supplementary information - nature · i ii iii iv v vi vii viii ix di stefano et al_ suppl. figure...
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S U P P L E M E N TA RY I N F O R M AT I O N
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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
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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
IP Lsd1WB LSD1
IP Lsd1WB CEBPα
IP Lsd1WB Hdac1
IP BRD4 -WB BRD4IP Brd4-WB CEBPα
Suppl. Fig. 1D
Sox2Sall4 Gdf3 Nanog
Lin28 Oct4Tfcp2l1
GAPDHBrd4 Lsd1
H3
Suppl. Fig. 3F
Cdk9 βTubulin
Hdac1
Suppl. Fig.3D PCNA
CEBPαLsd1
IP CEBPα -WB CEBPα
Suppl. Fig. 3G
IP CEBPα -WB PCNA
IP Lsd1-WB Lsd1
IP CEBPα -WB PARP
IP Hdac1 -WB Parp1
IP Lsd1-WB Parp1
IP Hdac1 -WB Pcna
IP Lsd1-WB Pcna
IP Hdac1 -WB Hdac1
-17
250-52-
55-
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43
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B cells
Bα’ ce
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B cells
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lls
B cells
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3h 6h 18h
C/EBPα
Input
IgG Brd4
Input
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Fig. 4E
Input
IgG C/EBPα
Input
IgG Lsd1
Input
IgG Lsd1 Input
IgG C/EBPα
Input
IgG Hdac1
Fig. 3C
70-
Di Stefano et al_ Suppl. Figure 8
50
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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