Supplemental Data

Supplemental Figure S1. Localization of RNF2 in spermatogenesis.

Supplemental Figure S2. Spermatogenic phenotypes of PRC1cKO testes.

Supplemental Figure S3. Increased apoptosis in spermatogonia of PRC1cKO testes.

Supplemental Figure S4. Gene ontology analysis and examples of differentially expressed genes in PRC1cKO spermatogonia.

Supplemental Figure S5. Retrotransposons were not derepressed in PRC1cKO spermatogonia.

Supplemental Figure S6. Occupancy Input, H2AK119ub, and BMI1 around TSSs in wild-type GS cells.

Supplemental Figure S7. Localization of SALL4 and RNF2 during spermatogenesis.

Supplemental Figure S8. ChIP-seq enrichment and gene expression profiles of 6 clusters of genes identified by k-means clustering.

Antibody list

Supplemental Table 1 (separate file) This table contains the gene list of PRC1-regulated genes based on RNA-seq analysis. This list also contains RNA-seq reads and P-values between PRC1ctrl and PRC1cKO at Thy1+ spermatogonia and c-Kit+ spermatogonia.

Supplemental Table 2 (separate file) This table contains the gene list of each cluster identified by k-means clustering analysis of PRC1-regulated genes based on ChIP-seq analysis. This list also contains ChIP-seq reads (±2 kb around TSS) in GS cells (RNF2, BMI1, H2Ak119ub) and cultured Thy1+ spermatogonia (SALL4).

A

DAPI RNF2 PLZFGCNA

#1 (PLZF+ undifferentiated spermatogonia)

#2 (PLZF- differentiating spermatogonia) #1#1

#2#2

B

DAPI RNF2 SCML2c-Kit

#2#2

#1#1

#1 (c-Kit- undifferentiated spermatogonia)

#2 (c-Kit+ differentiating spermatogonia)

RNF2

RNF2

DAPIPLZF

DAPIc-Kit

Wild-type

Wild-type

Supplemental Figure S1. Localization of RNF2 in spermatogenesis.(A, B) Immunostaining of testicular sections at P7. Scale bars, 20 μm. The regions with yellow squares are magnified in panels shown on the right. Scale bars in magnified panels, 5 μm.

6wE H2AK119ubGCNA

H2AK119ub

PR

C1c

KO

PR

C1c

trl

G

PR

C1c

trlP

RC

1cK

O

6wP7 H&E H&E H

D P7 F

A CB

n=6 PRC1cKO

n=6

0 3 6 9

12 15 18 ***

Litte

r num

ber (

n)

PRC1ctrl

PRC1cKOPRC1ctrl

Meiosis

H2AK119ubGCNA

H2AK119ub

PR

C1c

KO

PR

C1c

trl

Test

icul

ar w

eigh

t (m

g)/ b

ody

wei

ght (

g)

PRC1cKOPRC1ctrl0 1 2 3 4

***

0 20 40 60 80

100

Pop

ulat

ion

of g

erm

cel

lsw

ith H

2AK

119u

b (%

)

PRC1ctrlPRC1cKO

P7 6w

*** ***

PRC1cKOPRC1ctrl

***

510152025

Area

dim

ensi

on o

f tu

bule

s (x

100

μm

2 )

0

I 6wPRC1ctrl PRC1cKO

H1TDAPI

0 30 60 90

120 150

H1T

+ ce

lls /

tubu

le (n

)

***

PRC1cKOPRC1ctrl

Supplemental Figure S2. Spermatogenic phenotypes of PRC1cKO testes.(A) Litter sizes when PRC1ctrl or PRC1cKO males were mated with wild-type females. Data are represented as mean ± s.e.m. Six independent mice were examined. (B) Picture of PRC1ctrl and PRC1cKO testes at 6 months. (C) Testicular weight per body weight of adult testes at the ages of 2-9 months. Data are represented as mean ± s.e.m. Five and four independent mice were scored for the PRC1ctrl and PRC1cKO, respectively. (D, E) Immunostaining of H2AK119ub and GCNA, a germ cell marker, in testicular sections at postnatal day 7 (P7) and at 6 weeks old (6w). GCNA-positive cells are shown with arrowheads. Scale bar, 20 μm. (F) Quantitative data of the percentage of germ cells with H2AK119ub at P7 and 6w. Data are represented as the mean ± s.e.m. Three independent mice were examined for each time point. (G) Histological testicular sections stained with hematoxylin and eosin. Scale bars, 20 μm. (H) Area dimen-sion of tubules in testes at 6 weeks. Center bars are medians; bottom and top bars are, respectively, 10% and 90% of data points; and bottom and top boxes are, respectively, 25% and 75% of data points. Three independent mice were examined. (I) Immunostaining of H1T in testicular sections at 6 weeks. Scale bars, 20 μm. Quantitative data are shown in right-hand panels. Data are represented as mean ± s.e.m. Three independent mice were examined for each antibody. *** P<0.0001, unpaired t-test.

C

A

DAPIKi67

GCNA

DAPIPhospho-Histone H3

GCNA

PRC1cKOPRC1ctrl

PRC1cKOPRC1ctrlD

p-H

3+ c

ells

/ G

CN

A+

cells

(%)

Ki6

7+ c

ells

/ G

CN

A+

cells

(%)

0

20

40

60

80

n.s.

PR

C1c

KO

PR

C1c

trl

0 5

10 15 20 25 30 35 n.s.

PR

C1c

KO

PR

C1c

trl

DAPICleaved Caspase-3

GCNAPRC1cKOPRC1ctrl

0

10

20

30

PR

C1c

KO

PR

C1c

trl

Cle

aved

Cas

pase

-3+

tubu

les

/G

CN

A+

tubu

les

(%)

*

*

0

10

20

15

PLZ

F+ c

ells

/ C

leav

ed C

aspa

se-3

+ ce

lls (%

)

5

PR

C1c

KO

PR

C1c

trl

DAPICleaved Caspase-3

PLZFPRC1cKOPRC1ctrl

PLZF PLZF PLZF

CleavedCaspase-3

PLZF

B

Cleaved Caspase-3

Cleaved Caspase-3

Supplemental Figure S3. Increased apoptosis in spermatogonia of PRC1cKO testes.(A, B) Immunostaining of testicular sections at P7. Cleaved Caspase-3 is an apoptosis marker, GCNA is a germ-cell marker, and PLZF is an undifferentiated spermatogonia marker. Scale bars, 20 μm. Quantitative data are shown in right-hand panels. Data are represented as mean ± s.e.m. Three independent mice were examined. * P<0.05, unpaired t-test. (C, D) Immunostaining of testicular sections at P7. Scale bars, 20 μm. Quantitative data are shown in right-hand panels. Three independent mice were examined. Data are represented as mean ± s.e.m. n.s. not significant, unpaired t-test.

CleavedCaspase-3

C

B

GO:0007155~cell adhesion GO:0035295~tube development

GO:0043067~regulation of programmed cell death GO:0007389~pattern specification process

Upregulated genes in PRC1cKO Thy1+

GO:0007049~cell cycle GO:0051276~chromosome organization

GO:0007126~meiosis GO:0000279~M phase

GO:0051028~mRNA transport GO:0006396~RNA processing

GO:0007283~spermatogenesis

Downregulated genes in PRC1cKO Thy1+

GO:0001944~vasculature development GO:0035295~tube development

GO:0007155~cell adhesion GO:0007389~pattern specification process

GO:0006928~cell motion GO:0045935~positive regulation of nucleobase, nucleoside, nucleotide GO:0001822~kidney development

GO:0050886~endocrine process GO:0006916~anti-apoptosis

Upregulated genes in PRC1cKO c-Kit+

0 10 20 30 40 50 60

GO:0007049~cell cycle GO:0000279~M phase

GO:0048285~organelle fission GO:0051276~chromosome organization

GO:0006281~DNA repair GO:0007059~chromosome segregation

GO:0006396~RNA processing GO:0007126~meiosis

GO:0006260~DNA replication GO:0016570~histone modification

GO:0019941~modification-dependent protein catabolic process

GO:0007276~gamete generation GO:0007283~spermatogenesis

GO:0000226~microtubule cytoskeleton organization

Downregulated genes in PRC1cKO c-Kit+

A

D

E

0 1 2 3 4 5 6 7

Scml2

Dnmt1

Tex15

Syce2

Fancd2

Lin28a

Atr

Tex14

Tdrd9

Tex16

Tex10

Dmrt1

Tex30

Stra8

Brca2

Tex11

Trip13

Syce1

Rb1

Sohlh1

Plzf

Nanos1

Tex13

Sohlh2

Sycp1

Taf4b

Atm

Rad51

Blm

RPKM

c-Kit+ PRC1cKOc-Kit+ PRC1ctrl

0 1 2 3 4

0 2 4 6 8

0 1 2 3 4 5

Remaining representative genes among the downregulated genes in PRC1cKO c-Kit+ spermatogonia

Supplemental Figure S4. Gene ontology analysis and examples of differentially expressed genes in PRC1cKO spermatogonia.(A, B) Gene ontology analysis of genes upregulated in PRC1cKO spermatogonia. (C, D) Gene ontology analysis of genes downregulated in PRC1cKO spermatogonia. P value represents modified Fisher Exact P Value. (E) Expression profiles of representative genes downregulated in PRC1cKO c-Kit+ spermatogonia. 15 germline genes among the top 50 downregulated genes in c-Kit+ spermatogonia with the lowest P values are shown in Figure 2D, the remaining repre-sentative genes are shown here.

P value (-log10)

PLZF PLZF6w

DAPI

LINE-1 ORF1p

0 5,000 10,000 15,000

0

2

4

-2

-4

GSAT (major satellite)SATMIN (minor satellite)

L1MdT

MMERVK10c-int

L1MdGfL1MdA

IAPEY-int

0 5,000 10,000 15,000

0

2

4

-2

-4

L1MdT

MMERVK10c-int

L1MdGfL1MdA

IAPEY-int

GSAT (major satellite)

LINE-1 ORF1p

PR

C1c

KO

PR

C1c

trl

Mean of normalized read counts (n)Mean of normalized read counts (n)

Thy1+ undifferentiated spermatogonia

c-Kit+ differentiating spermatogoniaA

BP

RC

1cK

O /

PR

C1c

trl ra

tio (l

og2)

Dow

nreg

ulat

ed in

cKO

Upr

egul

ated

in c

KO

PR

C1c

KO

/ P

RC

1ctrl

ratio

(log

2)D

ownr

egul

ated

in c

KOU

preg

ulat

ed in

cKO

Supplemental Figure S5. Retrotransposons were not derepressed in PRC1cKO spermatogonia.(A) RNA-seq analysis using Repbase. GSAT (major satellite) and SATMIN (minor satellite) were modestly derepressed in Thy1+ undifferentiated spermtogonia, and GSAT was modestly derepressed in c-Kit+ undifferentiated spermtogonia of the PRC1cKO (shown as red dots; P<0.05, Wald test and >2 fold change). Reprotransposons such as LINE, IAP, and ERVK10c were not derepressed in PRC1cKO spermatogonia (shown as blue dots). (B) Immunostaining of testicular sections at 6 weeks (6w) with LINE1 ORF1 protein (ORF1p) and PLZF, an undifferentiated spermatogonia marker. Scale bars, 20 μm.

Upregulated genes in PRC1cKODownregulated genes in PRC1cKO

0

8e-10

1.5e-9

2.3e-9

3e-9

Aver

age

tag

dens

ity (p

er b

p) RNA-seq: Thy1+ChIP: H2AK119ub in GS ChIP: H2AK119ub in GS

RNA-seq: c-Kit+B

0

8e-10

1.5e-9

2.3e-9

3e-9

DChIP: BMI1 in GSRNA-seq: c-Kit+

0

0.5e-9

1e-9

1.5e-9

2e-9

C

0

1e-9

2e-9

3e-9

4e-9

RNA-seq: Thy1+AChIP: Input in GS

Distance from TSSs (kb)

-5 -2.5 TSS 2.5 5 -5 -2.5 TSS 2.5 5 -5 -2.5 TSS 2.5 5 -5 -2.5 TSS 2.5 5

Supplemental Figure S6. Occupancy Input, H2AK119ub, and BMI1 around TSSs in wild-type GS cells.(A-D) Occupancy of Input, H2AK119ub, and BMI1 around TSSs in wild-type GS cells. ChIP-seq data of GS are shown for indicated genes based on RNA-seq analyses..

SALL4 RNF2PLZF

SALL4PLZF

DAPI

P2, Wild-type

DAPISALL4

PLZF

DAPIRNF2PLZF

DAPI

SALL4 RNF2PLZF

SALL4PLZF

DAPI

P8, Wild-type

DAPISALL4

PLZF

DAPIRNF2PLZF

DAPI

SALL4 RNF2c-Kit

SALL4c-Kit

DAPI

P8, Wild-type

DAPISALL4

c-Kit

DAPIRNF2

c-Kit

DAPI

SALL4 RNF2DMC1

SALL4DMC1

DAPI

8w, Wild-type

DAPISALL4DMC1

DAPIRNF2DMC1

DAPI

A

B

C

D

Supplemental Figure S7. Localization of SALL4 and RNF2 during spermatogenesis.(A-D) Immunostaining of testicular sections at P2 (A), P8 (B, C), and 8 weeks (8w: D). DMC1-positive leptotene and zygotene spermatocytes are shown with arrowheads. Scale bars, 20 μm. The regions with yellow squares are magnified in right panels. Scale bars in magnified panels, 20 μm.

Supplemental Figure S8. ChIP-seq enrichment and gene expression profiles of 6 clusters of genes identified by k-means clustering. (A) Distribution of ChIP-seq enrichment in each cluster of genes identified by k-means clustering shown in Figure 5B. “Other” represents all other genes that do not belong to these clusters. Center bars are medians; bottom and top bars are, respectively, 10% and 90% of data points; and bottom and top boxes are, respectively, 25% and 75% of data points. (B) Distribution of RNA-seq RPKM enrichment during representative stages of spermatogenesis in each cluster of genes identified by k-means clustering shown in Figure 5B. Other represents all other genes that do not belong to these clusters. * P<0.05, ** P<0.001, unpaired t-test.

0

2

4

6

RPK

M+1

(log

2)

PRC1ctrl Thy1+ spermatogonia

0

2

4

6

RPK

M+1

(log

2)

PRC1ctrl c-Kit+

spermatogonia

RPK

M+1

(log

2)

Wild-type pachytenespermatocytes

0

2

4

6* **

RPK

M+1

(log

2)

Wild-type round spermatids

0

2

4

6* **

Gene expression during spermatogenesis

ARNF2 in GS

Clu

ster

1C

lust

er 2

Clu

ster

3C

lust

er 4

Clu

ster

5C

lust

er 6

Oth

er

0

2

4

6

RN

F2 e

nric

hmen

t (lo

g 2)

**

H2AK119ub in GS

-2

0

2

4

H2A

K119

ub e

nric

hmen

t (lo

g 2)

*n.s.

BMI1

enr

ichm

ent (

log 2)

BMI1 in GS

-1

1

3

5 **

SALL

4 en

richm

ent (

log 2)

SALL4 in Thy1+

0

2

4n.s.

*

ChIP-seq enrichment in GS or cultured Thy1+ spermatogonia

B

Clu

ster

1C

lust

er 2

Clu

ster

3C

lust

er 4

Clu

ster

5C

lust

er 6

Oth

er

Clu

ster

1C

lust

er 2

Clu

ster

3C

lust

er 4

Clu

ster

5C

lust

er 6

Oth

er

Clu

ster

1C

lust

er 2

Clu

ster

3C

lust

er 4

Clu

ster

5C

lust

er 6

Oth

er

Clu

ster

1C

lust

er 2

Clu

ster

3C

lust

er 4

Clu

ster

5C

lust

er 6

Oth

er

Clu

ster

1C

lust

er 2

Clu

ster

3C

lust

er 4

Clu

ster

5C

lust

er 6

Oth

er

Clu

ster

1C

lust

er 2

Clu

ster

3C

lust

er 4

Clu

ster

5C

lust

er 6

Oth

er

Clu

ster

1C

lust

er 2

Clu

ster

3C

lust

er 4

Clu

ster

5C

lust

er 6

Oth

er

Antibody listName Origin ApplicationsRabbit anti-H2AK119ub Cell Signaling IHCRat anti-GCNA Abcam IHCRabbit anti-PLZF Santa Cruz IHCMouse anti-PLZF Santa Cruz IHCMouse anti-γH2AX Millipore IHCRabbit anti-SALL4 Abcam IHC, IPGuinea Pig anti-H1T Gift from Dr. Mary Ann Handel IHCGoat anti-c-Kit R&D systems IHCMouse anti-RNF2 MBL IHC, IPRabbit anti-Ki67 Millipore IHCRabbit anti-phospho-H3 Cell Signaling IHCRabbit anti-LINE1-ORF1p Gift from Dr. Alex Bortvin IHCGoat anti-DMC1 Santa Cruz IHCRabbit anti-Cleaved Caspase-3 Cell Signaling IHC