Figure S4

Cell Stem Cell, Volume 20

Supplemental Information

RNA Helicase DDX5 Inhibits Reprogramming to Pluripotency by miRNA-Based Repression of RYBP and its PRC1-Dependent and -Independent Functions Huanhuan Li, Ping Lai, Jinping Jia, Yawei Song, Qing Xia, Kaimeng Huang, Na He, Wangfang Ping, Jiayu Chen, Zhongzhou Yang, Jiao Li, Mingze Yao, Xiaotao Dong, Jicheng Zhao, Chunhui Hou, Miguel A. Esteban, Shaorong Gao, Duanqing Pei, Andrew P. Hutchins, and Hongjie Yao

Figure S1 (Related to Figure 1)

2 1 D0

D3

D6

Ddx5 mRNA

D9 D12

4 3 2 1 0

Ctrl

OSKM

2 1 0

pMXs DDX5

Ctrl

OSKM Ctrl

FSC

Oct4-GFP

GFP

5.92%

pW-TRE-DDX5 Oct4-GFP

2.32%

80 60 40 20 0

Ctrl

pW-TRE DDX5

OSKM

G Cell number (×10,000)

F

E

pW-TRE DDX5

MEFs

MEFs

**

100

**

3

250 200

OSKM+shCtrl OSKM+shDdx5

150 100 50 0

D0 D2 D4 D6 D8 D10

Cell number (×10,000)

3

***

Relative expression level

4

0

D

Ddx5 mRNA

Ddx5 mRNA Relative expression level


5

C

Oct4-GFP colony number

B

A

250 200

OSKM OSKM+DDX5

150 100 50 0

D0 D2 D4 D6 D8 D10

Figure S2 (Related to Figure 2) A

B

Target site 1

Exon 2

Exon 1

Ddx5

NM_007840.3

Cas 9

target site Ddx5+/+ Ddx5-/-

Chr11

Ddx5-/- #2

PAM region

GACC GAGACCGCGGCCGGGATCGAGGGTGAGTGT CTGG CTCTGGCGCCGGCCCTAGCT CCCACTCACA GAGACCGCGGCCGGGATCGA

5‘


3’

Target site 2

Exon 1

Ddx5

Exon 2

NM_007840.3

Ddx5-/- #3 target site Ddx5+/+ Ddx5-/-

Chr11

Cas 9

PAM region

Ddx5-/- #4

CTCTGGAAAGAAGTTTGGAAATCCTGGGGAGAAA GAGACCTTTCTTCAAACCTTTAGGACCCCTCTTT GGAAAGAAGTTTGGAAATCC

5‘ 3’ 5‘


3’

C

D

/-

# x5 - 1 /Dd #2 x5 /#3


1.4

Dd

x5 Dd

Dd

x5 +

/+

mESCs

α-DDX5 α-β-Actin

F

Ddx5

Ddx5-/-

1.2 1 0.8 0.6 0.4 0.2 0

+/+

E

Ddx5+/+

Nanog

Oct4

Sox2

-/-

Ddx5-/- #2

150

0

Utf1

D0

Ddx5 #2

0 50K

G2

200

S G1

0

100K 150K 200K PI-A

50K

400

200

100

G2

G1

0 50K

100K 150K 200K PI-A

G1/S : 28.99 S : 39.54 G2/M : 25.18

G1/S : 26.33 S : 39.74 G2/M : 28.38

G

S

200 100

G2

100K 150K 200K PI-A

0

H

G1 50K

G1/S : 26.53 S : 40.31 G2/M : 26.4

Relatvie expression leve

4

OSKM+shDdx5#1 OSKM+shDdx5#2

3

**

** **

**

1

TW 1 T2 D 5 -/dx #1 5 -/D dx #2 5 -/ D dx -#3 5 -/#4

0

dx

D

D3

G2

G1/S : 29.81 S : 37.4 G2/M : 26.81

OSKM+shCtrl

2

S

100K 150K 200K PI-A

Ddx5 mRNA

log2(Fold Change)

W

D3

Ddx5 #3

Count

Count

Count

G1

D2

300

100

S

D1 -/-

300

100

Ddx5-/- #3

300

300

200

Ddx5-/- #1

-/-

Ddx5 #1

300

Ddx5+/+

450 Numbers of cells (10,000)

5‘ 3’

Count

Ddx5-/- #1

D6

D9

D12

Figure S3 ( Related to Figure 3) B MEFs

p #1 sh Ry bp #2

0.8

α-β-Actin

100

0.2

trl sh R

12 9 6 3 Ctrl

pMXs RYBP

500 400 300 200

2 0

** *** ***

Zeb2

Cdh1

Krt8

Epithelial genes

100

10

shRybp

200 150 100 50 0

D0 D2

D4 D6 D8 D10



250

shCtrl

200

50 0

Ctrl

pW-TRE RYBP

***

***

***

***

Zeb2

Cldn3

Krt8

Mesenchymal genes

L 250

100

1

K 300

150

MEFs OSKM+shCtrl OSKM+shRybp#1 OSKM+shRybp#2 Snai1

0.1

OSKM

200

Epithelial genes

Mesenchymal genes

J

pW-TRE RYBP

***

250

MEFs



0.01 0.001 0.0001

MEFs OSKM OSKM+RYBP Snai1

4

RYBP

I

***

6

Ctrl

H 10000 1000 100 10 1 0.1

8

0 Ctrl

OSKM

***

10

100

MEFs

G

Rybp mRNA


15

F

Secondary MEFs +Dox +Vc ***



***

0

yb p

sh C

yb p

yb p

sh C

sh R

sh R

E

Rybp mRNA 18

0

#2

#1

0

50

#2

0.4

150

yb p

α-RYBP

Secondary MEFs +Dox +Vc *** ***

#1

0.6

D Relative expression level

200

sh Ct rl sh Ry b

1


1.2

C

sh R

Rybp mRNA ** **

trl


A

OSKM-RYBP-iPSCs

OSKM OSKM OSKM+RYBP 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

X

150 100 50 0

D0 D2 D4 D6 D8 D10

Y

OSKM

B

OSKM Oct4-GFP colony number

60 40 20 0

Ctrl 4.57%

6 5 4 3 2 1 0

-1

-m

pri

-2

5b

5b

12 iR-

12 iR-

1E-05

***

1E-05 8E-06 6E-06 4E-06 2E-06 0E+00

rl

Ct

1 2 5# x5# d D sh

1.2

***

1.0

0.4 0.2 0.0

rl

t hC

s

1 0.8 0.6 0.4 0.2 0

C

ti-N

an

a

5b

12 9 6 3 0

NC

ti-

an

OSKM+anti-miR-125b

200 150 100 50 0

50 0

D0 D2 D4 D6 D8 D10

*** ***

***

60

**

40 20 0

***

300

endo-Sox2

O

200

100

0 D0 D2 D4 D6 D8 D10

100

endo-Oct4

Nanog

MEFs



250

150

80

a

N OSKM+anti-NC

200

MEFs OSKM+anti-NC OSKM+anti-miR-125b

100

1

iR-

-m nti

MEFs

M

b 25

2

OSKM+miR-125b

250

# x5

Dd sh OSKM D6

L

***

15

12

Rmi

nti

Rybp mRNA

K

1

# x5

Dd

sh


***



J

miR-125b

Ctrl DDX5 anti-NC anti-miR-125b

+ + -

2.34%

Oct4-GFP

OSKM+NC

0.6

MEFs

1.2

***

0.8

x Dd

sh

miR-29a

FSC

FSC

**

sh

-m

pri

1E-05

Ctrl

I

miR-125b

Cell number (× 10,000)

7

Relative expression level to U6

(×10-4)

0

GFP

H

miR-125b

25

Oct4-GFP

+ + -

+ +

+ +

Utf1

***

500 Oct4-GFP colony number

G

Ddx5-/-



Ddx5+/+

50

2.14%

1.34%

Oct4-GFP

GFP

F

75

E

miR-125b

Oct4-GFP

p=0.342

100

80

C -log2 (Relative expression)

D

**

100 Oct4-GFP colony number

log2 (Foldchange)

A

Figure S4 (Related to Figure 4)

400 300 200 100

0 anti-NC anti-miR-125b shCtrl shRybp

+ + -

+ + -

+ +

+ +


B OSKM D4

D8

Ddx5+/+ mESCs H2AK119ub1

D12

Ddx5-/- mESCs H2AK119ub1

sh C tr sh l D dx sh 5 C trl sh D dx sh 5 C trl sh D dx sh 5 C trl sh D dx 5

D2

α-H2AK119ub1 1

0.95

0.88

1.36

1.39

1591

1.56 1.46 2.06

5908

1335

α-H3

C

D Unique in Ddx5+/+ mESCs

Common in Ddx5+/+ & Ddx5-/- mESCs GO Biolog ica l Pr oce ss pat t ern specificat ion process regionalizat ion em bryonic organ m orphogenesis cell fat e com m it m ent ant erior/post erior pat t ern specificat ion m esoderm m orphogenesis m esoderm form at ion m esoderm developm ent

GO Biolog ica l Pr oce ss

-log10(Binom ial p value) 0 5 10 15 20 25 30 35 40 45 50 55 60

48.41 43.97 37.61 34.88 32.83 30.17

m esoderm developm ent m esoderm m orphogenesis cart ilage developm ent m esoderm form at ion odont ogenesis developm ent al m at urat ion m onocyt e aggregat ion form at ion of prim ary germ layer

63.43 60.33

E Unique in Ddx5-/- mESCs

GO Biolog ica l Pr oce ss

-log10(Binom ial p value) 0 1 2 3 4 5 6 7 8 9 10 11

cerebellar cort ex developm ent cerebellum developm ent m et encephalon developm ent response t o ret inoic acid cell fat e com m it m ent hindbrain developm ent cerebellar cort ex m orphogenesis t ongue m orphogenesis

9.91 9.79 9.53 8.81 7.53 7.42 7.40

11.73

-log10(Binom ial p value) 0

2

4

6

8

10

9.14 8.41 8.00 7.52 7.43 6.46 6.14

12 13.63


Jun Ddx5

Tril

10 kbp

Chr4: 95031534-95087422 +/+

210

H2AK119ub1

Ddx5

10 210

Ddx5-/- H2AK119ub1

10

Fragment near Tril TSS

*

IgG

0.08

0.04 0.02

*

0.06

Ddx5+/+

**

8 6 4 2 0

shCtrl

1

Bmp4

IgG shDdx5 RYBP shDdx5

0.4 0.2 shCtrl

0.04 D5

H2AK119ub1 Ctrl H2AK119ub1 RYBP

D5

*

0.12

10 5

D11


0.04 0

D5

D11

OSKM


25

**

**

20 15

5

5 D11

OSKM

0


* *

15 10

D5

IgG Ctrl IgG RYBP

20

10

0

shDdx5

0.08

D11

25 % Input

*

OSKM

shCtrl

Klf15

IgG Ctrl IgG RYBP

*

D5

2

OSKM

20 % Input

*

Bmp4

15

4

IgG shCtrl RYBP shCtrl

% Input

0

Egr4

0


0.04

D11

IgG Ctrl IgG RYBP

6

OSKM

0.08

OSKM

E

8

0

shDdx5

*

0.12 % Input

% Input

**

***

Rasd1


**

0.16

Ddx5-/-

10

0.6

Jun

0.08

Klf15

14

OSKM

Tril

0

**

12

OSKM

0.12

Ddx5+/+

mESCs

0.8

0

shDdx5


0.04

0

Ddx5-/-

% Input


10

Egr4

0.06

mESCs


C

*

0.02

0

Ddx5-/-

RYBP

0.08

0.04

mESCs

D

IgG


Ddx5+/+

Rasd1

Fragment near Rasd1 TSS

0.02

0

1

RYBP

% Input

0.06

% Input

% Input

RYBP

1 23

Med9

Tril

IgG

23

H2AK119ub1

Ddx5-/- H2AK119ub1

10

Fragment near Jun TSS

0.08

Ddx5

10 210

Ddx5-/- H2AK119ub1

1 kbp

Chr11: 59943181-59984944 +/+

210

H2AK119ub1

Jun

B

Rasd1

10 kbp

Chr6: 53795466-53840830 +/+

D11

D5

OSKM

Figure S7（Related to Figure 7）

0.2 Oct4

Nanog

Esrrb

Utf1

+ + -

+ +

+ + -

1

0.1 Flag shLuc shKdm2b RYBP

+ +

/-

DD

X5 -

DD

*

DD

*

*

1.6

X5 +

/+

Kdm2b

2

+ + -

+ + -

1#

E

1

1.15

1.31

1.34

α-KDM2B

1.2 0.8

α-DDX5

0.4

3#

/-

2#

/-

X5 DD

1#

X5 -

/-

DD

X5 -

DD

X5 +

/+

0 DD

Relatvie expression level

D

0.1 Flag shLuc shRybp KDM2B

10

DD

0

1

Kdm2b

α-β-actin

3#

0.4

10

Rybp

/-

0.6

100

Kdm2b

2#

1 0.8

Rybp

X5 -

100

1.2

OSKM

/-

Rybp mESCs Rybp-/- mESCs Relative expression level


1.4

C

OSKM

X5 -

B +/+


A

+ +

+ +

SUPPLEMENTAL LEGENDS Figure S1. Related to Figure 1. DDX5 Perturbs Reprogramming. (A) qRT-PCR analysis for Ddx5 mRNA expression at different time points of MEFs reprogrammed with OSKM. (B) qRT-PCR analysis of Ddx5 expression after overexpression with pMXs-DDX5 vector in MEFs. (C) qRT-PCR analysis of Ddx5 expression after overexpression with pW-TRE-DDX5 in MEFs. (D) Number of GFP+ colonies of MEFs transduced with OSKM and either empty plasmid or pW-TRE-DDX5 at day 10. (E) GFP+ cells analyzed by FACS in reprogrammed cells transfected with OSKM and either empty plasmid or pW-TRE-DDX5 at day 10. (F) Cell proliferation at different time points during OSKM reprogramming with control shRNA or Ddx5 shRNA. (G) Cell proliferation at different time points during OSKM reprogramming with either empty plasmid or Flag-DDX5. The data in (A-D) and (F-G) are reported as mean values ± SD with the indicated significance by using Student’s t-test analysis (*p < 0.05, **p < 0.01, ***p < 0.001). Data in (A-C) and (F-G) are plotted from 3 independent experiments each with 3 technical replicates. Data in (D) is plotted from 4 independent experiments each with 3 technical replicates. Figure S2. Related to Figure 2. Strategies of Generating Ddx5-/- mES Cell Lines and the Effects of DDX5 Loss-of-Function on Gene Expression, Cell Proliferation, Cell Cycle. (A) A schematic illustration of the Ddx5 knockout strategy using CRISPR/Cas9 in mESCs. (B) DNA sequencing diagram of Ddx5 proximal to target site in Ddx5+/+ and Ddx5-/- mESCs. (C) Western blot to examine Ddx5 knockout efficiency of three different Ddx5-/- mESCs. β-Actin was used as the loading control. (D) qRT-PCR analysis for the expression of pluripotent genes in Ddx5 +/+ and Ddx5-/- mESCs. (E) Cell proliferation of wild type and three different Ddx5 -/- mES cell lines at different time points. (F) Cell cycle assay of wild type and Ddx5-/- mESCs. Three different Ddx5-/- mES cell lines were used. (G) Heatmap of the expression of epigenetic-associated genes and other selected genes in wild-type and Ddx5-/- mESCs. Log2 relative gene expression is visualized as shades of red (higher than Ddx5 +/+ mESCs) and shades of blue (lower than Ddx5 +/+ mESCs) in Ddx5-/- mESCs. Two wild-type and four Ddx5-/- mESC lines were used. (H) qRT-PCR analysis for Ddx5 knockdown efficiency in MEFs transduced with shRNA for Ddx5 during reprogramming. The data in (D), (E) and (H) are reported as mean values ± SD with the indicated significance by using Student’s t-test analysis (*p < 0.05, **p < 0.01, ***p < 0.001). Data in (D), (E) and (H) are plotted from 3 independent experiments each with 3 technical replicates.

Figure S3. Related to Figure 3. Loss of RYBP Inhibits Somatic Cell Reprogramming. (A) qRT-PCR analysis for Rybp knockdown efficiency of MEFs reprogrammed with OSKM plus control shRNA or Rybp shRNA. (B) Western blot for RYBP knockdown efficiency using cell lysates from MEFs reprogrammed with OSKM plus control shRNA or Rybp shRNA. β-Actin was used as the loading control. (C) Number of GFP+ colonies in doxycycline-inducible secondary MEFs transduced with control shRNA or two shRNAs targeting Rybp at day 12. (D) qRT-PCR analysis of Rybp expression after pMXs-RYBP overexpression in MEFs. (E) Number of GFP+ colonies in doxycycline-inducible secondary MEFs transduced with empty vector or Flag-RYBP at day 12. (F) qRT-PCR analysis of Rybp expression after pW-TRE-RYBP over-expression in MEFs. (G) Number of GFP+ colonies of MEFs transduced with OSKM and either empty plasmid or pW-TRE-RYBP at day 10. (H) qRT-PCR analysis for the MET genes using RNA lysates from MEFs reprogrammed with OSKM and empty vector or RYBP at day 5. Untransduced MEFs were used for normalization. (I) qRT-PCR analysis for the MET genes using RNA lysates from MEFs reprogrammed with OSKM and shRNA control or 2 shRNAs for Rybp at day 5. Untransduced MEFs were used for normalization. (J) Cell proliferation at different time points during OSKM reprogramming with control shRNA or Rybp shRNA. (K) Cell proliferation at different time points during OSKM reprogramming with empty vector or RYBP. (L) Karyotype of a selected iPSC colony generated with OSKM and RYBP. The data in (A) and (C-K) are reported as mean values ± SD with the indicated significance by using Student’s t-test analysis (*p < 0.05, **p < 0.01, ***p < 0.001). Data in (A), (D), (F) and (H-K) are plotted from 3 independent experiments each with 3 technical replicates. Data in (C), (E) and (G) are plotted from 4 independent experiments each with 3 technical replicates. Figure S4. Related to Figure 4. miR-125b Is A Barrier for Reprogramming. (A) A MA plot with dots marked red and blue colors indicating miRNAs significantly regulated (p