fig s1 relative mrna expression mcf7 ps2 * * * * * ccng2 * * * * * * rprm * * * * klf6 * * * * * *...
TRANSCRIPT
Fig S1R
elat
ive
mR
NA
exp
ress
ion
MCF7
0.0
1.0
2.0
3.0
4.0
5.0
6.0 pS2
**
*
**
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0 CCNG2
**
*
*
**
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6 RPRM
*
* *
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2 KLF6
* **
***
0.0
0.5
1.0
1.5
2.0
2.5 CLDN4
*
*
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8 CERK
*
*
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0 EFEMP1
**
*0.0
0.5
1.0
1.5
2.0
2.5 ENC1
*
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6 BIK
*
*
***
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8 HBP1
**
*
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0 CXCR4
*
**
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 RAP1GA1
*
*
*
*0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 CDH3
*
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4BMP7
*
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6 MXD4
*
**
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0 MUC1
*
*
**
0.0
0.5
1.0
1.5
2.0
2.5
3.0 NDRG1*
*
*0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 RBL2
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 VE2E2+ICI
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8 BAK
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0 p21
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 PLK2*
0.0
0.5
1.0
1.5
2.0
2.5 DUSP4
***
4 hr 8 hr 24 hr 4 hr 8 hr 24 hr 4 hr 8 hr 24 hr 4 hr 8 hr 24 hr
Fig S1: Identification of estrogen-repressed genes in MCF7 cells. q-RT-PCR was used to confirm the estrogen-mediated repression of candidate genes identified from microarray data. The relative mRNA expression is depicted as ligand-mediated fold change compared to vehicle control. MCF7 cells were treated for 4, 8, or 24 hours with either vehicle, estrogen (E2), or a combination of E2 and ICI (E2+ICI). The data are an average of three replicates + SEM. A t-test analysis was performed in which the E2 treatment group was compared to the vehicle group and the E2+ICI group was compared to the E2 group (* p < 0.05).
Fig S2
Fig S2: Estrogen-mediated repression of selected genes in breast cancer cells. q-RT-PCR was used to confirm the estrogen-mediated repression of candidate genes identified from microarray data. The relative mRNA expression is depicted as ligand-mediated fold change compared to vehicle control. (A) ZR-75-1 and (B) T47D cells were treated for 4, 8, or 24 hours with either vehicle, estrogen (E2), or a combination of E2 and ICI. The data are an average of three replicates + SEM. A t-test analysis was performed in which the E2 treatment group was compared to the vehicle group and the E2+ICI group was compared to the E2 group (* p < 0.05).
Rel
ativ
e m
RN
A e
xpre
ssio
nZR-75-1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8 CERK
4 hr 8 hr 24 hr
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 RPRM
*
**
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 KLF6
*
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 CLDN4
***
0.0
0.2
0.4
0.6
0.8
1.0
1.2 EFEMP1
*
4 hr 8 hr 24 hr
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6 ENC1
4 hr 8 hr 24 hr
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 VE2E2+ICI
A
B
Rel
ativ
e m
RN
A e
xpre
ssio
n
T47D
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6RPRM KLF6
CERK EFEMP1 ENC1
CLDN4
*
*
*
*
**
**
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 VE2E2+ICI
4 hr 8 hr 24 hr 4 hr 8 hr 24 hr 4 hr 8 hr 24 hr
Fig S3
Fig S3: Identification of E2-repressed genes that are primary ERα targets. (A) MCF7 cells were treated with either vehicle, estrogen (E2), or a combination of E2 and cycloheximide (CHX). q-RT-PCR was used to calculate relative mRNA expression as ligand-mediated fold change compared to vehicle control. This fold change is reflected in the color intensity as shown in the legend.
A4h 8h
E2
CHX++ ++-+ +-
E2 Fold change
<0.3
0.3-0.5
0.5-0.7
0.7-0.9
1.3-1.5
1.5-1.9
>2
1.1-1.3
0.9-1.1
CLDN4
EFEMP1
CERK
KLF6
ENC1
BIK
HBP1
CXCR4
RAP1GA1
CDH3
BMP7
MXD4
NDRG1
RBL2
FLOT1
RND3
TENS1
IER3
Fig S4
Fig S4: Knockdown of HDAC1-10 and its effect on RPRM expression. MCF7 cells were transfected with non-specific siRNA (siNS) or siRNA against HDAC1-10 (siHDAC1-10) followed by either a vehicle or an E2 treatment for 12 hrs. The knockdown of each HDAC is shown in (A) and its effect on RPRM expression is shown in (B). q-RT-PCR was used to calculate relative mRNA expression as fold change compared to vehicle control. The data are an average of three replicates + SEM.
A
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
2.0
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
HDAC1 HDAC2 HDAC3 HDAC4 HDAC5
HDAC6 HDAC7 HDAC8 HDAC9 HDAC10
Rel
ativ
e m
RN
A e
xpre
ssio
n
siNS siHDAC1 siNS siHDAC2 siNS siHDAC3 siNS siHDAC4 siNS siHDAC5
siNS siHDAC6 siNS siHDAC7 siNS siHDAC8 siNS siHDAC9 siNS siHDAC10
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
12
V
E
B RPRM
Rel
ativ
e m
RN
A e
xpre
ssio
n
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
0.0
1.0
2.0
3.0
siNS siHDAC1 siNS siHDAC2 siNS siHDAC3 siNS siHDAC4 siNS siHDAC5
siNS siHDAC6 siNS siHDAC7 siNS siHDAC8 siNS siHDAC9 siNS siHDAC10
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
12
V
E
Fig S5
Fig S5: Localization and expression of the HDAC7 deletion and mutant constructs. (A) 293 cells were transfected with Flag-HDAC, Flag-HDAC7 (1-487), Flag-HDAC7 (438-912), and Flag-HDAC7 (H670A) and immunostained for Flag. (B) 293 cells were transfected with Flag-HDAC, Flag-HDAC7 (1-487), Flag-HDAC7 (438-912), and Flag-HDAC7 (H670A) and blot for Flag and β-actin.
B
HDAC7: +- - - -
-- + -HDAC7 (1-487): -
HDAC7 (438-912): -- - -+
HDAC7 (H670A): -- - +-
β-actin
Flag
100
75
45
50
150
MW (kDa)HDAC7 HDAC7(1-487) HDAC7 (438-912) HDAC7 (H670A)A
Tab
le 1
: q
PC
R P
rim
er a
nd
Pro
be
seq
uen
ces
Gen
eF
orw
ard
Pri
mer
Rev
erse
Pri
mer
Pro
be
β-a
ctin
CC
CT
GG
CA
CC
CA
GC
AC
GC
CG
AT
CC
AC
AC
GG
AG
TA
CA
TC
AA
GA
TC
AT
TG
CT
CC
TC
CT
GA
GC
GC
BA
K1
CA
CG
GC
AG
AG
AA
TG
CC
TA
TG
AC
CC
AA
TT
GA
TG
CC
AC
TC
TC
AT
TC
AC
CA
AG
AT
TG
CC
AC
CA
GC
CT
GT
BIK
TC
TT
GA
TG
GA
GA
CC
CT
CC
TG
TT
CA
TG
CC
AA
GA
AC
CT
CC
AT
GB
MP
7T
GT
AA
GA
AG
CA
CG
AG
CT
GT
AT
GT
CA
TC
AG
GC
GC
GA
TG
AT
CC
AC
CN
G2
GA
TC
GT
TT
CA
AG
GC
GC
AC
AC
AA
CC
CC
CC
TC
AG
GT
AT
CG
TC
DH
3A
GG
CT
GG
TT
GT
TG
TT
GA
AT
AA
GC
AC
AC
AG
CG
TG
GC
CA
AA
GA
GC
ER
KT
CG
GC
GC
TG
CA
TA
TC
GT
TC
AC
GG
AG
TA
GC
GA
AG
GA
GT
GT
CT
GG
CC
AT
GG
AT
GT
GT
CC
TC
AG
TC
CC
LDN
4C
TC
CT
GA
CT
CA
CG
GT
GC
AA
CG
TA
GG
AT
TC
CA
AG
CG
CT
GT
GC
AC
TC
TG
CG
AA
CG
TT
AA
GT
CC
GT
CC
OP
EB
/ K
LF6
AT
GC
CG
TT
CT
GC
AC
CC
TA
CA
GC
CC
TC
AA
AA
GA
CC
TT
CC
AC
AG
TT
GC
CT
CC
AG
GG
CC
TC
TC
CC
XC
R4
TC
AT
CA
CG
CT
TC
CC
TT
CT
GG
CA
TG
GA
CT
GC
CT
TG
CA
TA
GG
AD
US
P4
CG
AC
CG
GC
AA
AA
AT
AC
AC
GC
CG
GT
GG
AG
GA
GA
GT
GT
GT
TE
FE
MP
1A
CT
CT
GG
CG
CT
GG
TC
AA
GT
CC
GT
CT
GG
GA
CA
AT
GT
CA
CA
TT
CC
AC
GT
AC
AC
GC
AA
TG
CA
CT
GA
CG
GE
NC
1G
CT
GA
GA
CG
AC
CG
GT
TC
GT
GC
GG
TT
CT
CA
TG
CA
CA
CT
GA
CG
AC
TG
GC
CG
CA
AC
GC
AC
AE
Rα
AA
CC
GA
GA
TG
AT
GT
AG
CC
AG
CC
AG
GA
AC
CA
GG
GA
AA
AT
GT
GT
GT
CG
AA
GA
TC
TC
CA
CC
AT
GC
CC
TC
TF
LOT
1G
GC
CG
CC
TA
TG
AC
AT
CG
AG
CT
CC
TC
AA
TC
TG
CT
GC
TT
AG
TC
Fox
A1
GG
AT
GT
TA
GG
AA
CT
GT
GA
AG
AT
GG
AG
AC
CG
GG
AC
GG
AG
GA
GT
AG
AA
CC
AG
CG
AC
TG
GA
AC
AG
CT
AC
TA
CG
CA
HD
AC
1G
GA
CG
AA
GA
CG
AC
CC
TG
AC
AC
TT
CA
GA
AT
CG
GA
GA
AC
TC
TT
CC
TH
DA
C2
AA
AA
TT
TG
CG
CA
TG
TT
AC
CT
CA
TG
CT
AT
CC
GC
TT
GT
CT
GA
TG
CT
HD
AC
3T
CC
TG
CA
TT
AC
GG
TC
TC
TA
TA
AG
AA
GA
TA
AG
CG
GC
AC
AT
GT
CA
TG
CT
HD
AC
4C
AC
CC
CA
GA
GC
GG
AG
TG
TG
CC
TG
GA
CC
GT
AA
TT
TC
AG
AT
TH
DA
C5
CT
CG
GC
TC
TG
CT
CA
GT
GT
AG
AG
GG
CC
AC
TG
CG
TT
GA
TG
TT
GH
DA
C6
AA
TC
CA
AG
AG
AA
TT
GG
GT
GT
GT
CT
CC
AT
GG
TG
TT
GG
AG
CA
TG
TG
HD
AC
7G
CG
GC
AG
CT
TC
TC
CA
CA
AT
CA
GA
AT
CA
CC
TC
CG
CT
AG
CT
TH
DA
C8
CA
GC
CA
AG
AG
GG
CG
AT
GA
TC
AG
TG
GC
TG
GG
CA
GT
CA
TA
AH
DA
C9
GA
AT
GC
AC
AG
TA
TG
AT
CA
GC
TC
AG
TC
AC
CA
CG
GG
CA
TC
AT
CA
TC
HD
AC
10G
GA
TC
CA
GT
AT
CT
CT
TT
GA
GG
AT
GA
CA
GA
AG
CG
CC
CA
TG
CT
CA
TA
HB
P1
AA
GC
AG
AA
GC
TG
GG
AG
TC
GC
GC
CT
AT
GT
TT
GG
CA
CA
GA
TC
CT
CT
AG
TA
AC
CA
CC
AC
AT
GG
CT
CA
GC
IER
3G
CA
GC
CG
CA
GG
GT
TC
TC
CC
TC
TT
CA
GC
CA
TC
AG
GA
TC
TG
MLR
2/LC
oRT
CA
CC
TC
TG
GA
CC
TT
AC
TG
TC
AG
AG
CT
GC
CA
GC
AC
AT
GG
AC
TT
MU
C1
AG
AC
AG
CC
AA
GG
CA
AT
GA
GA
TG
CT
GG
TG
CT
GG
TC
TG
TG
TG
AC
AA
TG
GC
CA
GC
GC
AA
CC
AG
MX
D4
CC
CG
AA
CA
AC
AG
GT
CT
TC
AC
AA
CC
AG
TT
GC
TT
GA
GC
TG
CT
CA
N-C
oRA
GC
AT
TC
CA
TC
CC
TA
CG
GG
TG
GA
CC
CC
TT
CA
CC
AA
AG
CN
DR
G1
CG
CT
GA
GG
TG
AA
GC
CT
TT
GC
AC
AC
AG
CG
TG
AC
GT
GA
AC
AG
p21
GG
CG
TT
TG
GA
GT
GG
TA
GA
AA
TC
TC
TG
GA
GA
CT
CT
CA
GG
GT
CG
AA
TC
AT
GC
TG
GT
CT
GC
CG
CC
GT
PLK
2G
CA
AC
AC
TT
CC
CA
TG
GT
AC
TG
AG
TC
AG
AG
GG
AC
TC
TT
GG
CA
GC
TT
CA
AG
GC
AT
TC
AC
TG
CT
GC
TG
CT
AC
ApS
2C
CT
CC
C A
GT
GT
G C
AA
AT
A A
GG
T
CT
TC
T G
GA
GG
G A
CG
TC
G A
T
CT
GC
TG
TT
TC
GA
CG
AC
AC
CG
TT
CG
R
AP
1GA
1G
GA
CC
GA
AT
CT
GT
GT
AC
TG
CA
GT
GT
AT
GG
CA
GC
TT
GG
TG
GA
CT
TC
CG
CA
AC
AA
GG
AG
AT
CA
TG
TT
TC
AR
BL2
TT
TC
GT
TT
TG
CG
GA
GA
TG
CT
TG
CA
GG
CC
AA
GA
GA
GA
TC
TG
TG
RIP
140
TT
GG
AG
GG
AG
GC
TT
CA
TC
TG
GC
TT
TC
GT
TT
CT
GC
AG
TA
GG
AA
GT
AR
ND
3C
AC
AG
GC
AG
AC
GC
CA
GT
GT
AT
GC
CA
AG
GT
GG
CA
AC
GT
RP
RM
CG
GA
CG
AG
CG
TA
GC
CT
GT
AG
TG
AG
TG
AG
AG
CA
CG
CA
CA
TG
CG
CG
AT
CT
GC
AC
CA
CG
CG
CA
TT
AT
SA
FB
1C
CC
GA
AG
AT
GA
CT
CG
GA
TA
CA
AT
GT
AG
AA
GA
GA
GT
CC
AC
TA
AC
CC
AG
AA
AC
CA
CA
AC
TG
CT
GC
GA
CC
CT
TT
TC
CT
SA
FB
2G
AT
CT
CA
AG
AA
CC
TT
TT
CA
GC
AA
GT
AT
TC
GA
CA
TG
GT
GA
CG
AA
TC
CA
CC
AA
AG
TG
GT
AA
CG
AA
CG
CC
CG
CS
MR
TG
GT
CA
AG
TC
CA
AG
AA
GC
AA
GA
GA
TG
CT
TC
TA
TA
GG
TC
AT
AA
GG
CC
TG
TT
CT
GA
AC
AC
CC
AC
AA
CC
GG
AA
TG
AG
CT
EN
S1
CA
GC
AT
GT
GG
TC
GT
CA
TT
CA
CT
GA
GA
CG
TT
GG
TG
AA
AT
GC
AT
GT
* A
SY
BR
Gre
en a
ssay
was
use
d fo
r ge
nes
lack
ing
a pr
obe.
*
Tab
le 2
: C
hIP
Pri
mer
s
Gen
eF
orw
ard
Pri
mer
Rev
erse
Pri
mer
RP
RM
-47
76T
CA
CT
TC
AT
TT
GT
TG
AT
GG
AC
GT
TT
TG
AA
AT
CA
TG
TA
CT
TA
AA
CA
GG
GA
TA
TG
RP
RM
+10
6C
AT
GT
GC
GT
GC
TC
TC
AC
TC
AG
GT
CC
TT
CA
CG
AG
GA
AG
TT
GA
TR
PR
M +
4105
GG
TA
GA
TG
GT
CC
AT
GA
TT
TC
TG
AT
TT
GT
CT
TT
CC
CT
TC
CT
AA
TT
CA
GA
GT
TpS
2G
GC
CA
TC
TC
TC
AC
TA
TG
AA
TC
AC
TT
CT
GC
GG
CA
GG
CT
CT
GT
TT
GC
TT
AA
AG
AG
CG
ER
3440
CA
CC
CC
GT
GA
GC
CA
CT
GT
CT
GC
AG
AA
GT
GA
TT
CA
TA
GT
GA
GA
GA
T