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Cell Stem Cell
Supplemental Information
Quantitative Dynamics of Chromatin Remodeling
during Germ Cell Specification
from Mouse Embryonic Stem Cells
Kazuki Kurimoto, Yukihiro Yabuta, Katsuhiko Hayashi, Hiroshi Ohta, Hiroshi Kiyonari,
Tadahiro Mitani, Yoshinobu Moritoki, Kenjiro Kohri, Hiroshi Kimura, Takuya Yamamoto,
Yuki Katou, Katsuhiko Shirahige, and Mitinori Saitou
Kurimoto et al., Supplemental Figure S1
456789
1011121314 Prdm1
Prdm14Tfap2cKitDnd1Itgb3
Pou5f1NanogSox2EsrrbTbx3Klf4Tcfcp2l1
TFgf8Hoxb1Hoxa1Mesp1Hand1Cdx2Cdx1Snai1Cdh2
Fgf5Dnmt3aDnmt3bMycOtx2
Log 2
exp
ress
ion
leve
l
ESC EpiLC d2PGCLC
d6PGCLC
ESC EpiLC d2PGCLC
d6PGCLC
ESC EpiLC d2PGCLC
d6PGCLC
ESC EpiLC d2PGCLC
d6PGCLC
d2 PGCLC d6 PGCLCF
B
A
C D
E
Rep
2
Rep
2
Rep1 Rep1 d2 avg.
d6 a
vg.
Testis
Ovary
Prdm1 (+/+) Prdm1(EGFP-Blimp1/EGFP-Blimp1)
E12.5 gonad, Prdm1(+/EGFP-Blimp1)MVH GFP DAPI/MVH/GFP
BLIMP1
EGFP-BLIMP1
Prdm1 (EGFP-Blimp1/EGFP-Blimp1) Prdm1 (+/+)
αTUBLIN
100
50
kDa
E12.5 PGC
Male
Female
108640-2-4 108640-2-4
10
8
6
4
0
-2
-4
10
8
6
4
0
-2
-4108640-2-4
10
8
6
4
0
-2
-4
Ex1Ex5 Ex6 Ex7 Ex8
Wild type3.1k
0.7k
ATG
EGFP-Blimp1-Neo
Targeting vectorMC1-DTA-pA
pApGKNeo
pApGKNeo
NotI
SwaI
SwaI
AvrII
HindIIIHindIII
HindIII
HindIII
AvrII
AvrII
AvrII
EGFPSALA
0.1k
EGFP
PmlI
PmlI
PmlI
SwaI
5’ probe3’ probe
Prdm1Ex3/F Prdm1Ex3/R
Prdm1Int3/F Prdm1Int3/R(247 bp)
(901 bp)
EGFP-Blimp1 EGFP
PmlI
Prdm1Ex3/F Prdm1Ex3/R(154 bp)
Figure S1. Expression of Key Genes During In Vitro PGC Specification, and Generation and Analysis of EGFP-Blimp1 Knock-in Mice, Related to Figure 1 and Figure 7(A) The expression patterns of genes for germ cell specification/development, pluripotency, an early mesodermal program, and epiblast development during in vitro PGC specification are shown with standard deviations (SDs) of two biological replicates. (B) Schematic representation of the wild-type Blimp1 locus, the targeting vector, and the EGFP-Blimp1 knock-in allele. White boxes: non-coding exons; black boxes: coding exons; black triangles: loxP sequences; LA: long arm; SA: short arm; folded line: MC1-DTA-pA cassette for negative selection. Key restriction enzyme sites and primers for genotyping are shown. (C) Histological sections of the testes and ovaries of the wild-type and EGFP-Blimp1 homozygous knock-in mice stained by hematoxylin and eosin. EGFP-Blimp1 homozygous knock-in mice are healthy and fertile, with their testes and ovaries showing normal spermatogenesis and oogenesis, respectively, indicating that the EGFP-BLIMP1 fusion protein functions appropriately in vivo. Bar, 50 µm. (D) Immunofluorescence analysis of MVH (red) and EGFP-BLIMP1 (green) expression counterstained by DAPI in the embryonic gonads (top, male; bottom, female) at E12.5 of EGFP-Blimp1 heterozygous knock-in mice. Note that EGFP-BLIMP1 localizes specifically in the nuclei of MVH-positive PGCs. Bar, 20 µm. (E) Western blot analysis of EGFP-BLIMP1 and BLIMP1 expression in PGCs at E12.5 in EGFP-Blimp1 homozygous knock-in and wild-type mice, respectively. αTUBLIN was used as a control. (F) Contour graphs for scatter plot comparisons of EGFP-BLIMP1 peaks in the two biological replicates (Rep.1 and Rep. 2) of d2 and d6 PGCLCs (top left and right, respectively), and of averaged EGFP-BLIMP1 peaks in d2 and d6 PGCLCs (bottom).
Normalized EGFP-BLIMP1 (log2)
Nor
mal
ized
EG
FP-B
LIM
P1
(log 2
)
C D
E F
Kurimoto et al., Supplemental Figure S2
J
LESC EpiLC
genome wide H3K27me3 Rep1 (log2) genome wide H3K27me3 in ESC avg. (log2)-8 -4 0 44 -8-8 -4 0 -4 0 4-8 -4 0 4-8 -4 0 4 -8 -4 0 4 -8 -4 0 4 -8 -4 0 4 -8 -4 0 4
Rep
2 (l
og2)
Epi
LC a
vg.
d2 L
IF A
g av
g.
d2 P
GC
LC a
vg.
d6 P
GC
LC a
vg.
G ESC
Rep
2 (lo
g 2)
Rep
2 (lo
g 2)
EpiLC d2 PGCLC d6 PGCLC
20-2-4-6-8 20-2-4-6-8 20-2-4-6-8 20-2-4-6-8 20-2-4-6-8 20-2-4-6-8 20-2-4-6-8
20
-2-4-6-8
H3K9me2 IP level at TSS ±1Kb Rep1 (log2)
H3K27me3 IP level at TSS ±1Kb Rep1 (log2)
H3K9me2 IP level at TSS ±1Kb in ESC avg. (log2)
H3K27me3 IP level at TSS ±1Kb in ESC avg. (log2)
Epi
LC a
vg.
d6 P
GC
LC a
vg.
d2 P
GC
LC a
vg.
H
I
genome wide H3K9me2 Rep1 (log2) genome wide H3K9me2 in ESC avg. (log2)
-8
-4
0
-8 -4 0 4 -8 -4 0 4-8 -4 0 4-8 -4 0 4 -8 -4 0 4 -8 -4 0 4 -8 -4 0 4
4
-8
-4
0
4
0-22-44-66-88-1010-1212-1414-1616-18 Log2 num
ber of genom
ic loci
0-22-44-66-88-1010-1212-1414-1616-18 Log2 num
ber of genom
ic loci
Log2 number
of genes
0-11-22-33-44-55-66-77-88-9
Log2 number
of genes
0-11-22-33-44-55-66-77-88-9
Epi
LC a
vg.
d2 P
GC
LC a
vg.
d6 P
GC
LC a
vg.
d2 LIF Ag d2 PGCLC d6 PGCLC
ESC EpiLC d2 PGCLC d6 PGCLC
420-2-4-6-8-10 420-2-4-6-8-10 420-2-4-6-8-10 420-2-4-6-8-10 420-2-4-6-8-10 420-2-4-6-8-10 420-2-4-6-8-10 420-2-4-6-8-10 420-2-4-6-8-10
20
-2-4-6
ESC EpiLC d2LIF Ag d2 PGCLC d6 PGCLC
Rep
2 (l
og2)
Epi
LC a
vg.
d2 L
IF A
g av
g.
d2 P
GC
LC a
vg.
d6 P
GC
LC a
vg.
LINE 1ORF2
SINEB1
IAPChI
P-s
eq IP
leve
l (lo
g 2)
norm
aliz
ed b
y C
hIP
-QP
CR
(L
INE
1 O
RF2
)
H3K9me2ESCEpiLCd2 PGCLCd6 PGCLC3
210
-1-2-3-4
ChI
P-s
eq IP
leve
l (lo
g 2)
norm
aliz
ed b
y C
hIP
-QP
CR
(L
INE
1 O
RF2
)
LINE 1ORF2
SINEB1
IAP
ESCEpiLCd2 LIF Agd2 PGCLCd6 PGCLC
H3K27me3
10
-1-2-3-4-5-6
ChI
P-s
eq IP
leve
l (lo
g 2)
norm
aliz
ed b
y C
hIP
-QP
CR
(L
INE
1 O
RF2
)
ChIP Q-PCR ∆Ct (ChIP-Input)
H3K9me2SINE B1IAP
ESC
ESC
d6
EpiLCd2
d6
d2
EpiLC
2
1
0
-1
-2
-3
-44 3 2 1 0 -1 -2
y = -1.26x + 0.85R2 = 0.67
ChI
P-s
eq IP
leve
l (lo
g 2)
norm
aliz
ed b
y C
hIP
-QP
CR
(L
INE
1 O
RF2
)
ChIP Q-PCR ∆Ct (ChIP-Input)
SINE B1IAP
ESC
ESCd6EpiLC
d2d2 LIF Ag
d6
d2d2 LIF Ag
EpiLC
H3K27me31
0
0
-1
1 1
-2
2
-3
3
-4
4
-5
5-6
6
y = -1.04x + 0.60R2 = 0.94
Figure S2. Distribution of Histone H3, and Normalization and Comparison of ChIP-seq Data for H3K3me2 and H3K27me3, Related to Figure 3 and Figure 5(A) ChIP-Q-PCR analysis of histone H3 on the LINE L1 ORF2, SINEB1, and IAP for ESCs, EpiLCs, d2 and d6 PGCLCs (color codes as indicated). (B) The log2 IP/input-frequency plots of histone H3 for the genome (single-copy regions, 2 Kb sliding windows with 1 Kb overlaps, red) and around TSSs (within 1Kb) of the HCP, ICP, and LCP genes (color codes as indicated) during in vitro PGC specification. (C) ChIP-seq values of H3K9me2 on the repetitive elements normalized by the ChIP-Q-PCR values of H3K9me2 on the LINE L1 ORF2 for ESCs, EpiLCs, and d2 and d6 PGCLCs (color codes as indicated). (D) ChIP-seq values of H3K9me2 on SINE B1 and IAP normalized by the ChIP-Q-PCR values of H3K9me2 on the LINE L1 ORF2 plotted against ChIP-Q-PCR values for SINE B1 and IAP for ESCs, EpiLCs, and d2 and d6 PGCLCs (SDs, two biological replicates). (E) ChIP-seq values of H3K27me3 on the repetitive elements normalized by the ChIP-Q-PCR values of H3K27me3 on the LINE L1 ORF2 for ESCs, EpiLCs, d2 LIF Ag, and d2 and d6 PGCLCs (color codes as indicated). (F) ChIP-seq values of H3K27me3 on SINE B1 and IAP normalized by the ChIP-Q-PCR values of H3K27me3 on the LINE L1 ORF2 plotted against ChIP-Q-PCR values for SINE B1 and IAP for ESCs, EpiLCs, and d2 and d6 PGCLCs (SDs, two biological replicates). (G) Contour graphs for scatter plot comparisons of H3K9me2 around the TSSs (within 1 Kb) in the two biological replicates (Rep.1 and Rep. 2) of ESCs, EpiLCs, and d2 and d6 PGCLCs (left), and between averaged values in ESCs and those in EpiLCs, or in d2 or d6 PGCLCs (right). (H) Venn diagrams of genes with H3K9me2 detected within 1 Kb from TSSs in the two biological replicates of ESCs, EpiLCs, and d2 and d6 PGCLCs. (legend continued on next page)
15546 964297
EpiLC
16926 4134
d2 PGCLC
16581 116275
d6 PGCLCESC
135121175 128
K15840 105483
EpiLC
16611 24345
d2PGCLC
16622 212151
d6PGCLCd2 LIF Ag
16954 4216
ESC
16036427 454
A B0
1
2
3
4
5
ESCEpiLC
d2 PGCLCd6 PGCLC
LINE1ORF2
SINE B1 IAP
∆Ct (
ChI
P-In
put)
H3 IP/Input (log2)-3 -2 -1 0 1 2
Freq
uenc
y
ESC
EpiLC
d2 PGCLC
d6 PGCLC
Genome
TSS
HCPICPLCP
D
Kurimoto et al., Supplemental Figure S3
OCT4, ESC OnlyTop50%
OC
T4
ES
C
Epi
LC
d2 d6
H3K27acOCT4, ESC&EpiLCTop50%
OC
T4
ES
C
Epi
LC
d2 d6
H3K27acOTX2, EpiLC OnlyTop50%
OTX
2
ES
C
Epi
LC
d2 d6
H3K27ac
±10 Kb0 0 55
Figure S3. Normalization and Comparison of ChIP-seq Data for H3K4me3 and H3K27ac, and Relationships between H3K27ac Peaks and OCT4-binding Sites During In Vitro PGC Specification, Related to Figure 2 (A) Contour graphs for scatter plot comparisons of H3K4me3 in the two biological replicates (Rep.1 and Rep. 2) of ESCs, EpiLCs, d2 LIF Ag, and d2 and d6 PGCLCs (left), and between averaged values in ESCs and those in EpiLCs, d2 LIF Ag, d2 or d6 PGCLCs (right). (B) Venn diagrams of genes with H3K4me3 peaks around TSSs (< 2Kb) in the two biological replicates of ESCs, EpiLCs, d2 LIF Ag, and d2 and d6 PGCLCs. (C) Contour graphs for scatter plot comparisons of H3K27ac in the two biological replicates (Rep.1 and Rep. 2) of ESCs, EpiLCs, d2 LIF Ag, and d2 and d6 PGCLCs (left), and between averaged values in ESCs and those in EpiLCs, d2 LIF Ag, d2 or d6 PGCLCs (right). (D) Heat map representation of the relationships of H3K27ac peaks during in vitro PGC specification with (left) OCT4-binding sites specific to ESCs (top 50%), (middle) OCT4-binding sites common to ESCs and EpiLCs (top 50%), and (right) OTX2-binding sites specific to EpiLCs (top 50%) (Buecker et al., 2014).
A
B
C
ESC EpiLC d2 LIF Ag d2 PGCLC d6 PGCLC
Rep
2 (l
og2)
H3K4me3 Rep1 (log2) H3K4me3 ESC avg. (log2)8 106420-2 8 106420-2 8 106420-2 8 106420-2 8 106420-2 8 106420-2 8 106420-2 8 106420-2 8 106420-2
810
6420
-2-4
Epi
LC a
vg.
d2 L
IF A
g av
g.
d2 P
GC
LC a
vg.
d6 P
GC
LC a
vg.
ESC EpiLC d2LIF Ag d2PGCLC d6PGCLC
Rep
2 (l
og2)
H3K27ac Rep1 (log2) H3K27ac ESC avg. (log2)86420-2-4 86420-2-4 86420-2-4 86420-2-4 86420-2-4 86420-2-4 86420-2-4 86420-2-4 86420-2-4
86420
-2-4
Epi
LC a
vg.
d2 L
IF A
g av
g.
d2 P
GC
LC a
vg.
d6 P
GC
LC a
vg.
Log2 number
of peaks
0-11-22-33-44-55-66-77-88-99-10
Log2 number
of peaks
0-11-22-33-44-55-66-77-88-99-1010-11
9579598 679
ESC
10554 356502
EpiLC
9706315 587
d2 LIF Ag
8578 1533280
d2 PGCLC
9101 715587
d6 PGCLC
(Legend for Figure S2, continued)(I) Contour graphs for scatter plot comparisons of genome-wide H3K9me2 (2 Kb sliding windows with 1 Kb overlaps) in the two biological replicates (Rep.1 and Rep. 2) of ESCs, EpiLCs, and d2 and d6 PGCLCs (left), and between averaged values in ESCs and those in EpiLCs, d2 or d6 PGCLCs (right). (J) Contour graphs for scatter plot comparisons of H3K27me3 around the TSSs (within 1 Kb) in the two biological replicates (Rep.1 and Rep. 2) of ESCs, EpiLCs, d2 LIF Ag, and d2 and d6 PGCLCs (left), and between averaged values in ESCs and those in EpiLCs, d2 LIF Ag, d2 or d6 PGCLCs (right). (K) Venn diagrams of genes with H3K27me3 detected within 1 Kb from TSSs in the two biological replicates of ESCs, EpiLCs, d2 LIF Ag, and d2 and d6 PGCLCs. (L) Contour graphs for scatter plot comparisons of genome-wide H3K27me3 (2 Kb sliding windows with 1 Kb overlaps) in the two biological replicates (Rep.1 and Rep. 2) of ESCs, EpiLCs, and d2 and d6 PGCLCs (left), and between averaged values in ESCs and those in EpiLCs, d2 LIF Ag, d2 or d6 PGCLCs (right).
PGCLC PGCLC PGCLC
C
BA
ED
Kurimoto et al., Supplemental Figure S4
F G
Log 2
exp
ress
ion
leve
l
Log 2
exp
ress
ion
leve
l
pattern specification process
regulation of transcription,DNA-dependent
embryonic morphogenesis
chordate embryonic development
anterior/posterior pattern formation
regulation of cell proliferation
skeletal system development
heart development
1E-3 1E-9 1E-15
d2 LIF Ag/d2 PGCLC>2 (IP level)p value
Fgf8, Tbx3, Cdx2, Tdgf1, Nodal, Hoxb1, Hoxa4, Hoxc4
Myc, Tbx3, Vcam1, Cer1, Wnt3, Wnt4, Gata4, Hand1
Prdm1, Cdh1, Cebpa, Cited1, Gata2, Gjab5, Krt8, Pdgfrb
Cer1, Cdx1, Hoxa7, Hoxb7, Hoxc4, Hoxd3, Msgn1, Wnt3
Id1, Id2, Id3, Ascl1, Etv2, Isl1, Meis2, Msx1, Klf4, Nkx1-2
Ccnd2, Cdkn2a, Cdkn2b, Egf , Fgf16, Fosl2, Myc, Tgfa, Tgfb1
Trp63, Aes, Bmi1, Hexa, Hoxb7, Hoxd1, Hoxd3, Igfbp3, Myf
Fgf8, Hand1, Id1, Id2, Id3, Tdgf1, Tgfb2, Erbb3, Erbb4, Casp8, Cfc1
H3K27me3 IP level (log2)
H3K
27m
e3 IP
leve
l (lo
g 2)
d2 PGCLC
d6 P
GC
LCd2
LIF
Ag
Epi
LC
420-2-4-8-10
4
2
0
-2
-4
-8
-10
4
2
0
-2
-4
-8
-10
4
2
0
-2
-4
-8
-10
ESC EpiLC
d2 PGCLC d6 PGCLC
p value p value
p valuep value
Dnd1, Dazl, Dnmt3a, Spo11, Sycp3, Tdrd7, Mael, Piwil2, Piwil4Spag16, Spata4, Spata19, Spata24,Tsnax, Tssk5, Fabp9
Tlr4, Il15, Cd40, Fas, Foxp1, Tgfbr2, Satb1, ItgalCald1, Myh7, Actn2, Actn3, Ryr1, Pde4b, Camk2d
Plcz1, Pla2g3, Cyp39a1, Lipa, Plcb2, Pld4
Plcz1, Gabrb3, Gabra4, Gabre, Kcne1, Cacna1bion transportgamete generation
spermatogenesislipid catabolic process
positive regulation ofimmune system processmuscle system process
1E-2 1E-2
1E-5 1E-17 1E-29
1E-81E-51E-3 1E-4
Tlr6, Tlr12, Cxcl15, Defa5, Defb3, Defb4
Adh1, Akr1c13, Aldh1a1, Cyb5r2, Kdm5d
Cfd, Clec7a, F2r, Il17a, Il1f6, Il23a, Olr1
Slc11a2, Ftmt, Steap4, Slc11a1, Fthl17, Slc25a37
Daf2, C2, C9, IC4b, l18r1, Il18rap, Il1rl1defense response
proteolysis
inflammatory responseoxidation reduction
Adora2a, Nrtn, Neurod2, Neurog1, Hes5
Fgf2, Fgf9, Shh, Ihh, Wnt1, Vgf, Fgfr4
Hoxa7, Hoxb13, Gata2, Foxa1, Glis3, Dmrt1
Hoxc5, Dll1, Cxcr4, Gsx2, Lefty1, Nkx2-1, Pax1
Abcc8, Cacna1d, Grin2a, Kcna7, Slc10a4, Camk2b
Bmp6, Bmp8a, Bmp8b, Foxc1, Igf1, Igf2, Hoxd3, Tgfb2
Sox2, Tgfb1, Dscam, Hmx2, Itga8, Nkx2-5, Bdnf, Six3
Adora2b, Adcy1, Adcy3, Adcy5, Adrb3, Galr1
Aldh1a2, Grem1, Hoxa10, Hoxb4, Tbx1, Twsg1, Esrrb
1E-3 1E-12 1E-24neuron differentiation
cell-cell signalingregulation of transcription,
DNA-dependentpattern specification process
ion transport
skeletal system developmentregulation of cAMP biosynthetic
processsensory organ development
embryonic morphogenesis
embryonic morphogenesispattern specification process
gland developmentear development
heart developmentcell adhesion
neuron differentiation
regulation of transcription,DNA-dependent
skeletal system development
Hoxa1, Hoxb1, Bmpr1b, Nodal, Tdgf1, Otx2
Eomes, Bmi1, Fgf4, Fgf8, Gsc, Tbx3, Wnt3, Hes1
Myc, Cdx2, Cebpa, Gata4, Klf4, Msx1, Tgif1, Nkx1-2
Bmp5, Hoxa4, Hoxb7, Hoxc4, Nf1, Pax7, Pdgfra, Sox9
Cdh1, Ccnd1, Cd44, Igf1r, Lama1, Pitx2, Wnt4, Ar
Hand1, Gja1, Pax3, Tbx2, Tbx20, Mixl1, Notch1, Tnnl1
Itga3, Itgb6, Tmem8, Pcdh7, Pcdh8, Jam3, Kitl, Cd44, Thy1
Eyz1, Zic1, Hesx1, Kcnq4, Nkx3-2, Nr4a3, Six2, Celsr1, Jag1
Neurod4, Erbb2, Hoxd9, Hoxd10, Gli2, Hes1, Olig1, Nrp1
innate immune response
ESC EpiLC d2PGCLC
d6PGCLC
d2PGCLC
d6PGCLC
ES
Cge
nes
d6 P
GC
LCge
nes
6
7
8
9
10
11
12
13
14
Ezh2Rbbp4Rbbp7Aebp2Phf1EedSuz12Mtf2Jarid2Phf19
ESC EpiLC6
7
8
9
10
11
12
13
14
Mll1Mll2Mll3Setd1a
Frac
tion
of g
enom
ic re
gion
(fol
d ch
ange
of I
P le
vel
> 2)
(d2
PGCL
C/Ep
iLC<
0.5)
(d2
PGCL
C/Ep
iLC>
2)
0
0.9
0
0.06
d6/EpiLC>2d6/EpiLC<0.5
Gene
body TSS
1Kb
5Kb
10Kb
20Kb
50Kb
100K
b
>100
Kb 0
0.1
0
0.3
(d6
PGCL
C/Ep
iLC>
2)
(d6
PGCL
C/Ep
iLC<
0.5)
Distance from TSS
d2/EpiLC>2d2/EpiLC<0.5
540-560520-540500-520480-500460-480440-460420-440400-420380-400360-380340-360320-340300-320280-300260-280240-260220-240
Numberof genes
Figure S4. Analysis of the H3K27me3 Targets During In Vitro PGC Specification, Related to Figure 3(A) Relationships between the log2 gene expression levels and the log2 H3K27me3 (top), H3K9me2 (middle), and H3K4me3 (bottom) levels during in vitro PGC specification. (B) Fraction of the genomic region with fold H3K27me3 IP level changes > 2 plotted against the genomic loci in EpiLC to d2 PGCLC (top) and EpiLC to d6 PGCLC (bottom) comparisons. (C) GO analysis for the ESC, EpiLC, d2 and d6 PGCLC PRC2 targets. (D) Contour graphs for scatter plot comparisons of the log2 H3K27me3 levels between d2 PGCLCs and EpiLCs (top), d2 LIF Ag (middle), or d6 PGCLCs (bottom). (legend continued on next page)
Expression level (log2)
H3K
27m
e3Lo
g 2 le
vels
H3K
9me2
2
0
-2
-4
-6
-82
0
-2
-4
-6
-8
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
ESC EpiLC d2 PGCLC d6 PGCLCCorresponding to log2 Expr. 8
H3K
4me3
2
0
4
6
8
10
Corresponding to Log2 Expr. 8
Kurimoto et al., Supplemental Figure S5
D
F
B
A C
ESC EpiLC d2PGCLC
d6 ESC EpiLC d2 d6 ESC EpiLC d2 d6
HCP ICP LCPH3K4me3 level (log2)
H3K
27m
e3 le
vel (
log 2
)
3210
-1-2-3-4-5-6-7-8
987654321 987654321 987654321 987654321
ESCCorresponding to log2 Expr. 8
Corresponding to log2 Expr. 8
EpiLC d2 PGCLC d6 PGCLC
7180 7173 7366 7270
2031 2955 1769 25131560 644 1451 102884 83 269 44
0%10%20%30%40%50%60%70%80%90%
100%
662 581 698 619
309 588 260 489
1375 11641159
1228
94 107 323104
175 121 123 13672 123 39 145
3215 32442585
3165
315 2891030
331
EpiLC
d2 PGCLC
d2 PGCLC
d6 PGCLC
ESC
EpiLC
Neuron differentiationPattern specification process
Embryonic morphogenesis
Neuron differentiationPattern specification process
Embryonic morphogenesis
Neuron differentiationPattern specification process
Embryonic morphogenesisNeuron differentiation
Pattern specification processEmbryonic morphogenesis
Neuron differentiationPattern specification process
Embryonic morphogenesis
Neuron differentiationPattern specification process
Embryonic morphogenesisNeuron differentiation
Pattern specification processEmbryonic morphogenesis
Neuron differentiationPattern specification process
Embryonic morphogenesisNeuron differentiation
Pattern specification processEmbryonic morphogenesis
Fgf4, Nr4a3, Prox1, Zic5, Odz4
1297
501
1566
1252
307
1611
436
1123
1312
Gata2, Pbx3, Etv4, Otx2, ShhHoxc5, Notch2, Satb2, NogNr4a3, Prox1, Chrna9, Prkra
Nrtn, Nrp1, Jag2, Dscam, NrcamHoxa1, Hoxa5, Hoxc4, , Bmpr1b, Otx1, Hes7Eomes, Ovol2, Tgfb3, Twist1, Mixl1
Pax6, Olig2, Ascl1, Nrn1, Aldh1a2Hoxd13, Acvr1c, Pax7, Foxa2, Emx1Fgf8, Fgfr2, Wnt3, Esrrb, Lef1, Cyp26b1
Jag1, Dll1, Isl2, Olig1, Farp2
Nrtn, Nrn1, Aldh1a2, Hes5, Ascl1Fgf8, Hoxa1, Hoxd13, Pax6, Wnt3
Hoxb7, Hoxd3, Gata4, Msgn1, Satb2
Fgfr2, Fgf9, Itga4, Lef1, Mixl1
Pax3, Nrcam, Dscam, Cxcr4, Olig2Hoxa5, Hoxc4, Bmpr1b, Otx1, Acvr1cEomes, Tgfb3, Twist, Tgfbr2, Fgf10
Hes1, Hes5, Nrtn, Evx1, Pax6
Nrcam, Jag1 Olig1, Runx1, Dscam, Dll1
Pax3, Olig2, Cxcr4, Isl2, Nkx2-9Hoxa5, Hoxc4, Msgn1, Acvr1cFgf4, Eomes, Myo6, Rgma, Zbtb16
Otx1, Hoxb7, Hoxb8, Hoxd3, Bmpr1b, Gata4Satb2, Tgfbr2, Tgfb3, Twist1, Foxc1
Hoxa1, Hoxb6, Hoxc8, Cdx2, Tbx3Tgif1, Msx1, Mixl1, Hand1, Dkk1, Wnt3
1E-1 1E-221E-10P value
1E-1 1E-221E-10P value
1E-1 1E-221E-10P value
0-2020-4040-6060-8080-100100-120120-140140-160160-180180-200200-220220-240240-260260-280280-300300-20
Numberof genes
Arg
2M
thfd
2lC
dk5r
1G
naz
Nrp
1A
dora
2aR
tn4r
l1R
eln
Igfb
p5
∆Ct (
ChI
P-In
put)
∆Ct (α
H3K
27m
e3
- nor
mal
IgG
)
1st IP (αH3K4me3)
2nd IP (αH3K27me3)
2nd IP (normal IgG)
2nd IP (αH3K27me3 - normal IgG)
-101234
-10123
-2-1
2
01
43
-2
-2-3-4-5-6
-1
Hoxa
1 3’
UTR
Gapd
hHo
xa1
Hoxa
5Pc
sk9
Bivalent promotersK4
K27
Ed6 PGCLC
E11.5 PGCs(Sachs et al,
2013)
1012
1423
1462
1E-3 P-value1E-25Neuron differentiation
Pattern specification process
Embryonic morphogenesis
Neuron differentiation
Pattern specification process
Embryonic morphogenesis
Neuron differentiation
Pattern specification process
Embryonic morphogenesis
ESC EpiLC d2PGCLC
d6 ESC EpiLC d2 d6 ESC EpiLC d2 d6
HCP ICP LCP
5 6 7 66 5 4 5
21 1720 16
1 0 3 1
0%10%20%30%40%50%60%70%80%90%
100%
1 1 0 01 1 1 2
30 3025
27
0 08
0
0 0 0 00 0 0 0
28 2821
27
0 07
1
BivK4K27Neither
Figure S5. Analysis of Bivalent Genes During In Vitro PGC Specification, Related to Figure 4(A) Relationships between the log2 H3K4me3 levels and the log2 H3K27me3 IP levels during in vitro PGC specification. (B) Transitions of the numbers of HCP, ICP, and LCP genes (all genes including those with log2 expression levels > 8) that bear H3K4me3 (blue), H3K27me3 (green), bivalent modifications (orange), and no modifications (pale blue) during in vitro PGC specification. (C) Sequential ChIP-Q-PCR analysis of bivalent genes in EpiLCs. The IP efficiency of the 1st ChIP using the anti-H3K4me3 antibody and of the second ChIPs using the anti-H3K27me3 antibody or normal IgG were indicated as ∆Ct values from the input (upper three panels). The fold enrichment in the second ChIP was indicated as the ∆Ct value between the two 2nd IPs (anti-H3K27me3 antibody - normal IgG) (bottom panel). ChIP-Q-PCR was performed for the H3K27me3-only region (Hoxa1 3’ UTR, blue), H3K4me3-only promoter (H3K4me3 peak around the TSS of Gapdh, green), and 12 bivalent promoters (H3K4me3 peaks around the TSSs of Hoxa1, Hoxa5, Pcsk9, Arg2, Mthfd2l, Cdk5r1, Gnaz, Nrp1, Adora2a, Rtn4rl1, Reln, Igfbp5, red). Note that the bivalent promoters were enriched in both the 1st and 2nd IPs (upper two panels). Bars indicate SDs of the two biological replicates. The primers used are given in Supplemental Table S1. (D) Venn diagram representation of the transitions of bivalent genes (log2 expression levels < 8, numbers indicated) and their GO enrichment during in vitro PGC specification. (E) Venn diagram representation of the bivalent genes (log2 expression levels < 8, numbers indicated) and their GO enrichment in comparison between d6 PGCLCs and PGCs at E11.5 (Sachs et al., 2013). (F) Transitions of the histone modifications of the germline genes (log2 expression levels < 8).
Germline genes (Log2 Expr. < 8) Biv K4 K27 Neither
(Legend for Figure S4, continued)(E) GO analysis of genes bearing higher levels of H3K27me3 (> 2 fold) in d2 LIF Ag compared to d2 PGCLCs. (F) Expression of PRC2 components during in vitro PGC specification (SDs, two biological replicates). (G) Expression of H3K4 methyltransferases during in vitro PGC specification (SDs, two biological replicates).
PGCLC PGCLC
PGCLC PGCLC
B
A
C D
E
Kurimoto et al., Supplemental Figure S6
Rara,Smad3,Sirt4,Hes1,Id3,Per1,Foxo3, Fos,Jun, Klf5, ...Xrcc4,Dedd,Rbm5,Gli3,Src,Bcl2,Bcl3,Bcl6,Trp53,Gsk3b, …
Mettl4,Mbd1,Su39h2,Suv420h1,Prmt1,Prdm9,Mll3,Mll5, …
EpiSC > EpiLC
EpiLC > EpiSC
H3K27ac peaks (IP/Input>16) around non-housekeeping genes (within gene body or <15Kbp from TSS)
Bivalent in EpiLC
Bivalent in EpiSC
1713
1150
937
Neuron differentiation
Pattern specification process
Embryonic morphogenesis
Neuron differentiation
Pattern specification process
Embryonic morphogenesis
Neuron differentiation
Pattern specification process
Embryonic morphogenesis
1E-33
1E-2 1E-6 1E-11
GO Neurondifferentiation
GO Embryonicdevelopment
GO Patternspecification
EpiLC genes
0 10 20 30 40 50 60 70
64
41
50
20
37
18
18
62
Number of H3K27ac peaksaround non-house keeping genes
Freq
uenc
y
H3K27me3 enrichment level (log2)-5 543210-1-2-3-4
EpiLCEpiSC
All genes
Germlinegenes
EpiLCEpiSC
1E-3 1E-7 1E-11P value
1E-3 1E-7 1E-11P value
P value
Regulation of transcription, DNA-dependentRegulation of apoptosis
Methylation
Positive regulation of transcription, DNA-dependent
Cell adhesionMyc, Ar, Rarg, Foxd3, Nfib, Lef1, Klf12, ...
Cdh1, Cdh4, Cdh10, Fgf4, Itga8, Arhgap6, ...
EpiLC
EpiSC
EpiLC
EpiSC
EpiLC
EpiSC
H3K
27ac
H3K
4me3
H3K
27m
e3
Pou5f1 Fgf8 Id3 Rara Smad3 Mettl4Hoxa1Hoxb cluster
b1b2b3
EpiSC > EpiLCEpiLC > EpiSC
H3K27ac peaksdifferential betweenEpiLC and EpiSC
Figure S6. Comparison of the Chromatin States between EpiLCs and EpiSCs, Related to Figure 2, Figure 3, Figure 4, and Figure 6(A) Selected GO terms enriched in non-house keeping genes (log2 expression levels < 8 either in EpiLCs or EpiSCs, or >2 fold difference between EpiLCs and EpiSCs) associated with strong H3K27ac peaks differential between EpiLCs and EpiSCs (IP/input > 16, and > 2 fold difference) (within gene bodies or < 15 Kb from TSSs). (B) ChIP-seq tracks of H3K27ac, H3K4me3, and H3K27me3 for selected genes in EpiLCs and EpiSCs. (C) Number of strong H3K27ac peaks differential between EpiLCs and EpiSCs (color codes as indicated) around the non-house keeping genes classified in the indicated GO terms (neuron differentiation, embryonic development, pattern specification) and the EpiLC genes. The classification of GO terms was defined using AmiGO 2 (Ashburner et al., 2000; Carbon et al., 2009). (D) Venn diagram representation of the bivalent genes (log2 expression levels < 8, numbers indicated) and their GO enrichment in comparison between EpiLCs and EpiSCs. (E) The log2 H3K27me3 IP level-frequency plots for the germline genes in EpiLCs (magenta) and EpiSCs (blue). Pale purple and cyan lines represent the plots for all genes as references.
Kurimoto et al., Supplemental Figure S7
B
A
H
D E F G
KJ1E-2 1E-9
chordate embryonicdevelopment
embryonic morphogenesis
epithelium development
pattern specification process
cell-cell adhesionregulation of transcription,
DNA-dependent
P value
Hoxa1, Hoxb5, Tbx3,Otx2, Wnt3, Bmp4, Bmi1
Fgf4, Bmp4, Gli3, Lama5, Lefty1, Hoxc10
Acvr1c, Tgfbr3, Chd7, Krt8, Msx1
Gja1, Src, Id3, Grlf1, Cd44, Cyp7b1, Bcl2
Dnmt3a, Dnmt3b, Ehmt1, Myc, Stat3, Klf4, Meis2
Dlg1, Frem2, Itga4, Itga6, Nptn, Pcdh19
0.00000.00100.00200.00300.00400.00500.0060
Pro
babi
lity
-250b 0 250bPosition of Best Site in Sequence
Motif 4, P=1.8e-371Motif 19, P=9.9e-339
EGFP-BLIMP1, d2PGCLC Motif 2
T (TF0000006 )
Motif 6
Motif 4 Motif 19
0.00000.00100.00200.00300.00400.00500.00600.00700.0080
Pro
babi
lity
-250b 0 250bPosition of Best Site in Sequence
Motif 6, P=1.1e-274Motif 2, P=1.8e-249T, P=4.0e-40
TEGFP-BLIMP1, d2PGCLC T
BLIMP motif WWM-9(Doody et al)
BLIMP consensus(Kuo et al)
Exp
ress
ion
leve
l (lo
g 2)
C
I TFs bound genes(<15Kb from TSS)
EGFP-BLIMP1, d2PGCLCT
1742,297 566
Hoxa1,Tbx3,Ehmt1,Dlg5,Lrp1,Krt8
TFs bound genes
EGFP-BLIMP1
10855 1769 2737 462
2440352 520
130
3782 350 558 148
0%10%20%30%40%50%60%70%80%90%
100%
ALLgenes
d2 d6 T
LCPICPHCP
BLIMP1 T
BLIMP1 (d2)BLIMP1 (d6)T
Enr
ichm
ent
(pea
k nu
mbe
r/bp)
(BLI
MP
1)
Frac
tionT
0
0.1
0.2
0.3
0
0.2
0.4
0.6
0.8
1
64 256
-256
1024
-102
4
4096
-409
6
1638
4
-163
84
6553
6
-655
36
2621
44
-262
144
1048
576
-104
8576 -6
4
Distance from TSS (bp)
Fgf4SrcGja1Tdgf1Uhrf1Klf9
Mesp1Msgn1Hoxa1BmperKrt8Anxa3
ESC EpiLC d2PGCLC
d6PGCLC
4
5
6
7
8
9
10
11
12
13
14
ESC EpiLC d2PGCLC
d6PGCLC
4
5
6
7
8
9
10
11
12
13
14
embryonic morphogenesis
pattern specification process
anterior/posterior pattern formation
positive regulation of transcription,DNA-dependent
mesoderm development
neuron differentiation
Fgf8, Msgn1, Tbx3, Hoxa1
Tbx6, Mesp1, Efna1, Vegfc
Hoxa1, Hoxa2, Hoxa4, Hoxa5
Gata6, Irf1, Atxn7, Ablm1, Foxo3
Mesp1, Tbx6, Tbx3, Ext1, Nanog
Ascl1, Numb, Neurog1, Epha1
1E-2 1E-9P value
ESC
EpiLCd2 PGCLC
d6 PGCLC
d2 PGCLC
d6 PGCLC
H3K
27m
e3E
GFP
-BLI
MP
1
Fgf4Fgf3Hoxb4Hoxb5 Hoxb2
100Kb 100Kb
BLIMP1 bound genes T bound genes
Figure S7. Analysis of the BLIMP1 and T Target Genes, Related to Figure 7(A) ChIP-seq track transitions for H3K27me3- (top, blue) and BLIMP1-binding (bottom,red) in the 100 Kb regions around Fgf3 and Fgf4 (left) and Hoxb cluster (right). (B) Distribution of BLIMP1 (in d2 and d6 PGCLCs) and T [in EpiLC aggregates stimulated by BMP4 for 36 hrs (Aramaki et al., 2013)] peaks (color codes as indicated) in the genome represented by peak numbers per base pair plotted against the distances from the TSSs (red dotted line). (C) The promoter classes for genes bound by BLIMP1 and T. (D) The sequence motifs for BLIMP1 binding in comparison to those identified previously (Doody et al., 2010; Kuo and Calame, 2004). (E) Motif probability graph showing the position of the consensus motifs in the BLIMP1-binding sites. (F) The sequence motifs for T binding. A motif sequence from the JASPAR CORZ database (TF00006 as PazarID) (Mathelier et al., 2014) is shown as a reference. (G) Motif probability graph showing the position of the consensus motifs in the T-binding sites. (H) Expression of representative targets of BLIMP1 (left) and T (right) during in vitro PGC specification. (I) Venn diagram showing the overlap between genes (< 15 Kb from the TSSs) bound by BLIMP1 in d2 PGCLCs and those bound by T in EpiLC aggregates stimulated by BMP4 for 36 hrs. (J) GO analysis of BLIMP1 targets (core enrichment genes identified in Figure 7E). (K) GO analysis of T targets (core enrichment genes identified in Figure 7F).
8
SUPPLEMENTAL TABLES (Table S1-Table S6), see separate Excel documents
Table S1. Primers Used in This Study, Related to All Figures
Table S2. Antibodies Used in This Study, Related to All Figures
Table S3. Outlines and Mapping Statistics of ChIP-seq experiments, Related to All
Figures
Table S4. Gene Expression and Chromatin States Analyzed in This Study, Related
to All Figures
Table S5. Cell-type specific H3K27ac peaks and TF binding motifs in Such Peaks,
Related to Figure 2
Table S6. BLIMP1 and T Binding Sites During In Vitro PGC Specification, Related
to Figure 7
9
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Analysis of the Gene Expression for In Vitro PGC Specification
The gene expression data on an Affymetrix Gene Chip microarray reported in (Hayashi
et al., 2011; Nakaki et al., 2013) were used for the transcriptome analysis of in vitro
PGC specification (GEO numbers: GSM744093 and GSM744094 for ESCs,
GSM744095 and GSM744096 for EpiLCs, GSM1070847 and GSM1070848 for d2
PGCLCs, and GSM744101 and GSM744102 for d6 PGCLCs).
A gene list was created using Ensemble Genes with genes that are included in the
microarray probes for Affymetrix Mouse430_2. In the case that multiple probes were
assigned to a single gene, the probe that gave the highest average expression values was
selected. The promoter classes (HCPs, ICPs, and LCPs) were defined as reported
previously (Borgel et al., 2010).
Significantly expressed genes were defined as those showing an averaged log2
expression level > 8 in two biological replicates. Differentially expressed genes
(Figure 1C) were defined by significant expression levels and fold changes > 2 in at
least one pair-wise comparison among ESCs, EpiLCs, and d2 and d6 PGCLCs, and such
genes were further classified by the highest expression levels among the four key cell
types. Gene ontology analysis was performed using the DAVID gene ontology
functional annotation tool (http://david.abcc.ncifcrf.gov/) (Huang da et al., 2009a, b).
Generation of EGFP-Blimp1 Knock-in Mice and ESCs
All the animal experiments were performed under the ethical guidelines of Kyoto
University and RIKEN CDB. Noon of the day when the copulation plugs of mated
females were identified was designated as embryonic day (E) 0.5.
The targeting vector for the EGFP-Blimp1 knock-in allele, in which EGFP cDNA and a
linker sequence (the BspEI-HindIII sequence in the pEGFP-C1 plasmid; Addgene) were
inserted into the first ATG of the Prdm1 genes, was constructed using SalI-NotI and
SwaI-XhoI sites of the DT-A-pA/loxP/PGK-Neo-pA/loxP vector
(http://www.cdb.riken.jp/arg/cassette.html). The targeting vector was linearized using
the SalI site, and electroporated into the TT2 ESC line (Yagi et al., 1993).
Homologous recombination was screened by PCR using the primer set NeoGt-1/F and
Prdm1_N3659/R (Supplemental Table S1), and confirmed by Southern blot analysis
with the 5’- and 3’- probes (PCR amplicons by primers described in Supplemental Table
S1), which detected AvrII and HindIII sites of the Prdm1 locus, respectively. Random
integration was ruled out by Southern blot analysis using a probe targeting the Neo
locus. The homologous recombinant ESCs were injected into eight-cell-stage embryos
of ICR mice to generate chimeric mice. Chimeras with a high ESC contribution were
judged by coat color and mated with C57BL/6 females to generate Prdm1+/EGFP-Blmp1-Neo
10
mice, which were genotyped by PCR using Prdm1 Ex3/F and Ex3R primers
(Supplemental Table S1) and Southern blot analysis using tail tip DNAs. The
heterozygous mice were mated with the EIIa-Cre transgenic mice (Jackson Laboratory)
to excise the pGK-Neo-pA and generated Prdm1 heterozygous +/EGFP-Blmp1
mice, which
were genotyped by PCR using the Prdm1 Int3/F and Int3/R primers (Supplemental
Table S1). The heterozygous mice were intercrossed to generate homozygous Prdm1
EGFP-Blmp1/ EGFP-Blmp1 (EGFP-Blimp1 knock-in) mice (Accession No.
CDB0923K: http://www.cdb.riken.jp/arg/mutant%20mice%20list.html), from which
EGFP-Blimp1 knock-in ESCs were generated under the conditions described previously
(Hayashi et al., 2011). The EGFP-Blimp1 homozygous knock-in mice are healthy and
fertile.
Induction from ESCs of EpiLCs and PGCLCs
BVSC (Hayashi et al., 2011) and EGFP-Blimp1 knock-in ESC lines were induced into
EpiLCs as described previously (Hayashi et al., 2011) for 48 hrs (BVSC) or 36 hrs
(EGFP-Blimp1 knock-in). The EpiLCs were then cultured under a floating condition
by plating 2-3×103 cells per well of a lipidure-coated U-bottom 96-well plate (Thermo
Scientific) in GK15 medium (Hayashi et al., 2011) containing LIF (1000 U/mL) for
induction of d2 LIF aggregate (d2 LIF Ag), LIF and BMP4 (500 ng/mL) for d2
PGCLCs, and LIF, BMP4, BMP8a (500ng /mL), SCF (100 ng/mL), and EGF (50
ng/mL) for d6 PGCLCs, respectively. Since BMP8a was proved to be omittable for
PGCLC induction (data not shown), d6 PGCLC induction from the EGFP-Blimp1
knock-in ESCs was performed without BMP8a. d2 and d6 PGCLCs of the BVSC line
were purified with a fluorescence-activated cell sorter (FACS) (ARIA III; BD
Biosciences) by fluorescence of BV. For the EGFP-BLIMP1 knock-in line, since the
EGFP signal by the EGFP-BLIMP1 fusion protein was too low to be distinctively
detected by the FACS analysis, d2 PGCLCs were purified with FACS using an anti-Kit
antibody (Supplemental Table S2) and d6 PGCLCs using anti-SSEA1 and anti-Integrin
3 antibodies (Supplemental Table S2).
ChIP-seq Data Analysis and Normalization
Read data were mapped on the mouse mm9 genome by Bowtie v0.12.9 (Langmead et
al., 2009) and manipulated by Picard-tools v1.85 (http://piard.sourceforge.net), IGV
tools v2.3.5 (Robinson et al., 2011) and Samtools v0.1.18 (Li et al., 2009) for fitting to
the subsequent analyses. Read patterns were visualized by IGV (Robinson et al.,
2011).
The distribution of H3 was constant throughout the genome, around the TSSs, and on
the major repetitive elements (LINE1 ORF2, SINE B1, and IAP) during in vitro PGC
specification (Figure S2A and S2B).
11
Peak callings for EGFP-BLIMP1, T, H3K4me3, and H3K27ac were performed using
MACS v1.4.2 (Zhang et al., 2008) with default settings (P-value cutoff 10−5
). To
enable the comparison of peaks in different samples, we scanned all peaks throughout
the genome, and considered peaks detected in proximity (within 1 Kb) as a single peak.
Read densities (FPKM) of H3K4me3 within 500 bp from peak centers were normalized
as follows: We calculated the invariant set (Li and Hung Wong, 2001) of read densities
against the biological replication 1 of ESCs, then drew a linear regression line in log
scale with a slope predefined as 1.0, and used the Y-intercept value of this line for each
sample as a coefficient for normalization. The peak intensity was defined by the
IP/input ratio [normalized read density of the peak (within 500 bp from peak centers)
over Input (within 5 Kb)] averaged in the two biological replicates. H3K4me3 peaks
closest to TSSs (<2 Kb) were identified, and the level of TSS-associated H3K4me3 was
considered significant when the peak intensities were higher than the average of the 95th
percentile for significantly expressed genes in all four key cell types. The expression
and chromatin states are summarized in Supplemental Table S4.
H3K27ac exhibited few peaks that were consistently high in all samples, and thus could
not be normalized by the same method as used for H3K4me3. Therefore, read
densities of H3K27ac within 500 bp from the peak center were normalized using an MA
plot against the replication 1 of ESCs; the average log-transformed read densities were
divided into 30 fractions, and the modes of fold differences in the top 10% of fractions
were used as normalization coefficients. Peak intensities were defined as for
H3K4me3 (see above), and only high intensity peaks (>16) were used to identify
H3K27ac peaks associated with genes, which were defined by peaks in the gene body or
within 15 Kb from TSSs. The enrichments of transcription factor motifs in H3K27ac
peaks were analyzed using MEME-ChIP (MEME v4.9.1) (Machanick and Bailey, 2011).
Cell-type specific H3K27ac peaks are listed in Supplemental Table S5.
Normalization and determination of peak intensities for EGFP-BLIMP1 and T were
performed as for H3K4me3, except that reads within 250 bp from peak centers were
used. Peaks satisfying the following criteria were used in the subsequent analyses:
intensities of >16 and significantly higher than the background levels within 5 Kb from
the peaks (average and 3× standard deviation of ChIP intensities). The peaks of
EGFP-BLIMP1 and T associated with genes were defined by those within 15 Kb from
the TSSs. The EGFP-BLIMP1 and T peaks are listed in Supplemental Table S6.
The genome-wide absolute levels of H3K27me3 and H3K9me2 appear to change in a
dynamic fashion during both in vivo and in vitro PGC specification and development
(Hayashi et al., 2011; Nakaki et al., 2013; Seki et al., 2005; Seki et al., 2007).
Therefore, by using absolute IP efficiency measured by ChIP-Q-PCR, we normalized
12
the genome-wide levels of H3K27me3 and H3K9me2 in each cell type in order to
quantify their transitions during in vitro PGC specification.
ChIPs for H3K27me3 and H3K9me2 were performed with 2-3×104 cells (two biological
replicates for each cell type) using 2 g antibodies (Supplemental Table S2)
independent from the ChIP-seq analysis. The IP efficiency of repetitive elements in
the ChIP-ed DNAs was measured by Q-PCR as ∆Ct values from the input (LINE 1
ORF2, SINE B1, and IAP; Supplemental Table S1).
For ChIP-seq data, reads mapped at more than one location of the genome (mm9) were
incorporated for repetitive sequence analysis with “-M 1” and "―best” options at
bowtie mapping (these reads were excluded from all other analyses using the “-m”
option), and the IP/input ratio of the genomic loci corresponding to the ChIP-Q-PCR
analysis was calculated. Then, the read counts of the ChIP-seq data were normalized
so that the ratios of LINE 1 ORF2 levels among the cell types in the ChIP-seq data were
the same as those in the ChIP-Q-PCR data. This normalization gave good correlations
between the ChIP-seq and ChIP-Q-PCR values of other repetitive elements (SINE B1
and IAP), and of single-copy genes (Figure S2C-S2F and data not shown).
Although the H3K27me3 signals were enriched around the TSSs, it was difficult to
define the peaks due to the lawn-type distribution of H3K27me3 (Marks et al., 2012)
and the genome-wide changes of the H3K27me3 levels. Similarly, we did not detect
H3K9me2 peaks around many TSSs (Figure 5). Therefore, for H3K27me3 and
H3K9me2, we simply counted the ChIP-seq reads within 1 Kb from the TSSs to define
the modification levels associated with the genes. The normalized IP/input ratio
[normalized read density (within 1 Kb from the TSSs, or any genomic sites) over input
(within 5 Kb)] averaged in the two biological replicates was defined as the IP level.
The H3K27me3 IP level corresponding to log2 expression level 8 was determined by
averaging for TSSs of genes with log2 expression levels of 8 ± 0.5. Enrichment levels
of H3K27me3 were defined by fold differences from the IP level corresponding to the
log2 expression level 8. TSSs with significant changes of H3K27me3 were defined by
enrichment levels > 1 and fold changes > 2 in at least one pair-wise comparison among
ESCs, EpiLCs, and d2 and d6 PGCLCs, and such TSSs were further classified by the
highest enrichment levels among the four key cell types (PRC2 targets). Bivalent
genes were defined as genes with significant H3K4me3 levels associated with the TSSs
and H3K27me3 enrichment level around the TSSs > 1.
Because d2 PGCLCs and d2 LIF aggregates showed similar global distribution of
H3K27me3 (Figure S4D), IP levels were used to identify differential H3K27me3 levels
at the TSSs between the two cell types (Figure 3H).
13
The two biological replicates of the ChIP-seq data for each histone modification and
BLIMP1 binding exhibited good reproducibility (Figure S1F, S2G-S2L, and S3A-S3C).
Consistent with the general properties of each modification, in all the cell types, we
found a positive correlation between the levels of gene expression and the levels of
H3K4me3 peaks closest to the TSSs, and negative correlations between the levels of
gene expression and the levels of H3K27me3 and H3K9me2 around (±1 Kb) the TSSs
and between the levels of H3K4me3 and the levels of H3K27me3 (Figure S4A and
S5A).
Sequential ChIP-Q-PCR
A sequential ChIP-Q-PCR was performed using 1.8×107 EpiLCs, based on a protocol
reported previously (Truax and Greer, 2012). Prior to immunoprecipitation, 18 g of a
rabbit polyclonal antibody for H3K4me3 (Abcam, ab8580) was incubated with 90 l of
M280 Dynabeads Protein G for 1 hr at room temperature. As described above,
EpiLCs were fixed and lysed, and the chromatins were solubilized by sonication (with
1×106 cells/400 L of SDS-lysis buffer per tube, and 10 cycles of sonication) and
diluted in ChIP dilution buffer. The sonication products (in total 25.2 mL) were
pooled and divided into 100 L of the Input and 25.1 mL of the first ChIP sample. The
first ChIP samples were then subjected to immunoprecipitation as described above.
Purified ChIPed DNA was eluted in 100 l of elution buffer, and diluted in 2 mL of the
ChIP dilution buffer. Prior to the second immunoprecipitation, 2 g of a rabbit
polyclonal antibody for H3K27me3 (Abcam, ab108425) and a normal rabbit IgG (Santa
Cruz, sc-2027) were incubated with 5 L of M280 Dynabeads Protein G. The diluted
and purified ChIPed DNA was then divided into 100 L of the first IP product and 2 mL
of the second ChIP sample. 900 L of the second ChIP sample was subjected to
immunoprecipitation with Dynabeads-anti-H3K27me3 antibody complex or
Dynabeads-normal rabbit IgG complex. ChIPed DNA of the second
immunoprecipitation was purified and eluted in 30 L of the elution buffer (the second
IP product). Reverse crosslink and Proteinase K treatment followed by purification of
DNA were performed as described above. QPCR was performed with the primer pairs
described in Supplemental Table S1, using 1:10 diluted DNA of the Input, the first IP
product, and the second IP product.
Comparison of the ChIP seq data for EpiLCs with those for EpiSCs
ChIP-seq and Input data for H3K4me3, H3K27ac, and H3K27me3, and transcriptome
data by microarray analysis for EpiSCs (Factor et al., 2014; Hayashi et al., 2011) were
obtained from the NCBI database (GEO accession numbers: GSE57409 for ChIP-seq,
GSM744097 and GSM744098 for microarray), and processed as described above.
Criteria regarding the levels of H3K27me3 and H3K4me3 were also defined as
described using the gene expression levels obtained from the microarray data. The
peak signals of H3K27ac (MACS P-value < 10−5
) around genes (gene bodies and < 15
14
Kb from the TSSs) were calculated and normalized to median.
Gene Set Enrichment Analysis (GSEA)
To identify expression level changes caused by loss-of-function of Blimp1, single-cell
microarray data for PGCs and Blimp1-null cells with deficient Blimp1 transcripts
(Blimp1) at the E/MB stage were used (Kurimoto et al., 2008) (GEO numbers:
GSM280697 - GSM280704 for wild-type PGCs and GSM280749 - GSM280760 for
Blimp1-null, Blimp1 transcript-positive cells), and differences in the averaged log2
expression levels were calculated between wild-type PGCs and Blimp1-null Blimp1
transcript positive cells.
To identify expression level changes caused by gain-of-function of Blimp1, microarray
data on overexpression of the Blimp1 transgene in EpiLC aggregations (TF-PGCLCs)
were used (Nakaki et al., 2013) [GEO numbers: GSM1139220 and GSM1139230 for
day 1 Blimp1-induced TF-PGCLCs (d1B-PGCLCs), GSM1139215 and GSM1139225
for EpiLCs with a Blimp1 transgene (B-EpiLCs), GSM1139218 and GSM1139228 for a
parental clone treated with doxycycline for 1 day in aggregation (d1 Parental
aggregation), and GSM1139213 and GSM1139223 for EpiLCs of a parental clone
(Parental EpiLCs)], and the difference between the consequences of Blimp1
overexpression (d1B-PGCLCs - B-EpiLCs) and of aggregation formation (d1 Parental
aggregation - Parental EpiLCs) were calculated.
To identify expression level changes correlated with T, the differences in the log2
expression levels between d2 PGCLCs and EpiLCs were calculated. GSEA
(Subramanian et al., 2005) was performed using the above-described expression level
differences and lists of genes with EGFP-BLIMP or T peaks.
Immunofluorescence Analysis of Spread Cells
d6 PGCLCs and EpiLCs induced from BVSC ESCs were purified with FACS, mixed at
a 1:1 ratio, and spread on MAS-coated glass slides (Matsunami Glass) using Cytospin4
(Thermo Scientific) as reported previously (Nakaki et al., 2013). The spread cells
were then fixed by 4% paraformaldehyde, and incubated overnight in blocking buffer
(PBS containing 0.2% Tween 20, 0.1% BSA, and 1% normal goat serum) with rabbit
anti-Lamin B1 antibody (ab16048; Abcam). The cells were washed and incubated for
2 hrs in the blocking buffer with DAPI and anti-rabbit IgG conjugated with Alexa Fluor
633. Image data were obtained using a confocal laser scanning microscope (Olympus
FV1000).
Immunofluorescence Analysis of Cryosections
Gonads at E12.5 were fixed in 4% PFA for 2 hrs at 4ºC, then sequentially soaked in PBS
containing 10% and 30% sucrose, and embedded in OCT compound (Sakura Finetek).
15
The embedded samples were frozen at −80ºC and sectioned at a 10 m thickness using a
cryostat (Leica). The cryosections were incubated overnight in the blocking buffer
containing rat anti-GFP monoclonal (Nacalai Tesque) and rabbit anti-MVH polyclonal
antibodies (Abcam), washed three times in PBS containing 0.2% Tween 20, and then
incubated in the blocking buffer containing DAPI, goat anti-rat antibody conjugated to
Alexa Fluor 488, and anti-rabbit antibody conjugated to Alexa Fluor 568. Image data
were obtained using a confocal laser scanning microscope (Olympus FV1000).
Histology
Mouse testes and ovaries were fixed in Bouin’s fixative overnight and embedded in
paraffin wax. Sectioning was performed at a 6 μm thickness using a microtome
(Leica). The sectioned samples were mounted on MAS-coated glass slides
(Matsunami Glass) and stained with hematoxylin and eosin.
Western Blot Analysis
PGCs at E12.5 of EGFP-BLIMP1 knock-in mice and Stella-EGFP transgenic mice
(Seki et al., 2007), which is the wild-type for the Prdm1 locus, were purified with
Magnetic Activated Cell Sorting (MACS; Miltenyi Biotec) using anti-SSEA-1
microbeads. The proteins were separated by 6% SDS-Polyacrylamide gel
electrophoresis, blotted to an Immobilon-P Transfer Membrane (Millipore, Bedford,
MA), and incubated with rabbit anti-BLIMP1 polyclonal antibody that we generated or
mouse anti--Tubulin monoclonal antibody (Sigma-Aldrich). Immunodetection was
performed using goat anti-rabbit or sheep anti-mouse IgGs conjugated to HRP, and an
ECL-plus western blotting detection system (GE Healthcare). Luminescent signals
were detected using an LAS4000IR multicolor luminescent image analyzer (Fuji Film).
Accession Numbers
The ChIP-seq and normalized microarray data used in this study were deposited in the
NCBI database (GEO accession numbers: GSE60204 and GSE60018).
16
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