Supplementary Figure 1. Dendrogram based on (A) … · Supplementary Figure 2. 3D PCA plot based on (A) expression or (B) PRIMs of all protein-coding and noncoding genes. For SSCs,

Supplementary Figure 1. Dendrogram based on (A) expression or (B) PRIMs of all protein-coding and noncoding genes. Different cell types are distinguished by colors as in Figure 1. Left, dendrograms include all cell lines. Right, dendrograms without published data on SSCs, iPSCs, and MEFs. In-house data are highlighed in gray.

A

Update Figure S1

HSC_1

HSC_2

MEF

MEF_SY12_1

MEF_SY12_2

PGC_E12d5_LBJ13_1

PGC_E14d5_LBJ13_2

PGC_E14d5_LBJ13_1

PGC_E14d5_LBJ13_3

PGC_E12d5_LBJ13_2

PGC_E12d5_LBJ13_3

PGC_E13d5_LBJ13_3

PGC_E13d5_LBJ13_1

PGC_E13d5_LBJ13_2

MASC_OG1

MASC_OG2

ESC_BLA13

ESC_OG

iPS4

iPS1

MASC_Rosa3

ESC_129mix

MASC_Rosa5

MASC_VG5

MASC_VG8

GS_SHS15_1

GS_SHS15_2

SSC_OG2

SSC_VG

SSC_OG1

SSC_B6

PS_LBJ13

RS_LBJ13

PS_SHS15_1

PS_SHS15_2

RS_SHS15_1

RS_SHS15_2

RS_ES13_1

RS_ES13_2

0.00.51.01.52.02.53.0

Clu

ster

Den

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hclu

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dis

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na.o

mit(T

SS

.exprs

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[, −

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:10, 24, 27, 30, 48:5

0,

56:1

41)]

)))

Height

Transcriptome (RNA-seq)

HSC_1

HSC_2

MEF

MEF_SY12_1

MEF_SY12_2

PGC_E12d5_LBJ13_1

PGC_E14d5_LBJ13_2

PGC_E14d5_LBJ13_1

PGC_E14d5_LBJ13_3

PGC_E12d5_LBJ13_2

PGC_E12d5_LBJ13_3

PGC_E13d5_LBJ13_3

PGC_E13d5_LBJ13_1

PGC_E13d5_LBJ13_2

MASC_OG1

MASC_OG2

ESC_BLA13

ESC_OG

iPS4

iPS1

MASC_Rosa3

ESC_129mix

MASC_Rosa5

MASC_VG5

MASC_VG8

GS_SHS15_1

GS_SHS15_2

SSC_OG2

SSC_VG

SSC_OG1

SSC_B6

PS_LBJ13

RS_LBJ13

PS_SHS15_1

PS_SHS15_2

RS_SHS15_1

RS_SHS15_2

RS_ES13_1

RS_ES13_2

0.00.51.01.52.02.53.0

Cluster Dendrogram

hclust (*, "complete")

dist(cor(na.omit(TSS.exprs.chip[, −c(1:10, 24, 27, 30, 48:50,

56:141)])))

Height

RS_ES13.2 RS_ES13.1 RS_SHS15.2 RS_SHS15.1 PS_SHS15.2 PS_SHS15.1 RS_LBJ13 PS_LBJ13 SSC.3 SSC.1 SSC.4 SSC.2 SSC_SHS15.2 SSC_SHS15.1 MASC.4 MASC.3 MASC.5 ESC.2 MASC.6 iPS.1 iPS.2 ESC.1 ESC_BLA13 MASC.2 MASC.1 PGC_E13d5_LBJ13.2 PGC_E13d5_LBJ13.1 PGC_E13d5_LBJ13.3 PGC_E12d5_LBJ13.3 PGC_E12d5_LBJ13.2 PGC_E14d5_LBJ13.3 PGC_E14d5_LBJ13.1 PGC_E14d5_LBJ13.2 PGC_E12d5_LBJ13.1 MEF_SY12.2 MEF_SY12.1 MEF HSC.2 HSC.1

Height 3.0 2.5 2.0 1.5 1.0 0.5 0.0

MEFHSC_1HSC_2

PGC_E12d5_LBJ13_1PGC_E14d5_LBJ13_2PGC_E14d5_LBJ13_1PGC_E14d5_LBJ13_3PGC_E13d5_LBJ13_3PGC_E13d5_LBJ13_1PGC_E13d5_LBJ13_2PGC_E12d5_LBJ13_2PGC_E12d5_LBJ13_3

SSC_OG1SSC_B6

SSC_OG2SSC_VG

MASC_Rosa3ESC_129mix

MASC_Rosa5MASC_VG5MASC_VG8

iPS1iPS4

ESC_OGMASC_OG1MASC_OG2

PS_LBJ13RS_LBJ13

PS_SHS15_1PS_SHS15_2RS_SHS15_1RS_SHS15_2

RS_ES13_1RS_ES13_2

0.00.51.01.52.02.5

Clu

ster

Den

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ram

hclu

st (*

, "co

mpl

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)di

st(c

or(n

a.om

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S.ex

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[, −c

(1:1

0, 1

3, 2

4:27

, 30,

32,

33,

48:

50, 5

6:14

1)]))

)

Height

RS_ES13.2 RS_ES13.1 RS_SHS15.2 RS_SHS15.1 PS_SHS15.2 PS_SHS15.1 RS_LBJ13 PS_LBJ13 MASC.2 MASC.1 ESC.1 iPS.2 iPS.1 MASC.4 MASC.3 MASC.5 ESC.2 MASC.6 SSC.4 SSC.2 SSC.3 SSC.1 PGC_E12d5_LBJ13.3 PGC_E12d5_LBJ13.2 PGC_E13d5_LBJ13.2 PGC_E13d5_LBJ13.1 PGC_E13d5_LBJ13.3 PGC_E14d5_LBJ13.3 PGC_E14d5_LBJ13.1 PGC_E14d5_LBJ13.2 PGC_E12d5_LBJ13.1 HSC.2 HSC.1 MEF

MEFHSC_1HSC_2

PGC_E12d5_LBJ13_1PGC_E14d5_LBJ13_2PGC_E14d5_LBJ13_1PGC_E14d5_LBJ13_3PGC_E13d5_LBJ13_3PGC_E13d5_LBJ13_1PGC_E13d5_LBJ13_2PGC_E12d5_LBJ13_2PGC_E12d5_LBJ13_3

SSC_OG1SSC_B6

SSC_OG2SSC_VG

MASC_Rosa3ESC_129mixMASC_Rosa5

MASC_VG5MASC_VG8

iPS1iPS4

ESC_OGMASC_OG1MASC_OG2

PS_LBJ13RS_LBJ13

PS_SHS15_1PS_SHS15_2RS_SHS15_1RS_SHS15_2

RS_ES13_1RS_ES13_2

0.00.51.01.52.02.5

Cluster Dendrogram


dist(cor(na.omit(TSS.exprs.chip[, −c(1:10, 13, 24:27, 30, 32,

33, 48:50, 56:141)])))

HeightHeight

2.5 2.0 1.5 1.0 0.5 0.0


Update Figure S1

iPS1_H3d3OSKM.prim

PGC_E12d5_LBJ13.prim



HFTAC.prim

qHFSC.prim

aHFSC.prim

LSK_AM10.prim

MAC.prim

HSC_1.prim

HSC_2.prim

AGSC_HSS14.prim

PS_SHS15.prim

RS_SHS15.prim

Sperm_ES13.prim

RS_LBJ13.prim

PS_LBJ13.prim

RS_ES13.prim

MASC_OG_1_2.prim

MASC_OG_3.prim

MASC_Rosa.prim

iPS_MCV81_MTS08.prim

ESC_MTS07.prim

ESC_WJA12.prim

ESC_H3d3HA_BLA13.prim

iPS4_H3d3OSKM.prim

SSC_B6.prim

SSC_VG.prim

SSC_OG.prim

GS_SHS15.prim

MEF_H3d3OSKM_D0.prim

MEF_MTS07.prim

MEF_MTS08.prim

PiPS_MCV6_MTS08.prim


0.00.51.01.5

Clu

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Den

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hcl

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(*,

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")d

ist(

cor(

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it(T

SS

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[, −

c(1

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, 1

03

, 1

04

, 1

09

, 1

10

,

1

28

, 1

32

, 1

40

)]))

)

Height

iPS1_H3d3OSKM.prim




HFTAC.prim

qHFSC.prim

aHFSC.prim

LSK_AM10.prim

MAC.prim

HSC_1.prim

HSC_2.prim

AGSC_HSS14.prim

PS_SHS15.prim

RS_SHS15.prim

Sperm_ES13.prim

RS_LBJ13.prim

PS_LBJ13.prim

RS_ES13.prim

MASC_OG_1_2.prim

MASC_OG_3.prim

MASC_Rosa.prim

iPS_MCV81_MTS08.prim

ESC_MTS07.prim

ESC_WJA12.prim


iPS4_H3d3OSKM.prim

SSC_B6.prim

SSC_VG.prim

SSC_OG.prim

GS_SHS15.prim


MEF_MTS07.prim

MEF_MTS08.prim



0.00.51.01.5

Cluster Dendrogram


dist(cor(na.omit(TSS.exprs.chip[, −c(1:99, 103, 104, 109, 110,

128, 132, 140)])))

Height

PiPS_MCV8_MTS08 PiPS_MCV6_MTS08 MEF_MTS08 MEF_MTS07 MEF SSC_SHS15 SSC.1 SSC.3 SSC.2 iPS.2 ESC_BLA13 ESC_WJA12 ESC_MTS07 iPS_MCV81_MTS08 MASC.3 MASC.2 MASC.1 RS_ES13 PS_LBJ13 RS_LBJ13 Sperm_ES13 RS_SHS15 PS_SHS15 AGSC_HSS14 HSC.2 HSC.1 Macrophage LSK_AM10 aHFSC_LWH11 qHFSC_LWH11 HFTAC_LWH11 PGC_E14d5_LBJ13 PGC_E13d5_LBJ13 PGC_E12d5_LBJ13 iPS.1

Height 1.5 1.0 0.5 0.0

Epigenome at Promoters (PRIM) Epigenome at Promoters (PRIM)

iPS1_H3d3OSKM.prim




AGSC_HSS14.prim

HFTAC.prim

qHFSC.prim

aHFSC.prim

LSK_AM10.prim

MAC.prim

HSC_1.prim

HSC_2.prim

PS_SHS15.prim

RS_SHS15.prim

Sperm_ES13.prim

RS_LBJ13.prim

PS_LBJ13.prim

RS_ES13.prim

MASC_OG_1_2.prim

MASC_OG_3.prim

MASC_Rosa.prim

ESC_MTS07.prim

ESC_WJA12.prim


iPS4_H3d3OSKM.prim

SSC_OG.prim

SSC_B6.prim

SSC_VG.prim




0.00.20.40.60.81.01.21.4

Cluster Dendrogram


dist(cor(na.omit(TSS.exprs.chip[, −c(1:99, 103, 104, 109, 110,

117:119, 128, 132, 134, 140)])))

Height

iPS1_H3d3OSKM.prim




AGSC_HSS14.prim

HFTAC.prim

qHFSC.prim

aHFSC.prim

LSK_AM10.prim

MAC.prim

HSC_1.prim

HSC_2.prim

PS_SHS15.prim

RS_SHS15.prim

Sperm_ES13.prim

RS_LBJ13.prim

PS_LBJ13.prim

RS_ES13.prim

MASC_OG_1_2.prim

MASC_OG_3.prim

MASC_Rosa.prim

ESC_MTS07.prim

ESC_WJA12.prim


iPS4_H3d3OSKM.prim

SSC_OG.prim

SSC_B6.prim

SSC_VG.prim




0.00.20.40.60.81.01.21.4

Clu

ster

Den

drog

ram

hcl

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(*,

"co

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te")

dis

t(co

r(na.o

mit(

TS

S.e

xprs

.chip

[, −

c(1:9

9, 103, 104, 109, 110,

117:1

19, 128, 132, 134, 140)]

)))

Height

PiPS_MCV8_MTS08 PiPS_MCV6_MTS08 MEF SSC.3 SSC.2 SSC.1 iPS.2 ESC_BLA13 ESC_WJA12 ESC_MTS07 MASC.3 MASC.2 MASC.1 RS_ES13 PS_LBJ13 RS_LBJ13 Sperm_ES13 RS_SHS15 PS_SHS15 HSC.2 HSC.1 Macrophage LSK_AM10 aHFSC_LWH11 qHFSC_LWH11 HFTAC_LWH11 AGSC_HSS14 PGC_E14d5_LBJ13 PGC_E13d5_LBJ13 PGC_E12d5_LBJ13 iPS.1

Height 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

B

Supplementary Figure 2. 3D PCA plot based on (A) expression or (B) PRIMs of all protein-coding and noncoding genes. For SSCs, iPSCs, and MEFs, only in-house data were analyzed. Different cell types are distinguished by colors as in Figure 1.

Figure S1

-0.2

0.4

0.2

0

00.1

-0.1-0.2

-0.3

0.12 0.14 0.16 0.18

HSC.1 HSC.2

MEF

SSC.1/2/4 SSC.3

iPS.1

iPS.2 MASC.3/4/5/6 MASC.2

MASC.1

ESC.1

ESC.2

PC3

PC1 PC2


A B

0.14

-0.1

0

-0.2

0.4

0.1

0.2

0.3

0.150.160.170.180.190.2

0.2-0.2 0

PC3

PC1

PC2

Epigenome at Promoters (PRIM)

SSC.3 SSC.2

SSC.1 PiPS_MCV6 PiPS_MCV8

HSC.2 HSC.1 MEF

iPS.1

iPS.2 MASC.1 MASC.3

MASC.2

ESC_MTS07 ESC_WJA12

ESC_BLA13

020

4060

8010

0

ESCMASCPiPSiPSMEFHSCMacrophageq/a−HFSC, HFTACSSCPGCAGSCPachytene SpermatocyteRound SpermatidSperm

ESC MASC PiPS iPS

020

4060

8010

0


SSC PGC AGSC PS RS Spermatozoon

020

4060

8010

0


MEF HSC, LSK Macrophage q/a-HFSC, HFTAC

Pluripotent Germ Cells Somatic Cells / Multipotent Cells

Supplementary Figure 3. Correlation coefficients between in-house and published cell lines. (A) Expression of all protein-coding and noncoding genes. (B) PRIMs of all protein-coding and noncoding gene promoters with K4me3 and/or K27me3 modification. Black boxes denote data produced in-house.

1 0.93

1

0.96

0.92

1

0.94

0.95

0.93

1

0.9

0.88

0.91

0.89

1

0.9

0.88

0.91

0.89

0.97

1

0.88

0.86

0.89

0.87

0.89

0.89

1

0.88

0.86

0.88

0.86

0.86

0.86

0.97

1

0.86

0.88

0.86

0.87

0.86

0.86

0.95

0.95

1

0.86

0.88

0.86

0.87

0.86

0.86

0.94

0.95

0.98

1

0.87

0.88

0.86

0.87

0.86

0.86

0.94

0.94

0.98

0.98

1

0.87

0.89

0.87

0.87

0.87

0.87

0.95

0.95

0.98

0.97

0.98

1

0.88

0.87

0.88

0.87

0.87

0.87

0.97

0.96

0.96

0.96

0.96

0.96

1

0.86

0.88

0.86

0.86

0.86

0.86

0.94

0.94

0.98

0.98

0.97

0.97

0.96

1

0.84

0.87

0.83

0.85

0.85

0.85

0.91

0.91

0.96

0.96

0.95

0.95

0.93

0.95

1

0.86

0.88

0.85

0.86

0.85

0.85

0.93

0.94

0.96

0.96

0.96

0.96

0.96

0.96

0.95

1

0.87

0.88

0.86

0.87

0.87

0.87

0.95

0.95

0.96

0.96

0.96

0.96

0.96

0.95

0.95

0.98

1

SSC.1

SSC.2

SSC.3

SSC.4

SSC_SHS15.1

SSC_SHS15.2

MASC.1

MASC.2

MASC.3

MASC.4

MASC.5

MASC.6

ESC.1

ESC.2

ESC_BLA13

iPS.1

iPS.2

SSC.1SSC.2SSC.3SSC.4

SSC_SHS15.1

SSC_SHS15.2

MASC.1

MASC.2

MASC.3

MASC.4

MASC.5

MASC.6ESC.1ESC.2

ESC_BLA13iPS.1iPS.2

0.00 0.25 0.50 0.75 1.00

PearsonCorrelation

SSC

MASC

ESC

iPS

Gene Expression (RNA-seq)

1 0.93

1

0.91

0.91

1

0.9

0.84

0.86

1

0.84

0.81

0.8

0.79

1

0.76

0.79

0.75

0.65

0.85

1

0.75

0.73

0.74

0.77

0.76

0.68

1

0.82

0.83

0.81

0.76

0.85

0.85

0.77

1

0.87

0.84

0.83

0.86

0.88

0.8

0.84

0.9

1

0.81

0.8

0.79

0.81

0.86

0.8

0.82

0.88

0.95

1

0.68

0.69

0.67

0.65

0.74

0.73

0.67

0.77

0.78

0.79

1

0.84

0.81

0.8

0.8

0.86

0.81

0.79

0.87

0.9

0.88

0.8

1

0.84

0.83

0.81

0.82

0.86

0.8

0.8

0.89

0.93

0.9

0.76

0.88

1

0.78

0.78

0.78

0.74

0.76

0.73

0.71

0.78

0.8

0.77

0.66

0.77

0.77

1

0.82

0.81

0.81

0.78

0.78

0.73

0.73

0.8

0.82

0.77

0.67

0.8

0.8

0.82

1

SSC.1

SSC.2

SSC.3

SSC_SHS15

MASC.1

MASC.2

MASC.3

ESC_MTS07

ESC_WJA12

ESC_BLA13

iPS.1

iPS.2

iPS_MCV81_MTS08

PiPS_MCV6_MTS08

PiPS_MCV8_MTS08

SSC.1SSC.2SSC.3

SSC_SHS15

MASC.1

MASC.2

MASC.3

ESC_MTS07

ESC_WJA12

ESC_BLA13iPS.1iPS.2

iPS_MCV81_MTS08

PiPS_MCV6_MTS08

PiPS_MCV8_MTS08

0.00 0.25 0.50 0.75 1.00

PearsonCorrelation

SSC

MASC

iPS

PRIM=log2(H3K4me3/H3K27me3)

A B

Supplementary Figure 4. Genes grouped by promoter histone modifications. (A) Using peak detection results at promoters, all 32,581 protein-coding and noncoding

genes were assigned to each of four different categories: K4me3 only (green), only marked by K4me3 modification; K27me3 only (red), only marked by K27me3 modification; K4me3+K27me3 (yellow), marked by both K4me3 and K27me3 modifications; and no modification (grey), no significant K4me3 or K27me3 modification detected. For each cell type with at least three biological repeats from different cell lines or resources (Supplementary Table S1), results are presented as mean values and standard deviations. Number in brackets, sample size.

(B) GO enrichment in bivalent genes identified from different cell types. Red, over representation. Blue, under representation.

0%

20%

40%

60%

80%

100%

ESC

(3)

iPS

(3)

MA

SC (2

)

SSC

(4)

PGC

E12

.5

PGC

E13

.5

PGC

E14

.5

AG

SC

PS (2

)

RS

(3)

Sper

m

MEF

(3)

LSK

HSC

(2)

Mac

roph

age

qHFS

C

aHFS

C

HFT

AC

% T

otal

Pro

mot

er

K4me3 Only

K4me3+K27me3

K27me3 Only

No Modification

A B

Germline Soma Pluri-/Multi-potent

ESC

iP

S M

ASC

SS

C

PGC

E12

.5

PGC

E13

.5

PGC

E14

.5

AG

SC

PS

RS

Sper

m

MEF

LS

K

HSC

M

acro

phag

e qH

FSC

aH

FSC

H

FTA

C

ESC

iPS

MASC

SSC

PGC_

E12d

PGC_

E13d

PGC_

E14d

AGSC

PS RS Sper

mME

FLS

KHS

CMA

CqH

FSC

aHFS

CHF

TAC

Cell fate commitment, GO:0045165

Heart morphogenesis, GO:0003007

Pancreas development, GO:0031016

Eye morphogenesis, GO:0048592

Embryonic placenta development, GO:0001892

Regulation of organ formation, GO:0003156

Mesenchymal to epithelial transition, GO:0060231

Regulation of kidney development, GO:0090183

Bone morphogenesis, GO:0060349

Epithelial cell morphogenesis, GO:0003382

Development of primary male sexual characteristics, GO:0046546

Mammary gland morphogenesis, GO:0060443

Gut morphogenesis, GO:0048547

Neuron fate specification, GO:0048665

Hemopoiesis, GO:0030097

Cell-cell signaling involved in cell fate commitment, GO:0045168

Embryonic skeletal system development, GO:0048706

Ear development, GO:0043583

Lung development, GO:0030324

Anterior/posterior pattern formation, GO:0009952

Mesenchyme development, GO:0060485

Vasculature development, GO:0001944

Limb development, GO:0060173

5

Enri

chme

nt

-5

Depl

etio

n

Cell fate commitment, GO:0045165 Heart morphogenesis, GO:0003007 Pancreas development, GO:0031016 Eye morphogenesis, GO:0048592 Embryonic placenta development, GO:0001892 Regulation of organ formation, GO:0003156 Mesenchymal to epithelial transition, GO:0060231 Regulation of kidney development, GO:0090183 Bone morphogenesis, GO:0060349 Epithelial cell morphogenesis, GO:0003382 Development of primary male sexual characteristics, GO:0046546 Mammary gland morphogenesis, GO:0060443 Gut morphogenesis, GO:0048547 Neuron fate specification, GO:0048665 Hemopoiesis, GO:0030097 Cell-cell signaling involved in cell fate commitment, GO:0045168 Embryonic skeletal system development, GO:0048706 Ear development, GO:0043583 Lung development, GO:0030324 Anterior/posterior pattern formation, GO:0009952 Mesenchyme development, GO:0060485 Vasculature development, GO:0001944 Limb development, GO:0060173

ESC

iPS

MASC

SSC

PGC_E12d

PGC_E13d

PGC_E14d

AGSC

PSRSSperm

MEF

LSK

HSC

MAC

qHFSC

aHFSC

HFTAC



Pancreas development, GO:0031016

Eye morphogenesis, GO:0048592

Embryonic placenta development, GO:0001892

Regulation of organ formation, GO:0003156

Mesenchymal to epithelial transition, GO:0060231

Regulation of kidney development, GO:0090183

Bone morphogenesis, GO:0060349

Epithelial cell morphogenesis, GO:0003382

Development of primary male sexual characteristics, GO:0046546

Mammary gland morphogenesis, GO:0060443

Gut morphogenesis, GO:0048547

Neuron fate specification, GO:0048665

Hemopoiesis, GO:0030097

Cell-cell signaling involved in cell fate commitment, GO:0045168

Embryonic skeletal system development, GO:0048706



Anterior/posterior pattern formation, GO:0009952

Mesenchyme development, GO:0060485


Limb development, GO:0060173

5

Enrichment

-5

Depletion

Figure S2

Supplementary Figure 5. ESC-like bivalent promoter modification is incompletely established in iPS. (A) Overlap of bivalent genes identified by peak detection in ESCs, MEFs, and iPS cells. (B) GO enrichment in bivalent genes shared or unique in ESCs, MEFs, and iPS cells.

Presence and absence of promoter bivalency in certain cell types is denoted by “+” and “-”, respectively. Red, over representation. Blue, under representation.

(C) Comparison of global gene expression profiles between MEFs and iPS cells. Black dots, MEF bivalent genes identified by peak detection. dashed line, cut-off of two-fold (log2) expression differences between MEFs and iPS cells.

(D) Percentage of genes with expression increases (black) or decreases (grey) in iPS cells. MEF bivalent genes, 1,867 genes with both K4me3 and K27me3 modifications at promoters. MEF univalent & unmodified genes, 30,714 genes modified with either K4me3, K27me3, or neither of the two modifications at promoters. p-value = 0.0011 by Fisher’s exact test (one-sided).

MEF + - + + + - - iPS + + - - + + -

ESC + + + - - - +

A B

iPS MEF (1125) (1867)

ESC (5022)

607 396

2958

Bivalent Promoter

0%

2%

4%

6%

8%

10%

Bivalent Genes Univalent & Unmodified Genes

% S

elec

ted

Gen

es

Expression Change in iPS

Increase Decrease

p-value = 0.0011

0 2 4 6 8 10 12

02

46

810

12

MEF

iPS

Expression of MEF Bivalent Promoters

Development of primary sexual characteristics, GO:0045137 Heart development, GO:0007507 Lung development, GO:0030324 Brain development, GO:0007420 Ear development, GO:0043583 Gland development, GO:0048732 Eye development, GO:0001654 Urogenital system development, GO:0001655 Kidney development, GO:0001822 Vasculature development, GO:0001944 Endocrine system development, GO:0035270 Skeletal system development, GO:0001501 Pattern specification process, GO:0007389 Embryonic organ development, GO:0048568 Morphogenesis of an epithelium, GO:0002009

X0

X1

X2

X3

X4

X5

X6

X7

Development of primary sexual characteristics, GO:0045137

Heart development, GO:0007507


Brain development, GO:0007420


Gland development, GO:0048732

Eye development, GO:0001654

Urogenital system development, GO:0001655

Kidney development, GO:0001822


Endocrine system development, GO:0035270

Skeletal system development, GO:0001501

Pattern specification process, GO:0007389

Embryonic organ development, GO:0048568

Morphogenesis of an epithelium, GO:0002009

5

Enrichment

-5

Depletion

X0

X1

X2

X3

X4

X5

X6

X7

Development of primary sexual characteristics, GO:0045137

Endocrine system development, GO:0035270


Gland development, GO:0048732



Eye development, GO:0001654

Urogenital system development, GO:0001655

Heart development, GO:0007507

Brain development, GO:0007420


Kidney development, GO:0001822



Skeletal system development, GO:0001501

5

Enrichment

-5

Depletion

Figure S3

C D

Supplementary Figure 6. Distinguishing active and repressive promoter chromatin states by PRIMs in (A) SSCs, (B) MASCs, (C) ESCs, (D) MEFs, and (E) iPS. Based on peak detection results at promoters, all genes were assigned to each of four histone modification categories as in Figure S4A (left). Probability density distribution of PRIMs was made for each of the three categories, K4me3 only (green line), K4me3+K27me3 (yellow line), and K27me3 only (red line), and cut-off value between K4me3 and K4me3+K27me3 or K27me3 groups was selected by the crossover between K4me3 and K4me3+K27me3 plot (dashed line). X-axis, PRIMs. Y-axis, probability density. Cut-off value, SSC=2.7, MASC=2.2, ESC=2.2, MEF=2.1, iPS=2.0 (middle). Expression of genes in promoter chromatin states delineated by active (above PRIM cut-off) and repressive (at or below PRIM cut-off) marks is shown in boxplot (Right). The bottom and top of the boxes indicate the 25th and 75th percentiles, the central bars indicate medians and whiskers indicate non-outlier extremes. Dashed line, median expression value of all genes.

0%

20%

40%

60%

80%

100%

MASC

% To

tal P

rom

oter

K4me3 Only K4me3+K27me3 K27me3 Only No Modification

0%

20%

40%

60%

80%

100%

SSC

% To

tal P

rom

oter


0%

20%

40%

60%

80%

100%

ESC (MTS07)

% To

tal P

rom

oter


02

46

810

12

Gene

Expre

ssion

(log2

FPKM

)

02

46

810

120

24

68

1012

Gene

Expre

ssion

(log2

FPKM

)

02

46

810

120

24

68

1012

Gene

Expre

ssion

(log2

FPKM

)

02

46

810

12

A

B

C

Gene Expression

Repressive (PRIMlow)

Active (PRIMhigh)

Chromatin State

Gene Expression


Active (PRIMhigh)

Chromatin State

Gene Expression


Active (PRIMhigh)

Chromatin State

0.0

0.1

0.2

0.3

0.4

0.5

−5 0 5 10

K4K4K27K27

Dens

ity

Repressive (R) Active (A)

Promoter with Peak

K4me3 Only K4me3 + K27me3 K27me3 Only

2.7

PRIM

0.0

0.2

0.4

0.6

−5 0 5 10

K4K4K27K27


Dens

ity

2.2

PRIM

0.0

0.2

0.4

−5 0 5 10

K4K4K27K27


Dens

ity

2.2

PRIM PRIMlow PRIMhigh

PRIMlow PRIMhigh

PRIMlow PRIMhigh

0%

20%

40%

60%

80%

100%

MEF (MTS07)

% To

tal P

rom

oter


0%

20%

40%

60%

80%

100%

iPS_MCV81 (MTS08)

% To

tal P

rom

oter


02

46

810

12

Gene

Expre

ssion

(log2

FPKM

)

02

46

810

120

24

68

1012

Gene

Expre

ssion

(log2

FPKM

)

02

46

810

12

D

E

Gene Expression


Active (PRIMhigh)

Chromatin State

Gene Expression


Active (PRIMhigh)

Chromatin State

0.0

0.2

0.4

0.6

−5 0 5 10

K4K4K27K27

Dens

ity


2.1

PRIM

0.0

0.2

0.4

0.6

−5 0 5 10

K4K4K27K27De

nsity


2.0

PRIM PRIMlow PRIMhigh

PRIMlow PRIMhigh

Figure S4

Supplementary Figure 7. Promoter chromatin states, transcriptional activities, and biological functions of genes activated during reprogramming of SSCs (Class I). (A) Comparison of chromatin states (PRIMs) between ESCs and MASCs for all class I

promoters (grey dots). X-axis, PRIMs from ESCs. Y-axis, PRIMs from MASCs. Black dots, class I example genes displayed in Figure 3A. Dashed black line, cut-off between active and repressive chromatin states. Dark grey line, optimal curve fits to all class I promoters. r, correlation coefficient with all class I promoter PRIMs.

(B) Transcriptional activities of selected gene subsets. Genes are grouped by promoter PRIMs as in Figure 4A. The bottom and top of the boxes indicate the 25th and 75th percentiles, the central bars indicate medians and whiskers indicate non-outlier extremes. p-values were calculated using Wilcoxon tests. **, p-value < 0.01. *, p-value < 0.05. Dashed line, median expression value of all genes.

(C) GO enrichment in all Class I genes. Genes are grouped by promoter PRIMs as in Figure 4A. A, Active chromatin state. R, Repressive chromatin state. M, Histone modification (K4me3 or K27me3) identifiable. N, Histone modification (K4me3 or K27me3) unidentifiable. Bottom, number of genes in each group.

C

X0 X1 X2 X3 X4 X5 X6 X7Mesodermal cell fate commitment, GO:0001710Stem cell maintenance, GO:0019827Phosphate metabolic process, GO:0006796in utero embryonic development, GO:0001701Regulation of cell cycle, GO:0051726Positive regulation of ossification, GO:0045778Neuron development, GO:0048666Cell morphogenesis involved in differentiation, GO:0000904Respiratory system development, GO:0060541Blastoderm segmentation, GO:0007350Negative regulation of cell differentiation, GO:0045596Embryonic pattern specification, GO:0009880Vasculature development, GO:0001944Embryonic organ development, GO:0048568Morphogenesis of an epithelium, GO:0002009

5

Enri

chme

nt

-5

Depl

etio

n

MASC

SSC

A

R

M

N

R

R

N

M

A

A

R

A

N

N

-

Genes 100 40 107 25 239 19 206

(Mod

ified

) (S

tabl

e I)

(Act

ive)

X0

X1

X2

X3

X4

X5

X6

X7

Regulation of cell cycle, GO:0051726

Regulation of fibroblast proliferation, GO:0048145


Neuron fate commitment, GO:0048663

Venous blood vessel morphogenesis, GO:0048845




Stem cell maintenance, GO:0019827


Primary neural tube formation, GO:0014020


in utero embryonic development, GO:0001701

Placenta development, GO:0001890

Lung morphogenesis, GO:0060425

5

Enrichment

-5

Depletion

Regulation of cell cycle, GO:0051726 Regulation of fibroblast proliferation, GO:0048145 Pattern specification process, GO:0007389 Neuron fate commitment, GO:0048663 Venous blood vessel morphogenesis, GO:0048845 Embryonic organ development, GO:0048568 Cell fate commitment, GO:0045165 Vasculature development, GO:0001944 Stem cell maintenance, GO:0019827 Heart morphogenesis, GO:0003007 Primary neural tube formation, GO:0014020 Morphogenesis of an epithelium, GO:0002009 in utero embryonic development, GO:0001701 Placenta development, GO:0001890 Lung morphogenesis, GO:0060425

−4 −2 0 2 4 6 8

−4−2

02

46

8

Class I Promoters

ESC (PRIM)

MAS

C (P

RIM

)

No

No

r = 0.86

Repressive Active

Rep

ress

ive

Act

ive

Figure S7 A B

1.0 1.5 2.0 2.5 3.0

02

46

810

1214

SSC_avg MASC_avg ESC_avg

02

46

810

1214

Gen

e Ex

pres

sion

[log

2 FP

KM]


02

46

810

1214


02

46

810

1214


02

46

810

1214 Active Genes

Modified GenesStable I GenesAll Class I Genes

SSC MASC ESC

1.0 1.5 2.0 2.5 3.0

02

46

810

1214 Type (a) Promoters

Type (b) PromotersType (c) PromotersAll Class I Promoters

1.0 1.5 2.0 2.5 3.0

02

46

810


Type (b) PromotersType (c) PromotersAll Class I Promoters

MASCActive Genes MASCModified Genes

MASCStable I Genes All Class I Genes

** *

*

** **

**

*

** **

**

Supplementary Figure 8. Promoter chromatin states, transcriptional activities, and biological functions of gene repressed during reprogramming of SSCs (Class II). (A) Comparison of chromatin states (PRIMs) between ESCs and MASCs for all class II

promoters (grey dots). X-axis, PRIMs from ESCs. Y-axis, PRIMs from MASCs. Black dots, Class II example genes displayed in Figure 3A. Dashed black line, cut-off between active and repressive chromatin states. Dark grey line, optimal curve fits to all class II promoters. r, correlation coefficient with all class II promoter PRIMs.

(B) Transcription activities of selected gene subsets. Genes are grouped by promoter PRIMs as in Figure 4C. The bottom and top of the boxes indicate the 25th and 75th percentiles, the central bars indicate medians and whiskers indicate non-outlier extremes. p-values were calculated using Wilcoxon tests. **, p-value < 0.01. *, p-value < 0.05. Dashed line, median expression value of all genes.

(C) GO enrichment in all Class II genes. Genes are grouped by promoter PRIMs as in Figure 4C. A, active chromatin state. R, repressive chromatin state. M, histone modification (K4me3 or K27me3) is identifiable. N, histone modification (K4me3 or K27me3) is unidentifiable. Bottom, number of genes in each group.

C

X0

X1

X2

X3

X4

X5

X6

X7

Regulation of gene expression, epigenetic, GO:0040029DNA methylation involved in gamete generation, GO:0043046Spermatogenesis, GO:0007283piRNA metabolic process, GO:0034587Meiosis, GO:0007126Sexual reproduction, GO:0019953Morphogenesis of an epithelium, GO:0002009Pattern specification process, GO:0007389Skeletal system development, GO:0001501Stem cell division, GO:0017145Somatic stem cell division, GO:0048103Germ cell development, GO:0007281

5

Enrichment

-5

Depletion

Regulation of gene expression, epigenetic, GO:0040029 DNA methylation involved in gamete generation, GO:0043046 Spermatogenesis, GO:0007283 piRNA metabolic process, GO:0034587 Meiosis, GO:0007126 Sexual reproduction, GO:0019953 Morphogenesis of an epithelium, GO:0002009 Pattern specification process, GO:0007389 Skeletal system development, GO:0001501 Stem cell division, GO:0017145 Somatic stem cell division, GO:0048103 Germ cell development, GO:0007281

-

MASC

SSC

R

A

N

M

R

R

M

N

A

A

A

R

N

N

Genes 444 84 82 8 164 0 131

(Unm

odifi

ed)

(Sta

ble

II)

(Rep

ress

ive)

−4 −2 0 2 4 6 8

−4−2

02

46

8

Class II Promoters

ESC (PRIM)

MAS

C (P

RIM

)

No

No

r = 0.90

Repressive Active

Rep

ress

ive

Act

ive

Figure S8 A B

1.0 1.5 2.0 2.5 3.0

02

46

810


02

46

810

Gen

e Ex

pres

sion

[log

2 FP

KM]


02

46

810


02

46

810


02

46

810 Repress Genes

No Modified GenesStable II GenesAll Class II Genes

1.0 1.5 2.0 2.5 3.0

02

46

810


Type (b) PromotersType (c) PromotersAll Class II Promoters

1.0 1.5 2.0 2.5 3.0

02

46

810


Type (b) PromotersType (c) PromotersAll Class II Promoters

SSC MASC ESC

MASCRepressive Genes MASCUnmodified Genes

MASCStable II Genes All Class II Genes

** **

**

** **

**

* **

**

Supplementary Figure 9. K27ac modification in different cell types (blue) and transcription factor enrichment in ESCs (black) at selected genes. (A) Zic3 (Class I, MASCActive). (B) Nanog (Class I, MASCModified). (C) Zbtb16 (Class II, MASCRepressive). Core pluripotency regulators (Pou5f1, Sox2, Nanog) are highlighted in red. Pink region, Promoter. Blue bar at bottom, enhancer. ME, enhancers active in both MASCs and ESCs. SME, enhancers active in SSC, MASCs and ESCs. S, enhancers active only in SSCs. K27ac and transcription factor track range, 0 – 1.

SSC MASC

ESC Suz12

Zfx Nr5a2

Nanog Sox2

Pou5f1 Klf4

Esrrb Tfcp2l1

Nanog Zic3

ME ME SME ME ME ME ME SME ME S S S

Zbtb16

A B C

10 kb

Figure S7

Supplementary Figure 10. 3D PCA plot based on PRIMs of all promoters with K4me3+K27me3 bivalent histone modifications in SSCs. For SSCs, iPSCs, and MEFs, only in-house data were analyzed. Different cell types are distinguished by colors as in Figure 1.

0.220.20.180.16

-0.1

-0.2

0.14

0.40.2

0.3

0.2

0.1

0

0.1-0.1 0-0.2-0.3-0.4

SSC Bivalent Promoters (PRIM) in Different Cell Types

SSC.1 SSC.2 SSC.3

MASC.3 MASC.1/2

PC2

PC1

PC3 Figure S1

-0.2

0.4

0.2

0

00.1

-0.1-0.2

-0.3

0.12 0.14 0.16 0.18

HSC.1 HSC.2

MEF

SSC.1/2/4 SSC.3

iPS.1

iPS.2 MASC.3/4/5/6 MASC.2

MASC.1

ESC.1

ESC.2

PC3

PC1 PC2


A B

0.14

-0.1

0

-0.2

0.4

0.1

0.2

0.3

0.150.160.170.180.190.2

0.2-0.2 0

PC3

PC1

PC2

Epigenome at Promoters (PRIM)

SSC.3 SSC.2

SSC.1 PiPS_MCV6 PiPS_MCV8

HSC.2 HSC.1 MEF

iPS.1

iPS.2 MASC.1 MASC.3

MASC.2

ESC_MTS07 ESC_WJA12

ESC_BLA13

020

4060

8010

0


ESC MASC PiPS iPS

020

4060

8010

0


SSC PGC AGSC PS RS Spermatozoon

020

4060

8010

0


MEF HSC, LSK Macrophage q/a-HFSC, HFTAC

Pluripotent Germ Cells Somatic Cells / Multipotent Cells

Supplementary Figure 11. Histone modifications at selected genes in different types of germ cells. Green, K4me3 modification. Red, K27me3 modification. K4me3 track range, 0 – 1. K27me3 track range, 0 – 0.5.

Sox2

Cdx2 Gata6

E12.5 E13.5 E14.5

PGC

AGSC

PS

RS

Sperm

ESC

SSC

MEF

K4me3

K27me3

K4me3

K27me3

K4me3

K27me3

K4me3

K27me3

K4me3

K27me3

K4me3

K27me3

K4me3

K27me3

K4me3

K27me3

K4me3

K27me3

K4me3

K27me3

10 kb

A B C

Figure S8

Supplementary Figure 12. Analysis of SSC cell surface marks by flow cytometry.

Specific antibody

Control immunoglobulin

83.4%

Cou

nt

23.7% 89.4%

Figure S12

a6-integrin CD9 Thy-1 c-Kit

Documents

Supplementary Figure 1. Dendrogram based on (A) … · Supplementary Figure 2. 3D PCA plot based on (A) expression or (B) PRIMs of all protein-coding and noncoding genes. For SSCs,