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DNA repair [12]
Cellular response to DNA damage[86]
Chromatin modification[10]
Embryo development[52]
Cell cycle [56]
Cell adhesion[42]
Cell proliferation[78]
Cell death [76]
Developmental process[194]
Gene expression [163]
DNA repair [21]
Cellular response to DNA damage[86]
Chromatin modification[49]
Embryo development[79]
Cell cycle [101]
Cell adhesion[90]
Cell proliferation[129]
Cell death [134]
Gene expression [348]
Developmental process[361]
DNA repair [40]
Cellular response to DNA damage[86]
Chromatin modification[42]
Embryo development[75]
Cell cycle [100]
Cell adhesion[130]
Cell proliferation[137]
Cell death [178]
Developmental process[452]
Gene expression [338]
DNA repair [4]
Cellular response to DNA damage[86]
Chromatin modification[7]
Embryo development[10]
Cell cycle [21]
Cell adhesion[12]
Cell proliferation[16]
Cell death [22]
Developmental process[28]
Gene expression [44]
a b
c d
Supplementary Figure S1. (a-d) Distribution of CtBP bound regions in cancer-related gene ontologies. (a) Genes that have CtBP binding downstream of TSS, (b) Genes that have CtBP binding peaks at the distal upstream regions, (c) Genes that have CtBP binding peaks in their introns, and (d) Genes that have CtBP binding peaks at their exons. The GO terms are GO:0006281 DNA repair; GO:0006974 Cellular response to DNA damage; GO:0016568 Chromatin modification; GO:0009790 Embryo development; GO:0007049 Cell cycle; GO:0007155 Cell adhesion; GO:0008283 Cell proliferation; GO:0008219 Cell death; GO:0032502 Developmental process; and GO:0010467 Gene expression.
1
307
8.3e-150
MA0346.1 (NHP6B) , M01504 (Nhp6b) , Srf_secondary, Tcfap2e_secondary, Mtf1_secondary, M00218 (MYB.Ph3) , Tcf3_secondary, M01247 (Nanog) , Foxa2_secondary, Zfp105_primary, M01566 (Sfl1) , MA0377.1 (SFL1) , M01012 (HNF3) , MA0120.1 (id1) , Elf3_secondary, M01324 (Octamer) , M00092 (BR-C) , M01092 (TCF) , M01183 (BCL6) , Pou3f3_3235.2
310
5.1e-086
MA0062.2 (GABPA) , M01258 (GABP) , MA0076.1 (ELK4) , Gabpa_primary, M00016 (E74A) , M01163 (Elk-1) , M01165 (Elk-1) , M01078 (c-Ets-1) , M01167 (SAP-1a) , Ehf_primary, MA0062.1 (GABPA) , M00108 (NRF-2/GABP) , M00025 (Elk-1) , M00771 (Ets) , MA0026.1 (Eip74EF) , M01208 (FLI1) , M00341 (GABP) , M00032 (c-Ets-1(p54)) , M00224 (STAT1) , M01203 (PU1)
248
7.3e-055
M00196 (Sp1) , M00932 (Sp1) , M00931 (Sp1) , M00933 (Sp1) , M01273 (SP4) , M00255 (GC) , MA0079.2 (SP1) , M01303 (SP1) , M00982 (KROX) , MA0443.1 (btd) , Klf7_primary, Sp4_primary, Zfp410_secondary, M00008 (Sp1) , Egr1_primary, M01068 (UF1H3BETA) , Bcl6b_secondary, M01175 (CKROX) , Smad3_secondary, M01219 (SP1:SP3)
75
2.3e-048 Atf1_primary, Jundm2_primary, M00916 (CREB) , M01187
(STF1) , M00036 (v-Jun) , MA0018.1 (CREB1) , M00113 (CREB) , M01186 (STF1) , M00178 (CREB) , MA0018.2 (CREB1) , M00338 (ATF) , MA0374.1 (RSC3) , M00039 (CREB) , M00917 (CREB) , M00177 (CREB) , M01586 (TGA2) , M00179 (ATF2) , M00041 (ATF2:c-Jun) , M00691 (ATF1) , M00801 (CREB)
124 8.1e-045
M00652 (NRF-1) , MA0291.1 (DAL82) , M01243 (MTF1) , M01192 (HDG9) , Zfp161_primary, M01579 (Tbs1) , M01069 (GZF1)
62 4.1e-025
M00264 (Staf) , M00262 (Staf) , MA0088.1 (znf143) , M01260 (STAT1) , MA0137.2 (STAT1) , M00492 (STAT1) , M00457 (STAT5A) , M00460 (STAT5A) , M00224 (STAT1) , M00459 (STAT5B) , M01169 (Ikaros) , M00493 (STAT5A)
51
7.9e-013
M00793 (YY1) , M01035 (YY1) , M01057 (ERF2) , M00069 (YY1) , M00651 (NF-muE1) , MA0095.1 (YY1) , M00059 (YY1) , M00733 (SMAD4) , M01199 (RNF96) , MA0394.1 (STP1)
103
3.2e-002
M00171 (Adf-1) , M00923 (Adf-1) , M00695 (ETF) , M00982 (KROX) , M01057 (ERF2) , MA0079.2 (SP1) , M00933 (Sp1) , M01273 (SP4) , M01219 (SP1:SP3) , MA0410.1 (UGA3) , MA0123.1 (abi4) , M00321 (Muscle) , M00196 (Sp1) , M00958 (ABI4) , Egr1_primary, M00932 (Sp1) , M01253 (CNOT3) , M00915 (AP-2) , M00807
(Egr) , M01199 (RNF96)
82
2.5e-010
M00027 (AbaA) , Gabpa_secondary, M01172 (PU.1) , M01266 (ELF1) , M01219 (SP1:SP3) , MA0149.1 (EWSR1-FLI1) , MA0079.2 (SP1) , M00971 (Ets) , Sp4_primary, M00771 (Ets) , M00658 (PU.1) , Ascl2_secondary, M01122 (ZNF219) , MA0068.1 (Pax4) , M00339 (c-Ets-1)
, M00491 (MAZR) , M00957 (PR) , M01203 (PU1) , M01118 (WT1) , Irf3_secondary
Supplementary Figure S2. The Top 9 consensus binding motifs enriched under CtBP peaks identified from the top 1500 CtBP binding hotspots using MEME. Numbers indicate the frequency of the motifs within all the CtBP binding sequences and with the significance. Signif icance is ranked according to E-values as labeled. The E-value of a motif is based on its log likelihood ratio, width, numer of sites, the background letter frequencies, and the size of the training set. E-values are an estimate of the expected number of motifs with the given log likelihood ratio (or higher), and with the same width and site count, that one would find in a similarly sized set of random sequences. The consensus motifs were used to match transfac binding motifs using TOMTOM programming. The best matched binding motif is listed at right indicating the potential co-occupancy of CtBP with those factors.
2
00.20.40.60.81
1.21.41.6
CtrlCtBPKDIGG
* * ** **
* * * * * * * * * *
*
* * ** ** * * * * *
*
*
CtBPMCF-7
CtB
P C
hIP
N
orm
aliz
ed to
Input
0
0.04
0.08
0.12
0.16
0.2
* ** ** ***
**
** ** * ** **
*
* ****
CtrlCtBPKDIGG
MDA-MB-231CtBP
Supplementary Figure S3. CtBP in both MCF-7 and MDA-MB-231 cells before and after CtBP gene depletion by RNAi. (Top) Western blot of CtBP depletion and over-expression (CtBP1-GFP) in MCF-7 and MDA-MB-231 cells.(Bottom) ChIP assay of CtBP at genes indicated. Data shown is the average of 2 independent experiments and the error bars represent the standard deviation of the mean. P values are calculated by 2-sided student’s t-test. Single asterisk represents p<0.05 and double asterisk represents p<0.01. Intr 15 is a non-promoter region located in intron 15 of the BRCA1 gene locus used as a negative control.
ChIP validation assay of selected 30 gene promoters (Fig. 1c)
Intr
15
anti-CtBP
anti-Actin
CtBP
CtBPGFP
CtBP
CtBPGFP
anti-CtBP
anti-Actin
anti-CtBPanti-CtBP
Ctrl CtBP OE Ctrl CtBP OE
CtrlCtBP KD CtBP KDCtrl
MCF-7 MDA-MB-231
CtB
P C
hIP
Norm
aliz
ed to Input
Intr
15
3
0123456789
10
** ** *
*** ** * *
***
**
**
*
** *
* **
**
*
CtrlCtBPKD
MCF-7 mRNA
0
5
10
15
20
25
*
**
**
** ** ** * * *
**
*
*
* *
*
*
**
** * **
**
* *
CtrlCtBPKD
MDA-MB-231 mRNA
Supplementary Figure S4. Quantitative gene expression analysis of selected 30 CtBP target genes in MCF-7 and
MDA-MB-231 cells following with CtBP depletion by RNAi (CtBPKD). Here 53% and 80% of genes show
significant up-regulation (P< 0.05) and 16% and 0% of genes show significant down-regulation (P<0.05) in MCF-7
and MDA-MB-231 cells respectively. Data shown is the average of 3 independent experiments and the error bars
represent the standard deviation of the mean. P values are calculated by 2 sided student’s t-test. Single asterisk
represents p<0.05 and double asterisk represents p<0.01.
mR
NA
(qR
T-P
CR
) N
orm
aliz
ed to 1
8s
rRN
Am
RN
A(q
RT-P
CR
) N
orm
aliz
ed to 1
8s
rRN
A
4
0
0.5
1
1.5
2
2.5
**
*
*
*
*
** ** **
****
*
*
* * *
*
****
CtrlCtBPOE
MCF-7 mRNA
* *
* *
*
0
0.5
1
1.5
2
2.5
3
3.5CtrlCtBPOE
MDA-MB-231 mRNA
Supplementary Figure S5. Quantitative gene expression analysis of selected 30 CtBP target genes in MCF-7 and MDA-MB-231 after CtBP overexpression (CTBPOE). There are 16% and 0% of genes that show significant up-regulation and 43% and 6% genes that show significant down-regulation in MCF-7 and MDA-MB-231 respectively. The data shown is the average of 3 independent experiments and the error bars represent the standard deviation of the mean. P values are calculated by 2-sided student’s t-test. Single asterisk represents p<0.05 and double asterisk represents p<0.01.
mR
NA
(qR
T-P
CR
) N
orm
aliz
ed to 1
8s
rRN
Am
RN
A(q
RT-P
CR
) N
orm
aliz
ed to 1
8s
rRN
A
5
0
0.5
1
1.5
2 CtrlCtBP OE
CD44
CD24
** ** * ** *
* * * * * * * * * ** * * * * ** ** * ** ** ** ** **
*
0
1
2
3
4 CtrlCtBP KD
CD24
CD44N
orm
aliz
ed to 1
8s
rRN
A
**
** **** ** *
** * * * * * * *** * * * * * * * * * ** * * * **
0
0.4
0.8
1.2
1.6
UP
PR
O
DW UP
PR
O
DW UP
PR
O
DW UP
PR
O
DW UP
PR
O
DW UP
PR
O
DW UP
PR
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DW UP
PR
O
DW UP
PR
O
DW UP
PR
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DW UP
PR
O
DW UP
PR
O
DW UP
PR
O
DW UP
PR
O
DW UP
PR
O
DW UP
PR
O
DW UP
PR
O
DW UP
PR
O
DW
CtBP PRONSIgG PROCtBP UP/DWNSIgG UP/DW
CtB
P C
hIP
Norm
aliz
ed to Input
BPNT1 PLS1 ELF3 PIK3R3 GADD45B SIRT1 IGHMBP2 AGR2 MRPL13 DDIT3 WWP1 RASEF RAD9A VAV3 PRLR ANAPC5 MAT2B NDC80
**
** ** ** ** ** ** ** **** ** ** ** ** **
* *
*
MCF-7
MCF-7
MCF-7CtBP
Supplementary Figure S6. Quantitative ChIP and gene expression validation analysis of additional 41 CtBP target genes in cells overexpressing (CtBP OE) or depleted of CtBP KD) in MCF-7 cells. (Top and Middle panel). 14.6% genes show significant up-regulation and 48.7% of genes show significant down-regulation with CtBP overexpression. 60.9% genes show significant up-regulation and 12.1% of genes show significant down-regulation with CtBP depletion. (Bottom) panel shows validation of 18 CtBP target genes identified in CtBP ChIP-seq by CtBP ChIP assay. Both regions upstream of promoter (UP) and downstream of promoter (DW) were selected as negative controls for each individual gene. The gene expression data shown at top and middle are the average of 3 independent experiments and the ChIP data shown is the average of 2 replicates. The error bars represent the standard deviation of the mean. P values are calculated by 2-sided student’s t-test. Single asterisk represents p<0.05 and double asterisk represents p<0.01.
Norm
aliz
ed to 1
8s
rRN
A
6
DNA repair [9]
Cellular response to DNA
damage[12] Chromatin modification[8]
Embryo development[14]
Cell cycle [24]
Cell adhesion[16]
Cell proliferation[18]
Cell death [24]
Developmental process[57]
Gene expression [64]
DNA repair [4]
Cellular response to DNA
damage[12] Chromatin modification[6]
Embryo development[6]
Cell cycle [23]
Cell adhesion[6]
Cell proliferation[11]
Cell death [24]
Developmental process[34]
Gene expression [57]
279 CtBP targets with significant expression change upon CtBP knockdown
179 CtBP targets up-regulated significantly upon CtBP knockdown 100 CtBP targets down-regulated
significantly upon CtBP knockdown
DNA repair [8]
damage[17] Chromatin modification[10]
Embryo development[14]
Cell cycle [30]
Cell adhesion[12]
Cell proliferation[18]
Cell death [31]
Developmental process[81]
Gene expression [79]
Cellular response to DNA
a
b c
Supplementary Figure S7. Distribution of cancer specific gene ontology classes in CtBP bound genes identified as differentially expressed after CtBP gene depletion via micro-array analysis. Shown are the gene ontology distribution of (a) 279 CtBP target genes that show significant (p<0.05) change in expression following CtBP gene depletion. (b) CtBP target genes that are significantly (p<0.05) up-regulated by CtBP depletion. (c) CtBP target genes that are significantly (p<0.05) down-regulated by CtBP depletion.
7
Comet Assay
Ctrl RNAi
BRCA1 RNAi
+ ++ +++- --- --
P-value -5
=1.6 x 10
*
Ctrl H O2 2 H O2 2
recovery
0
100
200
300
400
500C
om
et A
ssay
Tail
mom
ent
Supplementary Figure S8. Analysis of change in tail moment in control and BRCA1 depleted
cells following DNA damage and recovery.
8
00.51
1.52
2.53
3.54
4.5
0
0.4
0.8
1.2
1.6
2
* *
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** *
*
**
**
**
* **
*
* * *
0
0.1
0.2
0.3
0.4
** ****
**
*
**
**
*
*
*
***
**
*
*
* ** **
CtrlCtBPKD
MDA-MB-231 H3Ac
MDA-MB-231 H4Ac
Supplementary Figure S9. ChIP assay of acetylated H3 and acetylated H4 at the selected 30 gene promoters in both MCF-7 cells and MDA-MB-231 cells. The data shown is the average of 2 independent experiments and the error bars represent the standard deviations. P values are calculated by 2-sided student’s t-test. Single asterisk represents p<0.05 and double asterisk represents p<0.01. Intr 15 is a non-promoter region located in intron 15 of the BRCA1 gene locus used as a negative control.
MCF-7 H3Ac
MCF-7 H4Ac
H3A
c C
hIP
N
orm
aliz
ed to Input
H3A
c C
hIP
N
orm
aliz
ed to Input
H4A
c C
hIP
N
orm
aliz
ed to Input
H4A
c C
hIP
N
orm
aliz
ed to Inpu
t
IGG
CtrlCtBPKDIGG
CtrlCtBPKDIGG
CtrlCtBPKDIGG
Intr
15
Intr
15
Intr
15
* * * * **
* * * ** *
**
* *
*
**
** *
**
*
*
*
*
*
*
**
*
*
*
*
*
*
*
*
*
*0
0.05
0.1
0.15
0.2
0.25
Intr
15
*
**
*
*
*
*
9
Low GlucoseHigh Glucose
Low GlucoseHigh Glucose
Low GlucoseHigh Glucose
00.5
11.5
22.5
33.5
4
AT
R
ER
CC
5
FAN
CD
2
RA
D5
1c
XR
CC
5
FAN
1
PALB
1
MU
S8
1
BR
IP1
0
0.5
1
1.5
2
2.5
3
3.5
4
AN
XA
CLD
N4
CST
6
KR
T1
8
NM
E1
ZN
F1
65
CLD
N9
GR
HL2
PAR
D6
B
0
0.5
1
1.5
2
2.5
3
3.5
4G
ATA
3
FOX
A
HE
S1
CE
BP
B
AM
OT
L2
RA
RG
DK
K1
TH
AP
OV
OL2
*
Genome Stability EMTSTEM/TIC
mR
NA
Fo
ld C
ha
ng
e
(No
rma
liz
ed
to
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8s
rRN
A)
*
0
20
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80
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G1 S G2/M
High Glucose
Low Glucose
Cell Cycle Analysis: MCF7 cells in High and Low Glucose Media (n=2)
Re
lativ
e %
b
a
c
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
p<.0004
p<.0021
*
*
**
**
Lo Glucose
Hi Glucose
Re
lative
Ch
IP H
3 A
ce
tyla
tio
n(N
orm
aliz
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to
in
pu
t a
nd
to
tal H
3)
g
a b c d e g hf i j k l
BRCA1
0–3 –1 1 2.7 3210 62 82 83 84 85 kb
a b c d e hf i j k l
Supplementary Figure S10. Carbohydrate restriction influences histone acetylation and
expression of DNA repair genes and other drivers of breast cancer independent of cell cycle
changes. (a) ChIP profile of relative histone 3 lysine acetylation after normalization to ChIP for
histone 3. Asterisks represent the p-values as indicated. Error bars represent the standard
errors from 5 independent biological replicates. (b) qRT-PCR analysis of gene expression in
MCF-7 cells cultured in high glucose and low glucose medium. The genes are functionally
subcategorized as genome stability, STEM/TIC and EMT related (See Figure 1c). * indicates p-
value <0.05, ** indicates p-value <0.01. Error bars represent the standard errors from 3
biological replicates. (c) Cell cycle analysis of MCF-7 cells cultured in both high glucose medium
and low glucose medium.
10
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
LA
LA
LA
LA
LA
LA
LA
LA
LA
LA
LA
LA
LA
LA
LA
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
NL
NL
NL
NL
NL
NL
NL
NL
NL
NL
NL
NL
NL
NL
NL
CL
CL
CL
CL
CL
CL
CL
CL
CL
CL
CL
CL
CL
CL
CL
ANXA3_EMT
−6
−4
−2
0
2
4
6
−6
−4
−2
0
2
4
6 p=2.12e−16
CDS1_EMT
−4
−2
0
2
−4
−2
0
2
p=2.02e−41
CLDN4_EMT
−6
−4
−2
0
2
−6
−4
−2
0
2
p=6.18e−35
CST6_EMT
−4
−2
0
2
4
−4
−2
0
2
4
p=1.44e−07KRT18_EMT
−4
−2
0
2
4
−4
−2
0
2
4
NME1_EMTp=1.4e−06
ZNF165_EMT
−4
−2
0
2
−4
−2
0
2
p=1.08e−11
CLDN9_EMT
−1
0
2
−
1
2p=1.58e−08
GRHL2_EMT
−3
−2
−1
0
1
2
3
−3
−2
−1
0
1
2
3 p=2.54e−28
PARD6B_EMT
−1
0
1
2
−1
0
1
2
p=5.31e−13
ATR_GS
−2
−1
0
1
2
−2
−1
0
1
2 p=2.65e−08
ERCC5_GS
−2
−1
0
1
2
3
−2
−1
0
1
2
3 p=0.000768RAD51C_GS
−2
0
2
4
6
−2
0
2
4
6p=4.36e−07
XRCC5_GS
FAN1_GS
−1.0
−0.5
0.0
0.5
1.0
1.5
−1.0
−0.5
0.0
0.5
1.0
1.5 p=0.0129
PALB2_GS
−2
−1
0
1
2
−2
−1
0
1
2 p=6.49e−08
BRIP1_GS
0
2
−2
0
2
p=2.58e−16
GATA3_STEM/TIC
−6
−4
−2
0
2
4
−6
−4
−2
0
2
4 p=3.1e−81
FOXA1_STEM/TIC
−10
−5
0
p=1.17e−140
HES1_STEM/TIC
−4
−2
0
2
−4
−2
0
2
p=4.72e−15
CEBPB_STEM/TIC
−4
−2
0
2
−4
−2
0
2
p=1.15e−08
RARG_STEM/TIC
−1
0
1
p=0.0494
DKK1_STEM/TIC
4
6
8
4
6
8 p=2.63e−13
−4
−2
0
−4
−2
0
22
AMOTL2_STEM/TIC THAP10_STEM/TIC OVOL2_STEM/TIC
−4
−2
0
2
−4
−2
0
2p=9.23e−53
1
2
3
1
2
3
00
p=7.91e−10
−3
−2
−1
−3
−2
−1
2
3
2
3
11
−2
−1
0
−2
−1
0
p=1.51e−15
−2
−1
0
1
2
−2
−1
0
1
2FANCD2_GS
p=1.48e−08
MUS81_GS
−1
0
1
2 p=0.0761
−4
−2
0
2
4
−4
−2
0
2
4p=3.89e−49
−2
−1
0
1
2
−2
−1
0
1
2 p=0.000178
a
(Creighton et al, 2010)
Supplementary Figure S11. CtBP targeted gene lists differentiate aggressive molecular
subtypes of breast cancer. (a) ANOVA profiling of individual genes from the EMT, genome
stability and STEM/TIC gene list described in Figure 1c. P-values shown were calculated by
comparing expression means across all subtypes.
11
EGFR-Signaling
Basal vs Luminal Subtype
Hypoxia
Cisplatin
Ductal Ca vs Normal Lobular
DNA Strand Elongation
NES=2.57
NES=2.30
NES=2.30
NES=2.35
NES=2.42
NES=2.51
a b
c d
e f
g
FDR<.0001 FDR<.0009
FDR<.005FDR<.004
FDR<.006FDR<.0001
Supplementary Figure S12. Genes changed by CtBP depletion influence mult iple pathways in
breast cancer tumor progression. (a-f) Gene Set Enrichment Assay (GSEA) of the genes
signif icantly (p<0.05) changed upon CtBP knockdown (Fig 2b). The Top 6 hits are listed. The
matched datasets are listed based on the NES, normalized enrichment score. FDR represents
the false discovery rate. (g) Overlap between CtBP target genes from ChIP-seq analysis and
genes upregulated in Luminal vs basal-like (mesenchymal) breast cancers. P-value was
derived by calculating the hypergeometric distribution based on 23660 refseq sequences.
CtBP bound genes
1823
83
P= 9.0E-37
(Charafe-Jauffret et al 2006)
Genes upregulated in Luminal vs mesenchymal
Breast Cancer447
364 1740
12
0
1
2
3
4
5
6
7
8
9
**
** ** **
*** **
**
*
*
*
*
CtrlMTOB
MCF-7 mRNA
Supplementary Figure S13. Selected 30 CtBP target gene expression in MCF-7 and MDA-MB-231 following MTOB treatment. There are 40% and 46% of genes that show significant up-regulation and 3% and 10% genes that show significant down-regulation in MCF-7 and MDA-MB-231 respectively. The data shown is the average of 3 independent experiments and the error bars represent the standard deviations. P values are calculated by student’s t-test. Single star represents p<0.05 and double star represents p<0.01.
00.51
1.52
2.53
3.54
4.5
*
*
*
* **
*
** ** * * * *
*
*
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CtrlMTOB
MDA-MB-231 mRNA
mR
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) N
orm
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ed to 1
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Am
RN
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orm
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13
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** ** ** * ** **
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*
*
MCF-7 CtBP
* * *
*** * * *
*
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*0
0.04
0.08
0.12
0.16
0.2
Supplementary Figure S14. ChIP assay of CtBP at selected 30 gene promoters in both MCF-7 cells and MDA-MB-231 cells following MTOB treatment. The data shown is the average of 2 independent experiments and the error bars represent the standard deviations. P values are calculated by student’s t-test. Single star represents p<0.05 and double star represents p<0.01. Intr 15 is the negative non-promoter region located in intron 15 of BRCA1 gene locus.
MDA-MB-231 CtBP
CtrlMTOBIGG
CtB
P C
hIP
Norm
aliz
ed to Input
CtB
P C
hIP
Norm
aliz
ed to Input
Intr
15
Intr
15
14
0
0.5
1
1.5
2
2.5
3
3.5
WWP1 CDH17BRCA1SIRT1 SMAD3RSF1 TOB2 TFF1
MTOB 1mM
MTOB 10mM
**
**
** *
*
**
0
0.1
0.2
0.3
0.4
0.5
0.6
UP PRO DW UP PRO DW UP PRO DW UP PRO DW UP PRO DW UP PRO DW UP PRO DW UP PRO DW
Ctrl CtBP PROMTOB CtBP PRO
CtBP UP/DWIgG UP/DW
WWP1 SIRT1 RSF1 TOB2 BRCA1 SMAD3 CDH17 TFF1
** **** ** **
*
* *
CtrlMCF-7
MCF-7CtBP
Supplementary Figure S15. Top panel, expression of 8 additional CtBP regulated genes in response to MTOB. Bottom, ChIP assay of CtBP binding on 8 CtBP target gene promoters in response to MTOB. Both upstream of promoter (UP) and downstream of promoter (DW) were selected as negative control region for each individual gene. The gene expression data at top is the average of 3 independent experiments and the ChIP data shown at the bottom is the average of 2 replicates. The error bars represent the standard deviations. P values are calculated by a 2-sided student’s t-test. Single asterisk represents p<0.05 and double asterisk represents p<0.01.
mR
NA
, qR
T-P
CR
Norm
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8s
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CtB
P C
hIP
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15
Gene Ontology Category TOTAL
GENES CtBP
Bound
LOG10(p)
GO:0044260 cellular macromolecule metabolic process
5948 752
-31.8147
GO:0006396 RNA processing
618 148
-28.2322
GO:0043170 macromolecule metabolic process
6533 791
-27.3157
GO:0010467 gene expression
3913 533
-26.7427
GO:0090304 nucleic acid metabolic process
3938
530
-25.172
GO:0044237 cellular metabolic process
7835
900
-24.9739
GO:0008152 metabolic process
8810
976
-22.6913
GO:0044238 primary metabolic process
7953
896
-21.2635
GO:0006807 nitrogen compound metabolic process
5043
623
-20.6194
Supplementary Table S1. Distribution of CtBP bound regions in cancer-related gene ontologies
16
Supplementary Methods
ChIP and ChIP-seq
All ChIP experiments were carried out as described 57. In brief, cells were cross-linked with 1% (w/v) formaldehyde for 5 min at room temperature. The cross-linking was quenched by 0.125 M glycine for 15 min. Then the cells were washed twice with PBS and collected. About 1x107 cross-linked cells, resuspended in 1 ml immunoprecipitation (IP) buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.5% (v/v) NP-40, 1.0% (v/v) Triton X-100, and freshly added proteinase inhibitor cocktail), and were sonicated for 13x20 s with 30 s break. Then the sonicated cells were centrifuged and the supernatant was used for immunoprecipitation. In most cases, the lysate from at least 2 million cells (up to 10 million) was incubated with each antibody overnight with rotation at 4 °C. The preblocked protein G beads were added to the lysate with rotation for 2 h at 4 °C. The beads were washed with IP buffer supplemented with 500 mM NaCl, IP buffer and then TE buffer, pH 8.0. Finally, the precipitated DNA–protein complex was incubated overnight with SDS and proteinase K at 65 °C . The DNA was purified by phenol-chloroform extraction and ethanol precipitation with the presence of glycogen. The purified DNA can be used for either real-time PCR(ChIP) or library construction(ChIP-seq). The ChIP-seq data of CtBP was stored as NCBI GSE36546.
ChIP-seq data analysis
The 36-mer short-read tags were mapped to the human genome(UCSC HG19), and only uniquely mapping read positions were considered. The procedure in searching for CtBP binding hotspots is as follows: Enrichment of tags in a 250 bp target window relative to a 200 kb surrounding window (local background) is gauged by the model based on the binomial distribution. Each tag is extended to the 150 bp length into its strand and assigned a z-score (explained below) using the target window and the background window both centered on the tag. An unthresholded hotspot is defined as a contiguous union of 250 bp windows whose z-scores are greater than 2. Once a hotspot is called, the z-score of the hotspot is given by the maximum z-score of the constituent tags. z-score calculation: If there are n observed tags and N tags that overlap the target window and the local background window, respectively, then the probability p of a tag in the background window overlapping the target window is given by the ratio [# of uniquely mappable tags for the 250bp window] / [# of uniquely mappable tags for the 200 kb window]. Because not all 36-mers in a window can be aligned uniquely to the reference genome, p differs greatly from genomic region to region. The expected number of tags overlapping the target window is then after this important step of adjusting for the differential mappability of short
reads. The standard deviation of the expectation is )1( pNp −=σ . The z-score for the
observed number of tags in the target window is defined by σµ−= nz . Peak detection and input
adjustment for ChIP-seq. The sequencing data from matching input samples are used for the processing of ChIP data, as a measure of background signal which might be significant. After normalizing the input data to match the number of tags in the ChIP data, the number of input tags is subtracted from the number of ChIP tags in the target window before calculating its z-score. In addition, ChIP hotspots were refined into 150 bp peaks using a peak-finding procedure. For each hotspot, a new z-score ‘peak z’ was computed similarly as for hotspot detection, but instead using
17
a 150 bp window within the hotspot and the hotspot as the target window and local background window, respectively. The peak z score was computed scanning through the hotspot with bp resolution for all possible 150 bp windows. A putative peak is defined as a 150 bp window whose peak z score is above 30. If nearby putative peaks overlap within a hotspot, the 150 bp window with the highest peak z score is selected to be the peak among the cluster of overlapping putative peaks. Therefore, a hotspot may not have a peak or may have multiple peaks. Repeat sequence filtering. In ChIP sequencing reads, artifacts are inevitably found and typically concentrated on relatively small regions. These artifacts were removed by filtering out sequence reads which overlap satellites, LINE (long interpersed repetitive elements), and STR (single tandem repeats) after extension of 150 bp. Repeat-masking data was downloaded from the UCSC genome browser site (http://hgdownload.cse.ucsc.edu/goldenPath/mm9/database/simpleRepeat.txt.gz). False discovery rate calculations. A final z-score threshold was imposed on hotspots for each dataset based on FDR (false discovery rate). To calculate FDR, random sampling of uniquely mappable tags was performed and a random dataset with the same number of tags was generated as for the observed dataset. Hotspots are called for the random dataset and their z-scores are calculated. Then the FDR for the observed hotspots at a given z score threshold z0 is estimated as [# of hotspots with z > z0 in the random data] / [# of hotspots with z > z0 in the observed data]. ‘FDR 0% hotspots’ are selected by setting a z score threshold that is just above the highest z score encountered in the random set.
Volcano plot of CtBP ChIP-seq binding profile
Read density for positions relative to transcription start sites (TSS). Shown is total number rd* of read tags summed over all Refseq annotated TSS normalized to the length L of the genome and the total number N of aligned reads (rd* = rd * L / N) such that rd*=1 approximately corresponds to an un-enriched distribution of reads.
Statistical calculation of Venn diagrams
To calculate if the overlapping of CtBP target gene list to other referenced gene lists, is statistical significant, a hypergeometric distribution is calculated to draw the statistical p value based on 37630 TSS in refseq(HG19, UCSC).
Comet assay
Comet assay were performed according to Olive et al 58. Briefly, a single-cell suspension was prepared using enzyme disaggregation. Keep cells in ice-cold medium or phosphate-buffered saline to minimize cell aggregation and inhibit DNA repair. Appropriate numbers of cells ere added into low-gelling-temperature agarose and a thin layer of cells will adherent to the Gel-Bond™ film. The cells were exposed to neutral lysis buffer (2% sarkosyl, 0.5M Na2EDTA, 0.5 mg/ml proteinase K (pH 8.0);equilibrate at 4 °C) for overnight at 37°C. Wash the film with electrophoresis buffer twice and submerge the film to the electrophoresis buffer in a chamber for electrophoresis. The cells were stained by SYBR Green and the pictures were obtained using flurecenc microsopy. The tail moment was used to tell the DNA damage extent. The tail moment is calculated by the following fomula: Tail moment=tail length x percentage of Tail DNA. Percentage of Tail DNA= aT×iT/(aT×iT+aH×iH), where aT represents the tail area, iT means average intensity of tail, aH represents the head area and iH means average intensity of Head.
18
Comet ScoreTM was used to analyze the comet pictures.
Immunofluorescence staining of cells
Cells were grown on cover-slips. Wash cells twice with PBS and fix in 3.5% paraformaldehyde. Wash cells with PBS for 3 times. Cells are permeablized in PBS supplemented with 10% goat serum and 0.3% Triton X-100 for 15mins. Wash cells twice by PBS. Then cells were treated with 3% H2O2 for 10mins and wash for twice. Block cells with blocking buffer(10% goat serum in 1xPBS) for 1hr. Incubate cells with primary antibody(1:50) in blocking buffer for 1.5hr. Wash cells for 3 times by PBS. For γH2AX staining, the cells were incubated with Alexa Fluor® 488 Goat Anti-Mouse IgG for 1hr. After washing for 3 times, mount cover slips using VECTASHIELD with DAPI . For CtBP1 and CtBP2 staining, the Cells were incubated with EnVision+ System- HRP Labelled Polymer Anti-mouse (Dako, Carpinteria, CA, K4000) for 1 hr. After washing for 3 times, cells were incubated with Liquid DAB+ (Dako) for 3 minutes, and wash twice again. Then the cells were counterstained in Hematoxylin (Vector) for 30 sec, twice. Sequentially wash cells with water(twice, 5mins), 95% ethanol(2min) and 100% ethanol(2min). Finally, dip cells in xylene and mount with Permount onto a slide for microscopy imaging.
Immunohistochemistry staining of tissues:
Formalin-Fixed, Paraffin-Embedded tissues were de-paraffinized by submerging the slides in Xylene twice for 15 minutes each. Tissues were then rehydrated by immersing slides into 100%, 95% and 70% ethanol twice for 5 minutes each sequentially. The tissues were next rinsed by immersing into distilled H2O for 3 minutes twice followed by a sequential 5 minute rinse in distilled H2O. A Biocare Medical Decloaking Chamber was then filled with 500mL of water. Tissue slides were placed in a plastic chamber and submerged in 100mL of 1mM EDTA pH 8.0. The plastic chamber was placed into the decloaking chamber and heated to 98°C for 4minutes. Tissue slides were allowed to cool to 70°C before removing from the decloaking chamber. Tissue slides were rinsed by submerging in 1X phosphate buffered saline (PBS) twice for 5 minutes each. Next tissue slides were immersed in 30% hydrogen peroxide (Calbiochem) diluted with distilled H2O to a final concentration of 3% for 10 minutes. Tissue slides were next rinsed in 1X PBS twice for 5 minutes each. Tissue slides were next placed in a humidity chamber and blocked with 500uL of 10% normal goat serum (NGS) (Vector Laboratories) in 1X PBS for 30 minutes at room temperature. Following the expiration of the 30 minute incubation time, the blocking solution was removed and approximately 200uL of appropriate primary antibody either αCtBP1 or αCtBP2 (Calbiochem) at a 1:50 dilution in 1% NGS was added to the tissue and incubated in a humidity chamber for 1 hour at room temperature. Following the 1 hour incubation with primary antibody, slides were washed by submerging slides in 1X PBS twice for 5 minutes each. Next 4-5 drops of secondary antibody conjugated with horse-radish peroxidase (HRP) (Dako EnVision + System-HRP Labelled Polymer Anti-Rabbit or Anti-mouse) was added to the tissues and incubated for 30 minutes at room temperature. Tissue slides were next washed by submerging with 1X PBS twice for 5 minutes each. Four to five drops of DAB in buffer (Dako Liquid DAB + Substrate Chromogen System) was next added to the tissues and incubated for 15 minutes at room temperature. After the incubation time had expired the DAB was removed and tissue slides were immersed in tap water twice for 5 minutes each. The tissue slides were then immersed into hematoxylin (Vector Laboratories) for 20 seconds. Hematoxylin was then rinsed from the tissue slides by immersing slides in tap water twice for 5 minutes each. Tissue slides were then washed by immersing slides into 95% ethanol for 2 minutes followed by a wash in 100% ethanol for 2
19
minutes. Tissue slides were then immersed in Xylene for 1 minute and subsequently mounted with a coverslip using Permount and dried overnight.
Scanning and Analysis of Tissue Slides Method:
Immunohistochemically stained tissue slides were converted to digital slide images by scanning the slides on an Aperio ScanScope XT slide scanner. High resolution digital slide images were then archived into Aperio’s digital pathology information management system “Spectrum”. Digital slide images were analyzed using Aperio’s IHC Nuclear Image Analysis algorithm to assess the nuclear staining for CtBP1 in MCF7 cells and quantify the intensity at individual cells. Values and colors are assigned to individual cells based on the intensity of nuclear staining with a classification of 0 and a blue color for no nuclear staining, 1+ and a yellow color for weak nuclear staining, 2+ and an orange color for moderate nuclear staining and 3+ and a red color for intense nuclear staining.
Tumor gene expression analysis from breast cancer datasets：
Analysis of tumor gene expression from breast cancer datasets was performed using BRB ArrayTools Version: 4.1.0 - Beta_3 Release. Affymetrix data sets were downloaded from the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/). Expression data were loaded into BRB ArrayTools using the Data Import Wizard. U133A probe sets for the individual gene signatures were identified by using the Affymetrix NetAffx Analysis Center Batch Query tool (http://www.affymetrix.com/analysis/index.affx). Expression data were filtered to exclude any probe set that was not a component of the signatures tested, and to eliminate any probe set whose expression variation across the dataset was P>0.05. Kaplan-Meier analysis was performed using the Survival Risk Prediction tool, specifying two risk groups, with fitting to a Cox proportional hazard model with P value ≤ 0.05.
Statistical analysis
All the error bars represent the standard deviations of the mean from at least 3 independent biological replicates unless otherwise indicated. Comparisons between two groups were done using unpaired Student’s t test and P<0.05 was considered statistically significant.
References
57. Nelson,J.D., Denisenko,O., & Bomsztyk,K. Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat. Protoc. 1, 179-185 (2006).
58. Olive,P.L. & Banath,J.P. The comet assay: a method to measure DNA damage in individual cells. Nat. Protoc. 1, 23-29 (2006).
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