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DOI: 10.1038/ncb3326

A

Ikzf3, Sox4, Erg,Gfi1b, Rag2, Rag1I

II

III

IV

V

VI

VII

VIII

IX

Ikzf1, Ebf1, Bcl6,Tcf4, Kdm5d, Fli1

Prdm9, Pou5f2, Arid3a,Klf7, Pou2f2

Lmx1a, Hoxb1, Mafg,Nr2f1, Pou1f1

Nr5a1, Wt1, Snai1, Tet2,Glis1, Fos, Smarca2

E2f1, Klf2, Dnmt1,H2afx, Etv1, Arid3b

Nr5a2, Klf1, Otx2, Snai3,Zic2, Zic5, Smad6

Hdac4, Klf8, Klf4, Prdm5, Jarid2, Rarg, Oct4, Sox2

Prdm14, Parp1,Sall4, Dnmt3b, Nr0b1, Kdm1a, Tbx3, Nanog

B cells

Bα’ cell

s

Day1

Day2

ESCs

10-10

10-20

10-30

pValue

Percent of genesfrom ontologyfound in cluster

immune system process

immune response

B cell activation

adaptive immune response

nucleosome organization

chromatin assembly

Chromatin organization

Epigenetic regulation of gene expression

NoRC negatively regulates rRNA expression

Negative epigenetic regulation of rRNA expression

Meiosis

Meiotic Recombination

channel activity

anion channel activity

ion channel activity

mesenchymal to epithelial transition

epithelial cell morphogenesis

epithelium development

cell migration

cell motility

cell adhesion

organelle fission

nuclear division

chromosome segregation

DNA replication

cell division

Cell cycle

canonical Wnt signaling pathway

Wnt signaling pathway

stem cell maintenance

stem cell proliferation

blastocyst formation

histone H4-R3 methylation

histone methylation

methylation

poly(A) RNA binding

RNA transport

Pre

cent

age

of G

O s

ets

foun

d in

clu

ster

5%

10%

15%

20%

25%

BI II III IV V VI VII VIII IX

Di Stefano et al_ Suppl. Figure 1

C

RNAseq countrelative to mean (log2)

RNAseq clusters

-2 0 21-1

D

H3

Oct4

Sox2

Lin28

Gdf3

Sall4

Nanog

Tcfp2l1

B cells

Day2

Day6

Day4

Day8

Rel

ativ

e ge

ne e

xpre

ssio

n (lo

g2)

First wave genes

Second wave gene Third wave genes

E

B cells

day 2

day 4

day 6

day 8

0

5

10

Oct4

B cells

day 2

day 4

day 6

day 8

0

5

10

15

Lin28

B cells

day 2

day 4

day 6

day 8

0

5

10

15

20

Tdh

B cells

day 2

day 4

day 6

day 8

0

5

10

15

Gdf3

B cells

day 2

day 4

day 6

day 8

0

2

4

6

8

Zfp296

B cells

day 2

day 4

day 6

day 8

0

5

10

15

20

Nanog

B cells

day 2

day 4

day 6

day 8

0

5

10

15

20

Sall4

B cells

day 2

day 4

day 6

day 8

0

5

10

Esrrb

B cells

day 2

day 4

day 6

day 8

0

5

10

Sox2

B cells

day 2

day 4

day 6

day 8

-4

-2

0

2

4

Rex1

Supplementary Figure 1 Characterization of Ba’ cell reprogramming into iPS cells. (A) Representative chimeric mouse obtained after blastocyst injection of aiPS clone. (B) Heatmap of RNA-seq data showing genes changing >2fold during reprogramming (FDR<1%, LRT test). (C) Gene Ontology (GO) analysis of protein clusters shown in panel A. The size of each circle represents the proportion of GO sets found in each

cluster; the intensity of the color represents the P-value, determined by a hypergeometric test. (D) Gene expression (qRT-PCR) of selected pluripotency genes. Values were normalized against Pgk expression. Error bars indicate s.d. (n=3 biologically independent samples). (E) Representative western blots for selected pluripotency transcription factors. See Suppl. Fig. 8 for uncut gel images.

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Protein Clusters

7497identified proteins

extracellular matrix protein (PC00102)protease (PC00190)cytoskeletal protein (PC00085)transporter (PC00227)

transmembrane receptor regulatory/adaptor protein (PC00226)

transferase (PC00220)oxidoreductase (PC00176)lyase (PC00144)cell adhesion molecule (PC00069)ligase (PC00142)nucleic acid binding (PC00171)signaling molecule (PC00207)enzyme modulator (PC00095)viral protein (PC00237)calcium-binding protein (PC00060)defense/immunity protein (PC00090)hydrolase (PC00121)transfer/carrier protein (PC00219)membrane traffic protein (PC00150)phosphatase (PC00181)transcription factor (PC00218)chaperone (PC00072)cell junction protein (PC00070)surfactant (PC00212)structural protein (PC00211)kinase (PC00137)storage protein (PC00210)receptor (PC00197)isomerase (PC00135)

CPANTHER classification

B cells

Bα’

d1

d2

ES

Pro

teom

ics

RNA-seq

0.2

0.4

0.6

0.8

1.0

0.6

0.7

0.8

0.9

1.0

Sam

ple

to s

ampl

e co

rrel

atio

n (P

ears

on c

oeffi

cien

t)

0.19 0.18 0.40 0.40 0.34 0.33 0.29 0.28 0.97 1.00

0.19 0.18 0.41 0.41 0.34 0.34 0.29 0.29 1.00

0.65 0.65 0.74 0.72 0.87 0.86 0.97 1.00

0.65 0.65 0.75 0.73 0.88 0.87 1.00

0.69 0.69 0.83 0.81 0.95 1.00

0.69 0.69 0.83 0.81 1.00

0.69 0.68 0.85 1.00

0.69 0.70 1.00

1.00

0.94 1.00

1.00 0.99 0.88 0.85 0.82 0.82 0.79 0.79 0.53 0.53

1.00 0.88 0.84 0.84 0.83 0.80 0.80 0.53 0.53

1.00 0.96 0.81 0.81 0.77 0.77 0.52 0.52

1.00 0.79 0.79 0.75 0.76 0.53 0.53

1.00 0.99 0.95 0.95 0.62 0.63

1.00 0.95 0.95 0.62 0.62

1.00 0.99 0.62 0.62

1.00 0.62 0.62

1.00

1.00 0.99

A

B

B cells

Bα’ cell

sDay

1Day

2ESCs

B cells

Bα’ cell

sDay

1Day

2ESCs

Di Stefano et al_ Suppl. Figure 2

-3

-1

1

3-3

-1

1

3-3

-1

1

3-3

-1

1

3-3

-1

1

3-3

-1

1

3-3

-1

1

3-3

-1

1

3-3

-1

1

3-3

-1

1

3

a

b

c

d

e

f

g

h

i

j

Supplementary Figure 2 Protein dynamics during reprogramming. (A) PANTHER classification for all the proteins identified by mass spectrometry in the samples tested. (B) Correlation between biological duplicates of RNA-seq and proteomic data. (C) C-means clustering of proteins changing >2 fold at any time points during reprogramming.

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B cells

RNAs

eq c

ount

rela

tive

to m

ean

(log2

)

Ciita

Ebf1Foxo1Gfi1bIkzf3

-2

0

2

4

Bα’ cell

sDay

1Day

2ESCs

A BWestern blots

C/EBPα IP

Input

IgG C/EBPα

C

Di Stefano et al_ Suppl. Figure 3

Hdac1

Parp1

B cells

Bα’ cell

s

Lsd1

C/EBPα

B cells

Bα’ cell

s

Brd4

Lsd1

Klf4

Gapdh

Input

IgG C/EBPα

C/EBPα

Parp1Pcna

Lsd1

Parp1Pcna

Hdac1

Parp1Pcna

Input

IgG Lsd1

Input

IgG Hdac1

F

D

C/EBPα IP Lsd1 IP HDAC1 IPG

B cells

3h 6h 18h

C/EBPα

Cdk9

β-Tub

Hdac1

Lsd1

C/EBPα

Pcna

Fraction #1 2 3 4 5 6 7 8 9

B cellsBα’ cells

Day1Day2H

3K27

ac

Brd4, B cellsBrd4, Bα’

ESCs

H

I

Ebf1

100kb

Foxo1

10kb

Gfi1b

5kb

Ikzf3

10kb

670KDa

0.0

0.5

1.0

1.5

Rel

ativ

e ex

pres

sion

Ciita

0.0

0.5

1.0

1.5

Ikzf3

0.0

0.5

1.0

1.5

Rag1

0.0

0.5

1.0

1.5

Ebf1

0.0

0.5

1.0

1.5

Foxo1

0.00.10.20.30.4

1.0

1.5

2.0

Gfi1b

B cells Bα’ cells Bα’ cells+VPA

*

***

**

**

Rag2 Rag1

20kb

Ciita

5kb

E

IP #1 IP #2 IP #3Number of peptides

C/EBPα

Lsd1

Hdac1

11 11 11

5 5 3

8 8 7

7.9 10-4.9

C/EBPα / IgGFd (log2) p value

10-3.4

10-2.1

2.2

1.1

Supplementary Figure 3 Gene silencing induced by C/EBPa, protein interactions and B cell specific gene enhancer activities during reprogramming. (A) Representative western blots of Brd4, Lsd1, Klf4 and Hdac1 in B and Ba’ cells. See Suppl. Fig. 8 for uncut gel images. (B) RNA-seq expression values for selected B cell specific genes. The data represent the average from two biologically independent samples. (C) Western blots of Cdk9 after induction of C/EBPa in B cells. See Suppl. Fig. 8 for uncut gel images. (D) Ba’ cell extracts were fractionated on Superose 6 10/300 GL column and Hdac1, Lsd1 and C/EBPa were probed by western blot. See Suppl. Fig. 8 for uncut gel images. (E) Peptide counts, P-value and enrichment over IgG of C/EBPa, Hdac1 and Lsd1, for the IP-mass spectrometry shown in

Fig. 3B. (F) C/EBPa co-immunoprecipitation experiment. Lsd1 or C/EBPa were probed by western blot. See Suppl. Fig. 8 for uncut gel images. (G) Co-immunoprecipitation of C/EBPa, Lsd1 and Hdac1. Parp1 and Pcna (negative controls) were probed by western blot. See Suppl. Fig. 8 for uncut gel images. (H) Screenshots of H3K27ac histone decoration and Brd4 binding by ChIP-seq at enhancers of selected B cell transcription factors. (I) Gene expression of selected B cell genes as measured by qRT-PCR in B cells (data from Fig. 3F), B cells treated for 18h with E2 (Ba’ cells) and B cells treated for 18h with both E2 and the Hdac1 inhibitor VPA. Error bars indicate s.d. (n=3 biologically independent samples). Statistical significance was determined using a two-tailed unpaired Student’s t-test (*P<0.05, **P<0.01).

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shCtrl shBrd4shLsd1

A

0 50K 100K 150K 200K 250K

FSC-A10 0

10 1

10 2

10 3

10 4

10 5

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5

CD

19

shC

trl-G

FP

C/EBPα-hCD4

96.6% 0.30%

0 50K 100K 150K 200K 250K

10 0

10 1

10 2

10 3

10 4

10 5

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5

CD

19

shB

rd4-

GFP

FSC-A C/EBPα-hCD4

97% 0.22%

0 50K 100K 150K 200K 250K

10 0

10 1

10 2

10 3

10 4

10 5

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5

CD

19

97.4%

FSC-A C/EBPα-hCD4

0.17%

shLs

d1-G

FP

B

shRNAs sorting strategy

0.14 3.61

1.3194.9

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5 0.095 3.03

1.0595.8

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 50.080 4.03

2.3993.5

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5

B cells B cells + JQ1B cells + S2101

Annexin V

SY

TOX

AA

Dva

nced

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5

Brd

U-A

PC

B cells B cells + JQ1B cells + S2101

Empty channel

64.5% 47.5%57.7%

C

E

Di Stefano et al_ Suppl. Figure 4

D

shCtrl

shLs

d10.0

0.5

1.0

1.5

Rel

ativ

e ge

ne e

xpre

ssio

n

shCtrl

shBrd4

0.0

0.5

1.0

1.5

Rel

ativ

e ge

ne e

xpre

ssio

n

Lsd1 qRT-PCR Brd4 qRT-PCR

DMSO S21

010

20

40

60

80

100

DMSO S2101n.s.OSKM reprogramming

Oct

4GFP

+ co

loni

es

F

10kb

C/EBPα, Bα’

C/EBPα, B cells

ESCs

Brd4, B cells

Brd4, Bα’

H3K2

7ac

ChIP

-seq B cells

Bα’ cells

C/EBPα, Bα’

C/EBPα, B cells

ESCs

Brd4, B cells

Brd4, Bα’

H3K2

7ac

ChIP

-seq B cells

Bα’ cells

Egln3

Rarg

DMSO E2 and JQ1 Doxy and JQ1

G

H

Supplementary Figure 4 Effect of Lsd1 and Brd4 inhibitions on iPS reprogramming. (A) Representative flow cytometry analysis of B cells treated with JQ1 or S2101 for 24 hours using Pacific Blue™ Annexin V/SYTOX® AADvanced™ Apoptosis Kit. (B) Representative BrdU (6h pulse) FACS staining of B cells treated with JQ1 or S2101 or DMSO as a control. (C) shRNA sorting strategy. (D) Gene expression by qRT-PCR of Lsd1 and Brd4 after specific knockdown in B cells. Error bars indicate s.d. (n=3 biologically independent samples). (E) Representative alkaline phosphatase positive iPS colonies obtained from reprogramming of B cells after Lsd1 and Brd4

knockdown. (F) Oct4-GFP and alkaline phosphatase positive iPS colonies obtained from reprogramming of B cells (OSKM alone without C/EBPa pulse) treated with S2101 or DMSO as a control. Error bars indicate s.d. (n=3 biologically independent samples). Statistical significance was determined using a two-tailed unpaired Student’s t-test (n.s. P>0.05). (G) Genome browser screenshots of Rarg and Egln3 loci showing C/EBPa, Brd4 and H3K27ac ChIP-seq data. (H) Representative alkaline phosphatase positive iPS colonies obtained from reprogramming of Ba’ cells induced with OSKM and treated with JQ1 during C/EBPa (E2) or OSKM (Doxy) induction.

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Di Stefano et al_ Suppl. Figure 5

Id1

ATAC-seq

ChIP-seqH3K27ac

ChIP-seq C/EBPα, Bα’

B cells

Bα’ cells

Day1

Day2

ESCs

B cells

Day1

Day2

ESCs

Ifitm6

Bα’ cells

B

Embryo developmentEmbryonic organ development

Developmental process

ACluster I Cluster II

10-1

5

10-1

0

10-5

100

Immune responseGranulocyte migration

Myeloid leukocyte differentiation

p-value p-value

p-value

Cluster III

10-2

0

10-1

5

10-1

0

10-5

100

Cell migrationCell motility

Inflammatory response

p-value

Cluster IV

Motif Logo

JASPAR: Spi1 (MA0080.3)

HOCOMOCO: IRF2 (M00172)

JASPAR: CEBPA (MA01002.3)

JASPAR: STAT2::STAT1 (MA0517.1)

JASPAR: RUNX1 (MA0002.2)

HOCOMOCO: IRF1 (M00171)

Ets

Irf

Cebp

Stat

Runx

Irf

Name in Fig5B Closest database motif Name in database

HOCOMOCO: NR5A2 (M00262)

JASPAR: ESSRA (MA0592.1)

HOCOMOCO: LEF1 (M00191)

JASPAR: SOX9 (MA0077.1)

JASPAR: POU2F2 (MA0507.1)

JASPAR: Klf5 (MA0599.1)

Lrh1

Essr

Lef1

Sox

Oct

Klf

10-4

0

10-3

0

10-2

0

10-1

010

0

Immune system processLeukocyte activation

Granulocyte migration

10-5

10-4

10-3

10-2

10-1

100

10kb 10kb

C

Aver

age

fragm

ent

cove

rage

(RP

M)

0.8

0.6

0.4

0.2

peak center +2kb-2kb

cluster Icluster IIcluster IIIcluster IV

1.4

1.0

0.6

0.2

Klf4cluster Icluster IIcluster IIIcluster IV

peak center +2kb-2kb

Brd4

EChIP-seq in ESCs

C/EBPαpeaks

197 430410603

Klf4peaks

F

C/EBPα, Bα’ cells

D Klf4

4C-seqB cells

ESCs

Bα’ cells

G

PU.1, B cells

PU.1, Bα’ cells

-90Kb enhancer

-280Kb enhancer

50kb

RP

KM

(rel

ativ

e to

B c

ells

)

Cluster III Cluster IVCluster IICluster I

+3kb-3kb +3kb-3kb +3kb-3kb +3kb-3kb

10

2

0

-2

-4

0

3h6h24h

10’30’3h12hC/EBPα

MNase

0

20

40

0

25

100

0

50

100

Supplementary Figure 5 ATAC-seq cluster analysis. (A) Gene ontology enrichment for genes associated with ATAC-seq peaks in each cluster shown in Figure 5A (nearest gene relative to the peak). P-values were determined by a hypergeometric test. (B) Genome browser screenshots of Id1 and Ifitm6 loci showing C/EBPa and H3K27ac ChIP-seq, as well as ATAC-seq data. (C) Selected over-represented DNA motifs shown in Figure 5B discovered (de novo) in ATAC-seq peaks, and similar motifs found in the JASPAR or HOCOMOCO database. (D) Genome browser screenshot of the Klf4 locus showing C/EBPa and PU.1 ChIP-seq data, and 4C data using the newly

discovered -90kb enhancer as view point (black triangle at the bottom). The second highlighted region (right) correspond to the second -280kb enhancer, as shown in Figure 5E. (E) Comparison of our ATAC-seq data (Fig. 5A), with Brd4 (GSE36561) and Klf4 (ref. 56) ChIP-seq data in ES cells. (F) Venn diagram showing the overlap between C/EBPa ChIP-seq peaks in Ba’ cells and Klf4 ChIP-seq peaks in ES cells. (G) Average plots of C/EBPa ChIP-seq (top) and MNAse-seq signal (bottom) in the C10 pre-B cell line at different timepoints after induction of C/EBPa, for each ATAC-seq cluster (Fig. 5A). Profiles were normalized to B cells and centered on the median.

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Di Stefano et al_ Suppl. Figure 6

5kb 5kb

Rarg Lefty2

Oct4

Sox2

Nanog

Klf4

Brd4

ChIP-seq in ESCs

Rel

ativ

e ex

pres

sion

(log

2)

Rarg

B cells

Bα' cell

sDay

1Day

2ESCs

6

9

12

15Lefty1

6

9

12

15

B cells

Bα' cell

sDay

1Day

2ESCs

A B

Bα’ cells vsB cells

GMPs vsB cells

850

Bα’ cells vsB cellsGMPs vs

B cells722

C upregulated genes

downregulated genes

EBα' cellsGMPsESCsMEFs

Cluster I Cluster II Cluster III Cluster IV

Aver

age

fragm

ent

cove

rage

(RP

M)

peak center

+2kb-2kb peak center

+2kb-2kb peak center

+2kb-2kb peak center

+2kb-2kb

F

Rarg Lefty2 Tet2 Ifitm6 Id1

B cells

Bα’cells

GMPs

ESCs

MEFs

G

1759 4086

2152 1101

5kb 5kb 40kb 2kb 5kb

1000

500

I

II

III

IV

−2 −1

Fragment counts relative to mean (log2)

0 1 2

B cells

Bα’GMPs

ESCs

D

−0.95 −0.90 −0.850.

00.

10.

20.

3Canonical variate 1

Can

onic

al v

aria

te 2

CMPGMP

B cells

MF

GranMon

LT-HSC

ST-HSCMPP

CD4MEP

EryA

NK

CD8 EryB

Bα’ cells

5

3

1

peak center +2kb-2kb

cluster Icluster IIcluster IIIcluster IV

Aver

age

fragm

ent

cove

rage

(RP

M)

C/EBPα ChIP-seqH

Supplementary Figure 6 C/EBPa induced changes in chromatin accessibility at myeloid and ES cell loci. (A) Genome browser screenshots of the Rarg and Lefty2 loci showing ChIP-seq data for Oct4, Nanog, Klf4 and Brd4 in ESCs (ref. 56, 57 and GSE36561). (B) Gene expression profile by RNA-seq for Rarg and Lefty1 during iPS reprogramming. The data represent the average from two biologically independent samples. (C) Comparison of GMPs and Ba’ cells for the number of upregulated and downregulated genes (>2fold) between B and Ba’ cells as well as between B cells and GMP, indicating the

number of genes that overlap. (D) Canonical component analysis (CCA) of RNA-seq from B cells and Ba’ cells, together with RNA-seq from different hematopoietic cell populations (ref. 58). (E) Heatmaps of ATAC-seq data from clusters I to IV of B cells, GMPs, Ba’ cells, ESCs. (F) Average peak intensities of ATAC-seq data from clusters I to IV of GMPs, Ba’ cells, ESCs and MEFs (ref. 44). (G) Genome browser screenshots of selected genomic loci displaying ATAC-seq data. (H) Average plot of C/EBPa ChIP-seq signal in GMPs for each ATAC-seq cluster.

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0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5

0-10 3 10 3 10 4 10 5

0

-10 3

10 3

10 4

10 5

Oct

4-G

FP

OSKM OSKM+TFIIDOSKM+TFIID

+JQ1OSKM+TFIID

+S21012.71% 20.9% 7.84% 32.8%

Empty channel

Di Stefano et al_ Suppl. Figure 7

slow

fast

0.0

0.5

1.0

1.5

2.0

Rel

ativ

e ge

ne e

xpre

ssio

n (lo

g2) Klf4 in GMPs

A

BqRT-PCR

GMP slowGMP fast

**

Kdm1a

Hdac1

Brd4Med

1Cdk

9Ceb

pa

Cebpb

Trp53

010002000300040005000

10000

20000

30000

Rel

ativ

e ex

pres

sion

CSFE

Slowcycl.

Fastcycling

100

101

102

103

104

0

200

400

600

800

100

101

102

103

10410

010

110

210

310

410

0

101

102

103

104

Sca1

c-K

it

100

101

102

103

104

CD

16/C

D32

CD34

GMPs

1.5%14.3%

C

ETet2

qRT-PCR

shCtrl

shTet2

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Supplementary Figure 7 Comparison of fast and slow cycling GMPs. (A) FACS plots showing sorting strategy to obtain GMPs and their separation into fast and slow cycling fractions after CSFE treatment. (B) Klf4 expression as determined by qRT-PCR in fast and slow cycling GMPs. Error bars indicate s.d. (n=3 biologically independent samples). Statistical significance was determined using a two-tailed unpaired Student’s t-test (**P<0.01). (C)

Array expression values for selected genes in fast and slow cycling GMPs. The data represent the average from two biologically independent samples. (D) Tet2 knockdown efficiency tested by qRT-PCR. Error bars indicate s.d. (n=3 biologically independent samples). (E) Representative Oct4-GFP FACS analysis of OSKM-induced MEFs overexpressing TFIID and treated with JQ1 or S2101.

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Suppl. Fig. 3B, C

Parp1Hdac1

IP CEBPα -WB CEBPα IP CEBPα-WB LSD1

Klf4

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Suppl. Fig. 1D

Sox2Sall4 Gdf3 Nanog

Lin28 Oct4Tfcp2l1

GAPDHBrd4 Lsd1

H3

Suppl. Fig. 3F

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Supplementary Figure 8 Uncut gels.

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Di  Stefano  et  al_Suppl.  Table  2

Gene B  cells Ba'  cells Day1 Day2 ESCsSirt1   24.70275 27.23866 25.46961 26.54176 28.35141Stx8   26.8133 25.78702 25.43324 27.02448 25.54576Dcaf13   25.02057 28.36722 28.79546 29.19405 30.42817Mkrn2   25.2734 24.22111 24.44819 25.5344 26.42498Atg3   28.54859 27.55685 28.53241 28.94388 27.5876Huwe1   27.74928 29.57995 29.38354 30.64386 31.68534Xrcc5   26.16631 27.92961 27.53529 28.66051 30.04875Uhrf1   29.13824 30.59902 30.22031 29.73002 31.70755Trim33   25.54421 27.52574 26.53638 27.20658 28.10385Atg7   28.70343 27.14042 28.4678 29.6404 26.30808Arrb1   28.12383 25.44546 26.19578 28.05901 24.23697Plk1   24.74554 27.64426 27.57963 26.2345 29.81607Nedd4   25.4834 29.35289 30.33005 30.18005 32.64503Fbxo22   27.85522 25.60917 27.27744 28.01333 29.16994Ube2q1   24.48448 27.64395 27.82395 27.28807 27.78896Ltn1   24.97531 27.54043 27.92525 28.42498 28.48326Bid   27.77276 25.6609 27.3468 28.1997 27.91658Senp3   25.23601 27.31095 27.73338 28.47647 30.40767Trim28   32.05651 32.93272 32.59655 32.56633 34.43291Trim30a   26.36436 25.46102 24.4115 26.1353 24.21135Rnf213   33.08924 29.53486 31.33844 33.43363 31.60491Psmd4   28.5357 30.09909 30.02147 30.02265 31.02364Ubox5   25.54592 24.13976 25.1692 25.20078 25.37965Ubr5   25.97307 28.40239 29.09079 29.83672 29.27233Eif4e2   24.71424 27.16535 27.44242 27.68783 28.28967Mycbp2   25.65503 24.56877 25.71953 27.95303 24.91644Sash1   27.54677 25.3172 27.50339 27.36829 24.05951Casc3   24.90638 26.14075 26.24807 26.37039 27.42977

Supplementary Table 1 List of genes in the independent component analysis shown in Fig. 1F.

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Di  Stefano  et  al  _Supplementary  Table  3

Primer  Table

qPCR  primersGenes Forward  primer Reverse  primerTdh CAGACTGAAGATAAAAGGCAG GCATCTGTTCTTCTGATACCZfp296 CCATCTCAGAATCCAAAGAG TATCTAGGTGTTGTGTGTCTGGNanog CAGTTTTTCATCCCGAGAAC CTTTTGTTTGGGACTGGTAGLin28a TGTTCTGTATTGGGAGTGAG CCATATGGTTGATGCTTTGGSall4 AAGAACTTCTCGTCTGCC AGTGTACCTTCAGGTTGCGdf3 CGTCTTAAGGAAAATCATCCG GGCAGACAAGTTAAAATAGAGGPou5f1 GTCCCTAGGTGAGCCGTCTTT AGTCTGAAGCCAGGTGTCCAGSox2 ATGAGAGATCTTGGGACTTC TCTATACATGGTCCGATTCCZfp42 GTTCGTCCATCTAAAAAGGG TAGTCCATTTCTCTAATGCCCEsrrb AAAGCCATTGACTAAGATCG AATTCACAGAGAGTGGTCAGPgk ATGTCGCTTTCCAACAAGCTG GCTCCATTGTCCAAGCAGAAT18s AACCCGTTGAACCCCATT CCATCCAATCGGTAGTAGCGCdh1 CATGTTCACTGTCAATAGGG GTGTATGTAGGGTAACTCTCTCEbf1 ACACAATTCATTCCCCGAAA AAGTCAACGGTTTTGCATCCFoxo1 AAGAGCGTGCCCTACTTCAA CTCCCTCTGGATTGAGCATCGfi1b TAATTCCTGGGCAAAAGAG TGTTTGATTGTGTTCCAGCTIkzf3 CTTTTCTTCAGAACCCTGAC CAATTGCTTGCTAATCTGTCCRag1 GAAGCTTCTGGCTCAGTCTACATCT ACCTCATAGCGCTGCAGGTTCiita CTGGACAAGAATGTCATCTG TTGACTCTTATGGGCTATGGKlf4 CATTAATTGTGTCGGAGGAAG CCGTTTGGTACCTTTAGAACLefty1 TGTGTGCTCTTTGCTTCC GGGGATTCTGTCCTTGGTTTLsd1 TCATTCAGCTGCAAGAAAAG TCCTCCTGAGTTTTCACTATCBrd4 CTGATGTCCGATTGATGTTC AGAGGACACTGTAACAACTG

ChIP  primersGenes Forward  primer Reverse  primerEbf1 CAGCAACCAAAACCTAGCAA TCCCACTATTTATTCCCACACiita ACCTTGGGAGTATGCACTGG AATTGGGTGACCACAGAAGCRag1 TCTCGCTCTCCTGTCAGTCA CCGAGCAGAGACGTTAGCTTGfi1b TCCCCAGAAATCATGTCAGA GCTATTTCTGCCAAGGGTGAFoxo1 CTGGTCAAGCTCTTGCCTGT GGATTGCAAGTTCTCCTCCAIkzf3 GCCAAAGAAACACAGGCAAT CCTCAAGAGCTGCTCACCTTcontrol  (gene  desert) TCAGAAAGGAATCAATCAATCAAA   ATGCCCTCTTCTGGTGTGTC

4C  primersGenes Reading  primer Non-­‐reading  primer

Klf4  promoterAATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTGACAGGACAAGCGCGTAC

CAAGCAGAAGACGGCATACGAGAGATACCTTTCACCAGGGAT

Klf4  enhancer  AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTCGCTTTATGTTCTGCCAGTAC

CAAGCAGAAGACGGCATACGATGTCACAGCCCCAGTAGTG

Supplementary Table 2 List of proteins annotated with the GO term “protein degradation”.

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Supplementary Table 3 List of primers used.

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