�de …

GO:0007067~mitosis

GO:0055029~nuclear DNA-directed …

GO:0030097~hemopoiesis

GO:0016481~nega�ve regula�on of …

48 Down

0 1 2 3 4 5 6

GO:0046872~metal ion bindingGO:0005773~vacuole

GO:0043068~posi�ve regula�on of programmed …GO:0016310~phosphoryla�on

serine/threonine-protein kinaseGO:0043067~regula�on of programmed cell death

GO:0051085~chaperone mediated protein …GO:0030336~nega�ve regula�on of cell migra�on

GO:0005624~membrane frac�onGO:0005765~lysosomal membrane

GO:0005261~ca�on channel ac�vityGO:0031410~cytoplasmic vesicle

GO:0051270~regula�on of cell mo�onGO:0031669~cellular response to nutrient levels

nucleo�de-bindingGO:0006897~endocytosis

GO:0045860~posi�ve regula�on of protein …GO:0048002~an�gen processing and …

GO:0050801~ion homeostasisGO:0043492~ATPase ac�vity, coupled to …

GO:0060113~inner ear receptor cell differen�a�onGO:0031228~intrinsic to Golgi membrane

GO:0006914~autophagygtp-binding

GO:0001568~blood vessel developmentGO:0042325~regula�on of phosphoryla�on

GO:0004713~protein tyrosine kinase ac�vityGO:0022843~voltage-gated ca�on channel ac�vity

GO:0051339~regula�on of lyase ac�vityGO:0007276~gamete genera�on

GO:0070663~regula�on of leukocyte prolifera�onGO:0048640~nega�ve regula�on of …

GO:0006635~fa�y acid beta-oxida�on

24 HI UP

0 1 2 3 4 5 6

transcrip�on regula�onGO:0003700~transcrip�on factor ac�vity

GO:0031981~nuclear lumenGO:0045941~posi�ve regula�on of …

GO:0060429~epithelium developmentGO:0016331~morphogenesis of …GO:0009615~response to virus

gtp-bindingGO:0016481~nega�ve regula�on of …

24 LO Down 0 1 2 3 4

GO:0051270~regula�on of cell mo�onGO:0031012~extracellular matrix

GO:0051960~regula�on of nervous system …GO:0001942~hair follicle development

GO:0042325~regula�on of phosphoryla�onGO:0007272~ensheathment of neurons

GO:0050770~regula�on of axonogenesisGO:0043392~nega�ve regula�on of DNA binding

GO:0016477~cell migra�onGO:0008219~cell death

nucleo�de phosphate-binding region:GTPGO:0030335~posi�ve regula�on of cell migra�onGO:0050768~nega�ve regula�on of neurogenesis

GO:0050801~ion homeostasisGO:0001501~skeletal system development

GO:0043068~posi�ve regula�on of programmed …GO:0045860~posi�ve regula�on of protein …

GO:0001701~in utero embryonic developmentcalcium transport

GO:0005765~lysosomal membraneGO:0048812~neuron projec�on morphogenesis

GO:0006811~ion transportGO:0000904~cell morphogenesis involved in …GO:0035272~exocrine system development

24 LO UP

0 10 20 30

GO:0031981~nuclear lumentranscrip�on regula�on

zincGO:0016481~nega�ve regula�on of transcrip�on

GO:0030324~lung developmentGO:0045944~posi�ve regula�on of transcrip�on …

GO:0046872~metal ion bindingGO:0030326~embryonic limb morphogenesis

GO:0042054~histone methyltransferase ac�vityGO:0001707~mesoderm forma�on

GO:0048864~stem cell developmentGO:0001944~vasculature development

atp-bindingGO:0004386~helicase ac�vity

GO:0008138~protein tyrosine/serine/threonine …Aminoacyl-tRNA synthetase

mitosisGO:0019866~organelle inner membrane

GO:0002520~immune system developmentGO:0009982~pseudouridine synthase ac�vityGO:0055123~diges�ve system development

GO:0006839~mitochondrial transportGO:0048667~cell morphogenesis involved in …GO:0014020~primary neural tube forma�on

ubl conjuga�on pathwayGO:0016310~phosphoryla�on

GO:0034062~RNA polymerase ac�vityGO:0004725~protein tyrosine phosphatase …

GO:0060324~face developmentGO:0046131~pyrimidine ribonucleoside …

GO:0006306~DNA methyla�onGO:0016477~cell migra�on

24 HI Down

a b

cd

e f

Figure-S2 (Surani)

Figure S2 Functional categories of differentially expressed genes upon BLIMP1 expression in P19ECs. (a-f). Gene ontology analysis of all genes with significant

expression changes (FDR < 0.005) in P19ECs upon BLIMP1 expression for both induced and repressed genes for each of the 3 comparisons performed.

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0 1 2 3 4

log2 (Fold Enrichment)

AllBLIMP1 targets

Positive regulation of protein phosphorylation

Wnt receptor signaling pathwayEndocytosis

Post-embryonic developmentIn utero embryonic development

Kidney developmentOrgan morphogenesis

Heart development

Anterior/posterior pattern formation

Canonical Wnt receptor signaling pathwayPositive regulation of cell migration

Positive regulation of gene expressionVasculogenesis

Homophilic cell adhesionAxon guidance

Regulation of Rho protein signal transduction

Embryonic skeletal system development Odontogenesis of dentine-containing tooth

Negative regulation of canonical Wnt receptor signaling pathwayEmbryonic skeletal system morphogenesis

0 2 4 6 8

Guanyl ribonucleotide binding

Transcription regulator activityRegulation of transcription, DNA-dependent

Regulation of RNA metabolic processDNA bindingTranscription

Regulation of transcription

Transcription factor activity

Cell morphogenesis involved in neuron differentiation

Cell morphogenesisCell projection organization

Neuron projection developmentGTP binding

Cellular component morphogenesisGuanyl nucleotide bindingg

Neuron differentiation

Cell morphogenesis involved in differentiationAxonogenesis

Neuron developmentNeuron projection morphogenesis

Middle ear morphogenesisMotor axon guidance

Axon guidance

AllBLIMP1 targets

log2 (Fold Enrichment)

Figure-S3 (Surani)

a bRepressed Genes Induced Genesc

−3 −2 −1 0 1 2 3

E8.5 PGCs vs BLIMP1 KO

peaksscores

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

Blim

p1 ta

rget

enr

ichm

ent (

log

)

log (Fold Change)

2

2

Repressed Induced

d

50 kb

Wnt5b Fbxl14

30 -

1 _

20 kb

Has2 Has2as1 _

50 kb

Spata13

30 -

1 _

100 kb

Meis2AK012325AK144485

2810405F15Rik

1 _

10 kb

AK019124 Klf9 Mir1192

30 -

1 _

50 kb

Etv1

22 -

1 _

30 -

28 -

e

f

−4 −2 0 2 4

−0.2

0.0

0.2

0.4

0.6

0.8

E7.5 PGCs vs E7.5 somapeaksscoresintersect.

BLI

MP

1 ta

rget

enr

ichm

ent (

log

) 2

log (Fold Change)2

Figure S3 RNAseq analysis during PGC specification; integrative analysis of PGC transcriptome, BLIMP1 induced changes in P19EC profiles, and BLIMP1targets. (a and b). Gene ontology analysis showing functional categories of genes repressed and induced respectively, between E7.5 PGCs and somatic cells and the genes from the comparison that are bound by BLIMP1. (c). Relative enrichment of BLIMP1 binding regions and the scores associated with genes differentially expressed between E8.5 PGCs and E7.5 Prdm1 (encoding BLIMP1)-KO PGC–like cells. (d). Correlation analysis of differentially expressed genes during PGC specification and upon BLIMP1 expression in P19ECs. The Pearson correlation coefficients are indicated in the bottom half for each pair-wise comparison and each point on the plot indicates the differential expression of a gene in the comparisons indicated on the x- and y-axes respectively by the juxtaposition to the squares along

the diagonal. (e). Relative enrichment of BLIMP1 binding regions associated with genes that are differentially expressed both upon BLIMP1 expression in P19ECs as well as during PGC specification. The x-axis indicates the log2 (fold change) and the y-axis indicates the log2 of the BLIMP1 target enrichment at each fold change-interval of differentially expressed genes over the average target frequency of the whole expression data set. Peaks: the enrichment of peaks associated with genes in each interval differential expression expression level interval Scores; the enrichment of binding scores calculated for genes in each interval. Intersect: The enrichment of peaks associated with genes differentially expressed in both comparisons, in each interval of differential expression. (f). ChIP sequencing tracks from the UCSC browser of genes showing example genomic loci of genes bound by BLIMP1 and repressed in both PGCs and P19ECs upon BLIMP1 expression.

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e

−4 −2 0 2 4 6−4 −2 0 2 4 6

−0.2

0.0

0.2

0.4

0.6

0.8

−0.2

0.0

0.2

0.4

0.6

0.8

Targ

et e

nric

hmen

t (lo

g )

log (FC)2 log (FC)2

2

BLIMP1BLIMP1/AP2g

PRDM14PRDM14/AP2g

BLIMP1/AP2g/PRDM14

Score p-ValueMotif

BLIMP1

IRF2

SP1-Q6

AP2

44.02

27.00

23.94

16.72 9.17E-5

2.06E-8

2.40E-10

0

16.17 5-E75.61FRN

AP2gAP2BLIMP1

a b

Distance From Peak Centre Distance From Peak Centre Distance From Peak Centre

Freq

uenc

y pe

r 50

bp

80.67

0

PRDM14

OCT4

SP1 (CACD)

AP2

SOX

81.05

56.81

56.59

45.25

0

0

0

0

AP2gAP2PRDM14Score p-ValueMotifc d

Distance From Peak Centre Distance From Peak Centre Distance From Peak Centre

Freq

uenc

y pe

r 50

bp

Figure-S4 (Surani)

Figure S4 AP2γ motif analysis on BLIMP1 and PRDM14 binding regions. (a). TRANSFAC motif scanning of the BLIMP1 binding regions. The enrichment scores and p-values for the enrichment of each motif are indicated. (b). The distribution of the BLIMP1, AP2 alpha- and the AP2γ motifs on the BLIMP1 binding regions. (c). TRANSFAC motif scanning of the PRDM14 binding regions. d). The distribution of the

PRDM14 motif, AP2 alpha and AP2g motifs around the centre of the binding regions. (e). Relative enrichment of BLIMP1, and PRDM14 targets on differentially expressed genes between E7.5 soma and PGCs filtered by the association of an AP2γ motif in the peak region and the combinatorial association of the peak regions to the differentially expressed genes.

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Figure-S5 (Surani)

AP

2g.n

oBLI

MP

1.no

PR

DM

14

BLI

MP

1.A

P2g

PR

DM

14.A

P2g

BLI

MP

1.no

PR

DM

14.n

oAP

2g

BLI

MP

1.A

P2g

.PR

DM

14

AP

2g

PR

DM

14.n

oBLI

MP

1.no

AP

2g

BLI

MP

1.P

RD

M14

PR

DM

14

BLI

MP

1

response to protein stimulusphospholipid biosynthetic processsomitogenesisneural crest cell migrationphosphatase activityregulation of gene expressioncellular response to protein stimulusprotein tyrosine/serine/threonine phosphatase activityprotein kinase C bindingnegative regulation of fat cell differentiationpositive regulation of peptidyl−serine phosphorylationneuron fate commitmentnegative regulation of BMP signaling pathwaypositive regulation of cell deathtranscription corepressor activityprotein tyrosine phosphatase activitydouble−stranded DNA bindingcell fate commitmentneural tube closurecellular protein localizationdorsal/ventral neural tube patterningprotein kinase activitytransferase activity, transferring phosphorus−containing groupsprotein phosphorylationphosphorylationkinase activityphosphoprotein phosphatase activityaxonogenesisnegative regulation of canonical Wnt receptor signaling pathwaycanonical Wnt receptor signaling pathwaymammary gland developmentadult locomotory behaviorprotein serine/threonine kinase activitydephosphorylationpositive regulation of protein phosphorylationembryonic skeletal system developmentregulation of cell cycleembryonic skeletal system morphogenesiscytoskeleton organizationcentral nervous system developmentphosphoprotein bindingsubstrate adhesion−dependent cell spreadingactin filament cappingpost−embryonic developmentplatelet activationRho guanyl−nucleotide exchange factor activityregulation of Rho protein signal transductionheart loopingureteric bud developmentdetermination of left/right symmetryforebrain developmentcarbohydrate metabolic processembryonic digit morphogenesispositive regulation of canonical Wnt receptor signaling pathwaypositive regulation of epithelial cell proliferationprotein tyrosine kinase activityhair follicle morphogenesisproximal/distal pattern formationhomophilic cell adhesionbranching involved in ureteric bud morphogenesissmoothened signaling pathwaypositive regulation of ERK1 and ERK2 cascadeendocytosisGTPase activator activityguanyl−nucleotide exchange factor activitycell migrationdorsal/ventral pattern formationtransferase activityDNA bindingzinc ion bindingprotein bindingregulation of transcription, DNA−dependenttranscription, DNA−dependentpositive regulation of gene expressionpositive regulation of transcription, DNA−dependentpositive regulation of transcription from RNA polymerase II promotermulticellular organismal developmentsequence−specific DNA binding transcription factor activitynegative regulation of transcription from RNA polymerase II promoteraxon guidanceskeletal system developmentblood vessel developmentinduction of apoptosis by extracellular signalspositive regulation of apoptosisin utero embryonic developmentintegrin bindingpositive regulation of cell−substrate adhesionvasculogenesiscell proliferationheart developmentchromatin bindingactin bindingkidney developmentembryonic limb morphogenesislung developmentodontogenesis of dentine−containing toothcalcium ion bindingpalate developmentmesoderm formationWnt receptor signaling pathway, calcium modulating pathwayembryonic hindlimb morphogenesisdevelopmental growthangiogenesisregulation of catalytic activityprotein complex bindingpositive regulation of cell migrationorgan morphogenesiscell−cell signalinganterior/posterior pattern formationpositive regulation of neuron differentiationpositive regulation of cell proliferationsequence−specific DNA bindingregulation of transcription from RNA polymerase II promotersmall GTPase mediated signal transductionWnt receptor signaling pathwayreceptor bindingnegative regulation of transcription, DNA−dependentskeletal system morphogenesisphosphatidylinositol binding

−4 −2 0 2 4

Log2 Fold Enrichment

AP

2g.n

oBLI

MP

1.no

PR

DM

14

BLI

MP

1.A

P2g

BLI

MP

1.no

PR

DM

14.n

oAP

2g

PR

DM

14.A

P2g

BLI

MP

1.A

P2g

.PR

DM

14

AP

2g

BLI

MP

1

BLI

MP

1.P

RD

M14

PR

DM

14.n

oBLI

MP

1.no

AP

2g

PR

DM

14

Rab GTPase bindingresponse to ethanolcell deathRho GTPase bindingRho GTPase activator activitysomatic stem cell maintenanceT cell receptor signaling pathwaygerm cell developmentSH3 domain bindingmicrotubule−based movementmicrotubule motor activitylipid catabolic processdamaged DNA bindingregulation of Rab GTPase activityRab GTPase activator activityzinc ion transportDNA−dependent ATPase activityregulation of endocytosisnegative regulation of epithelial cell proliferationATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanismendocytosishistone deacetylase bindingRho protein signal transductionneuromuscular junction developmentkinase activityphosphorylationprotein phosphorylationtransferase activity, transferring phosphorus−containing groupsprotein serine/threonine kinase activityprotein kinase activityintracellular signal transductiondephosphorylationubiquitin−protein ligase activitytranscription coactivator activityactin cytoskeleton organizationprotein polyubiquitinationresponse to DNA damage stimuluszinc ion bindingtransferase activitywound healingnegative regulation of MAP kinase activityMAPKKK cascadeprotein glycosylationdendrite developmentintracellular protein kinase cascadeprotein kinase bindingGTPase activator activityguanyl−nucleotide exchange factor activitypeptidyl−serine phosphorylationregulation of Rho protein signal transductionRho guanyl−nucleotide exchange factor activityglucose homeostasisplanar cell polarity pathway involved in neural tube closure

−4 −2 0 2 4Value

Color Key

Log2 Fold Enrichment

a b

Figure S5 GO-Term clustering of genes differentially associated with BLIMP1, AP2γ and Prdm14 during PGC specification. Clustering analysis of the GO-terms associated with genes that are repressed (a). or induced (b). in PGCs

compared to neighbouring somatic cells at E7.5 and bound by different combinations of either BLIMP1, AP2γ or Prdm14. The dark blue colour indicates that fewer than 4 genes were mapped to the indicated GO-term.

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Figure-S6 (Surani)

Rho protein signal transduction

Homophilic cell adhesion

Cell migration

Cell adhesion

Actin cytoskeleton organization

Actin filament organization

Microtubule−based movement

Negative regulation of MAP kinase activity

Intracellular protein kinase cascade

MAPKKK cascade

Cell cell signaling

Somatic stem cell maintenance

Spermatogenesis

Germ cell development

Chromatin modification

Organ morphogenesis

Embryonic skeletal system development

Proximal/distal pattern formation

Embryonic limb morphogenesis

Regulation of cell migration

Chromatin modification

Positive regulation of cell migration Substrate adhesion dependent cell spreading

Integrin binding

Positive regulation of cell proliferation

Phosphatidylinositol binding

Anterior/posterior pattern formation

Positive regulation of ERK1 and ERK2 cascade In utero embryonic development

Cell−cell signaling Cell-cell junction

Homophilic cell adhesion

a

b

Siglec5

Kdm4b

Mll5

Prmt7

Hdac7

Cdh4

Ptprm

Kif21a

Hmgn5

Lats2Gna12Itgb3

Tns3 Ednra

Ophn1

Thbs4

Dync2h1

Dtx3l

Foxa3

Bre Smarcal1

Hdac6 Eya2

Hdac4

Rps6ka1

Jak1

Ss18Bhlha15

Prdm14

Mcf2

Map2k6

Mtap7Dnd1

Rps6ka3

PRDM14

Klf2

Banp

Mtor

Dab2ip

CremLimk2

Kit

Dlgap5 Bmp6

Mark1

Tnc

Kif5a

Immp2l

Klf10

Col4a3bpHes3 Zfx

Nr0b1Agfg1

49*

Nanos3Morc1Sox2

D1Pas1

Ldb2

GsrNanog

Wdtc1

Suz12

Phf21aRbl1

Kdm3a

Cbx7

Azi1

Pcgf2

Ss18l1

Keap1

Rdh10

Pdgfa

Diap1

Robo1PtenAche

Hbegf

Rad51cVangl2

Tcfap2c

Bcl2l1

Acox1

Hist1h1a

Tcp11

Kif27

Arhgap17

Pvr

Kif15

Cntnap2

Clasp2

Spg7

Usp33

Lrrc16a

Pik3cb

Kif16b Gsk

3a

Nlgn2

Kif18b Cer

cam

Col9a1

Srgap1 Map

4k2Sorl1Btbd9

Rps6ka2

Oxsr1

Npnt

Ninj1Dlg1

Diap3

Diap2 Parva

Aplp1

Tube1

Fam175a

BLIMP1

Ash1lFgf8

Zfp39

Sfrp1

Vav3

Sirt1

Ift81

Taok2Rgmb

Pdlim7

Lrrk2Map3k3

Gna13

Tgfbr3Ppm1l

Dab1

Apoe

Pkn1

Dusp7

Suv420h1

Mib1

Fgfr2 Map4k1

Ptpn6 Map3k1

Ppp1r9b

Dok1

Cblc

Gdf3Ift88Myb

Mea1

Ncor2Esrrb

Odf2

Gpx4Dzip1

Fhod3

Mknk1

Itga9Cdh13

Kif24 Gpr56Ttyh1

Sdk1

Celsr1Prdm1

Nf1Col18a1

Tesk2Zfp36

Gal3st1

Gpld1

Twist1

AP2g

Sirpa

*4932438A13Rik

In utero embryonic development

BMP signaling pathway

Bcl9

Pdzd2

Clstn3Akt2

Tiam1

Cyr61

Hipk2Tnks

PRDM14

Chd9

L3mbtl3

Bmi1 RaraPrdm6 Pax3

Rcor1Kdm6b

Pcdhga12

Lama5

Pbx2

Fgf4

Clstn2

Snai1

Ctnnbip1

Src

Gli2

Wnt8a

Itga6

Pcdhgc3

Notch1

AP2g

Ptpru

HiraCreb3Sp6ItgavSuv420

h1Sox9

Macf1

Hip1

Snx24Sbf2

Wnt3

Hoxa10

Wnt5b

Tbx3Myo1e

Jub

Clstn1

Itgb5

Erbb2ip

Irs2

Pth1r

Arnt2

Pggt1b

Fgf3

Fgf15 Lifr

Snx15

Trip6

Spata13

Dll1

Itgb1bp1 Robo2

Wbp7

Smo Crkl

Vil1

Podxl

Myh10

Cdx4

Dsg2

Pcdh19

Pvrl3

Tyro3Antx

r1

*C230081A13Rik

Usp3

Dsc2

Rpgrip1l

Tshz3

Pbx1Cited2 Gja1

Mllt3Etnk2

Dlk1

Dact1

Dnmt3a

Satb2

Itga5

Smarcc2

Pcdh8

Epc1

Pik3r1

Mertk

Hhex

Cd44

Tnik

Arrb1

Htra1 Twsg1Prkd1

Ada

St5 Snx8Hip1r

Cdx1

Fbn2

Gata4Cfc1

Heg1Bcl2l11

Tial1

Pdf

Snx33

Hoxb9

Hoxa2

Hoxa4Dusp3

Hoxb5

Celsr3

Stat1

Dvl1

Hoxb6

Snx7

Fzd6

Igf2

Timp2

Hoxd9

Fzd1

BLIMP1

Cxcl12

Hoxb7

Cd47

Hoxb2

Hoxb1Hoxb8

Hoxa1Barx1

Sox4

Hoxa3

Dab2

Fstl3Bmper Tax1bp3

MycCcnd1

Wnt10a

Pard3

Bmp4

Wnt6

Hipk1

Lrp4

Dixdc1Gas1

Hoxb3

Tbx20Tpm1

Fzd2Wnt3a

Tgfb1

Zmiz1 Hoxb4Pdgfra

Itpr3

Panx1

Nphp3

Wnt receptor signaling pathway

Negative regulation of BMP signaling pathwayDorsal/ventral pattern formation

Dppa3

*C23

Uhrf1

Dnmt3b

Figure S6 A transcription factor network for PGC specification. BLIMP1, AP2γ and Prdm14 binding to differentially expressed genes at E7.5 between PGCs and soma. (a). Repressed genes. (b). Induced genes. The yellow nodes indicate the BLIMP1, PRDM14 and AP2γ peak and

associations. The smaller nodes indicate genes bound by the factors, with the binding associations indicated by lines connected to the yellow nodes. The colours of the gene-nodes indicate functional categories as shown.

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Figure S7 (Surani)

Correlation Coefficient

Prdm14

AP2g

Blimp1

Figure S7 A compendium of mESC transcription factor integrated profiles. (a). A full hierarchical clustering analysis of the genome-wide BLIMP1 and AP2γ binding patterns together with binding patterns of transcriptional regulators from mESCs. The red lines above the heat-maps indicate the main clusters, showing the pluripotency cluster (Oct4 etc), polycomb-cluster (Ring1b etc), self-renewal/proliferation cluster (N-Myc etc), and a genome-architectural cluster (CTCF etc). The combinatorial binding pattern analysis was performed by generating a unified data matrix based

on 165,607 unique peak regions, indicating for each factor whether it was bound or not. Subsequently the hierarchical clustering and Pearson’s correlation coefficients shown in the heat map were used to investigate global relationships. The colours indicate level of correlation for all pairwise comparisons as indicated on the figure. Note that BLIMP1 associates most strongly with the self-renewal cluster and has high correlation with polycomb factors. PRDM14 associates with the pluripotency cluster whereas AP2γ binding does not correlate highly with mESC transcriptional regulators.

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Figure-S8 (Surani)

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Cytokines

Blimp1 AP2g Prdm14

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MycKdm4b Dnmt3b Uhrf1

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BLIMP1 and AP2γ no PRDM14 80.02 53BLIMP1 and PRDM14 no AP2γ 926.95 1740PRDM14 and AP2γ no BLIMP1 365.44 628BLIMP1 and AP2γ and PRDM14 36.22 240BLIMP1 alone 2047.80 1058PRDM14 alone 9351.39 8072AP2γ alone 807.31 368Unbound 20658.86 22115Total 34274 34274

p-value = 0 (Chi-square test)

# of Bound GenesExpected Observed

BLIMP1 and AP2γ 17.00 100BLIMP1 and PRDM14 146.90 230PRDM14 and AP2γ 162.10 568BLIMP1 and AP2γ and PRDM14 1.60 35

p-value < 0.0001 (Permutation test, after 10.000 permutations)

# of Bound Regions

Observed

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Ap2g/Blimp1

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Figure S8 Statistical testing for binding overlap between BLIMP1, Ap2g and PRDM14 and co-expression of BLIMP1, AP2γ and PRDM14 induces PGC-like cell fate in vitro. (a). Number of observed and expected overlap in genomic binding sites of BLIMP1, AP2γ and PRDM14, and a scatterplot showing the observed against expected overlap in genomic binding sites of: 1. AP2γ and BLIMP1, 2. BLIMP1 and PRDM14, and 3. AP2γ and PRDM14. The p-value for the enrichment of overlap in binding sites is p < 0.0001 for all comparisons. (b). A contingency table for the calculation of a chi-

square p-value for the overlap of genes bound by different combinations of BLIMP1, AP2γ and PRDM14. The calculated p-value based on this table was p < 1x10-299. The total number of genes (34274) represents the number of unique gene identifiers in Ensembl that were a basis for the gene annotation of the respective transcription factor binding sites. (c). RT-qPCR analysis of sorted fluorescent PGCLCs on Day2 and 4 of either cytokine or doxycycline induction, as well as EpiLCs. The experiment is the second of two experiments performed. The first experiment is shown in Figure 8.

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Supplementary Tables Legends

Table S1 Microarray Expression Data. This table shows the genome wide differential gene expression in P19EC cells upon transient transfection of BLIMP1. The first four columns show the Entrez Gene ID, the gene symbol and the Illumina Probe ID as well as the gene definition. The next 3 columns give the false discovery rate (FDR) for each comparison and the last 3 columns show the log2 fold change (LFC) for each comparison.

Table S2 BLIMP1 Peaks. A list of the high-confidence binding regions of BLIMP1.

Table S3 BLIMP1 Peaks with Gene Annotation. Gene annotations for the high-confidence binding regions of BLIMP1.

Table S4 Single Cell PGC RNAseq Expression Data. This table shows the genome wide differential gene expression in nascent PGCs (E6.5, E7.5 and E8.5) and somatic neighbours (E7.5 soma) as well as Blimp1 null PGC-like cells (E7.5 Blimp1KO), assayed by single cell RNAseq. The first column shows the RefSeqID, the second column the gene symbol, the third column shows the gene localization and strand. The values indicate the expression levels in log2(reads pr. million).

Table S5 AP2g Peaks. A list of the high-confidence binding regions for AP2g.

Table S6 AP2g Peaks With Gene Annotation. Gene annotations for the high-confidence binding regions of AP2g.

Table S7 Primer Sequences. The table shows the primer sequences of primers used for RT-qPCR as well as ChIP-qPCR experiments in 3’ to 5’ direction.

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