Fig. S1
CBPKIX/KIX
CBP
+PKA
GST-CREBbZIP
-PKA
WT
Buffer C
CBPKIX/KIX
WT
CBPKIX/KIX
WT
Buffer C
GST-CREBbZIP GST-pCREBbZIP
Input
Anti phosphoCREB blot
Anti CREB
blot
p300
Sup. Fig. S1. CREB-dependent pull-down of CBP is blocked by the KIX mutation. GSTCREBbzip (CREB aa 1-283 lacking the bzip domain) or GST-P-CREBbzip (phosphorylated in
vitro with protein kinase A) was incubated with buffer or nuclear extracts prepared from WT or
CBPKIX/KIX MEFs. Western blots using CBP or p300 specific antisera were used to detect CBP/
p300; phospho-CREB specific antiserum (5322) confirmed Ser133 phosphorylation of the GST
fusion protein.
Fig. S2
Fig. S3
No Cre-adenovirus
Triple-KIX/flox mutant
flox control
FI
Triple-KIX/flox mutant
After Cre-adenovirus
flox control
EtOH
CREB-P
CREB
Western blot
Sup. Fig. S2. Comparable growth
rates of primary MEFs with or without
functional KIX domains. Growth of flox
and triple-KIX/flox MEFs with and
without Cre-adenovirus infection are
shown. Cells were maintained on a
3T9 protocol.
Sup. Fig. S3.
Normal CREB Ser133
phosphorylation in MEFs lacking a functional KIX
domain. Nuclear extracts of MEFs (flox control
and triple-KIX/flox mutant) treated for 30 min.
with EtOH vehicle or FI were blotted with phosphospecific (5322) or non-discriminating (244) CREB
antisera.
Areg
Nr4a3
CREB hypomorph
P=0.0362
relative expression
normalized to Actb
50
25
EtOH
FI
400
300
200
100
0
CREB null
wild type
I
20
10
relative expression
normalized to Actb
CREB hypomorph
CREB null
J
relative expression
normalized to Actb
relative expression
normalized to Actb
WT Hypo
wild type
CREB null
P<0.0001
L
wild type
CREB hypomorph
Crem (KID)
P=0.0003
K
0
P=0.0143
EtOH
FI
wild type
250
CREB null
EtOH
FI
Crem Icer (bZIP)
22.5
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
500
9
8
7
6
5
4
3
2
1
0
35000
30000
25000
20000
15000
10000
5000
0
EtOH
FI
wild type
EtOH
FI
A
re A
g re
m g
C
re C uta
m re n
Ic m t
er Ic
m er
ut
an
N
r4 N t
a3 r4
m a3
ut
an
t
relative expression
normalized to Actb
EtOH
FI
30
8
7
6
5
4
3
2
1
0
P=0.045
H
EtOH
FI
750
Crem (KID)
Crem Icer (bZIP)
wild type
CREB hypomorph
1000
P=0.0217
40
0
wild type
Crem Icer (5' UTR)
F
500
P=0.0658
G
EtOH
FI
P=0.0621
(RLU)
relative expression
normalized to Actb
EtOH
FI
75
wild type
CREB hypomorph
Nr4a3
E
100
0
wild type
1400
1200
1000
800
600
400
200
0
P=0.1594
Areg
D
C
EtOH
FI
relative expression
normalized to Actb
wild type
B
Crem Icer (5' UTR)
relative expression
normalized to Actb
EtOH
FI
900
800
700
600
500
400
300
200
100
0
Luciferase activity
350
300
250
200
150
100
50
0
relative expression
normalized to Actb
A
relative expression
normalized to Actb
Fig. S4
CREB null
P=0.0002
WT
WT Hypo
Null
WT
Null
99
99
NS
53
53
CREB
ATF-1
CREB
Hypo
38
38
anti-CREB (253)
anti-CREB/ATF-1 (5322)
Sup. Fig. S4. CREB and CRE dependence of cAMP-inducible genes. A-J. Expression of cAMP-responsive genes (A-H) and
Crem activating isoforms (I,J) in primary MEFs homozygous for a hypomorphic beta-isoform allele of CREB (Blendy et al., 1996),
A-C,G,I) or homozygous for a null allele of CREB (Mantamadiotis et al., 2002), D-F,H,J). Crem Icer (bZIP) primers (G,H) are
located in the bZIP domain and detect both Crem Icer and some longer isoforms of Crem. Crem Icer (5’ UTR) primers (C,F) and
Crem (KID) primers (I,J) are specific for Crem Icer and activating forms of Crem (e.g. Crem tau), respectively. Note increased
expression of activating forms of Crem in CREB mutant cells (I,J) as previously reported (Hummler et al. 1994). The increase in
EtOH signal of Crem Icer (bZIP) in CREB mutant MEFs may also indicate increased levels of activating isoforms such as Crem
tau (G,H). MEFs were treated for 90 min with ethanol vehicle (EtOH) or 10M forskolin/ 100M IBMX. Expression was
normalized to beta actin and the median wild type EtOH signal was set to 1 for each panel. (mean + s.e.m.; one MEF each
genotype, N=3 (A-C,G,I) or 3 MEFs each genotype, N=8-12 (D-F,H,J). P value shown is from one-tailed t-test on FI-treated wild
type and mutant MEFs. K. Western blot of CREB hypomorph, null and control MEFs demonstrating loss of full length CREB
protein, upregulation of CREB beta-isoform in CREB hypomorph MEFs and presence of CREB family member, ATF-1. Antibody
5322 detects CREB and ATF-1 (Groussin et al. 2000). Non-specific (NS). L. Promoters (including transcription start site) of Areg
(286 bp, 1 full site CRE), Crem Icer (231 bp, 4 full/half site CREs, (Molina et al. Cell 1993)) and Nr4a3 (345 bp, 4 half site CREs)
were cloned into the pGL3 Basic luciferase vector. CREs were mutated (2-3 bases of each CRE were changed), confirmed by
sequencing, and parental and mutant vectors were transfected into wild type MEFs with SV40 Renilla as an internal control.
MEFs were treated for 6-8hr with 10M forskolin/ 100M IBMX (FI) or ethanol vehicle (EtOH). (mean + s.e.m.; N=3-12).
Fig. S5
MSCV
GFP
MSCV
TORC2
GFP
99 kDa
TORC2
Sup. Fig. S5. TORC2 overexpression in MEFs.
Whole cell extracts from MEFs infected with
MSCV-GFP or MSCV-TORC2 GFP virus. Western
blot probed with TORC2 specific antibody.
Fig. S6
Luciferase activity (RLU)
7000
flox control
Triple-KIX/flox
6000
5000
4000
3000
2000
Sup. Fig S6. Triple-KIX/flox MEFs do not
have a general defect in transactivation.
Activity of Gal-Ets-1 and Gal-HIF-1 in flox
control and triple-KIX/flox MEFs normalized
to cotransfected SV40 Renilla luciferase
activity (mean + s.e.m., N=5).
1000
0
Gal-Ets-1
Gal-HIF-1
Gal-CREB-R314A
3000
Gal-CREB-S133A
5X Gal-luc activity
Gal-CREB
Gal-CREB
Gal-CREB-S133A
Gal-CREB-R314A
Gal-CREB-S133A&R314A
4000
No plasmid DNA
COS7 cells (two independent experiments)
5000
Gal-CREB-S133A/R314A
Fig. S7
Gal4-CREB
2000
Endogenous CREB
1000
CREB (244) western
FI
Et
O
H
FI
Et
O
H
FI
Et
O
H
FI
Et
O
H
0
Sup. Fig. S7. Mutation of Ser133 and R314 strongly attenuate Gal-CREB function in Cos7 cells, as in
MEFs. Cos7 cells were transiently transfected with the indicated Gal-CREB vectors, 5X Gal-luciferase,
and SV40 Renilla luciferase. Treatment was for 6 hr with EtOH or FI. (mean + s.e.m, for two
independent experiments). Western showing Gal-CREB and mutant proteins are expressed at similar
levels in Cos7 cells.
Crem Icer
Nr4a3
60
EtOH
FI
50
40
30
20
10
0
GFP
TORC-DN
relative ChIP signal
normalized to input
relative ChIP signal
normalized to input
Fig. S8
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
EtOH
FI
GFP
TORC-DN
GFP ChIP
GFP ChIP
Sup. Fig. S8. CREB target genes are a direct target of TORC-DN. ChIP using GFP-specific
antibody (Invitrogen). WT MEFs were infected with MSCV retrovirus expressing GFP alone or
TORC-DN (TORC1 1-44 fused to GFP). ChIP signal normalized to input.
Fig. S9
IP: CBP/p300
CBP/p300
200 kDa
GSTTORC2
GST
Far-Western
CBP
p300
Western
Sup. Fig. S9 Blots of CBP and
p300 immunoprecipitated from
293T cell extracts with mouse
monoclonal antibodies (anti-p300
RW128 and anti-CBP AC238 and
C-1). Far Western assay blots
were probed with purified
recombinant
GST-TORC2 or
GST protein followed by antiGST antibody (Santa Cruz
SC-459) and then anti-rabbit Ig
linked to HRP. IP Westerns were
blotted with rabbit polyclonal
antibodies to the N- and C-termini
of CBP or p300 as indicated.
C
EtOH
FI
wild type
CREB null
Hlf
B
60
EtOH
FI
50
40
30
20
10
0
wild type
CREB hypomorph
P=0.3006
Hlf FLAG ChIP
D
150
125
100
75
50
25
R
31
4A
S1
33
A
/R
31
4A
S1
33
A
0
G
FP
FL
A
G
-C
R
EB
relative ChIP signal
(average of
EtOH and FI signals)
EtOH
FI
12000
10000
8000
6000
4000
2000
0
ve
ct
or
P=0.4612
14000
H
lf
pr
om
ot
er
H
lf
(m
ut
an
tC
R
E)
14
12
10
8
6
4
2
0
Hlf
Luciferase activity (RLU)
Fig. S10
A
Sup. Fig. S10. The Hlf CRE region is a direct target of FLAGCREB, but Hlf expression is not affected by CREB deficiency.
A,B. Expression of Hlf in MEFs null for CREB (A) or
expressing a hypomorphic allele of CREB (B). MEFs were
treated for 90 min with ethanol vehicle (EtOH) or 10M
forskolin/ 100M IBMX (FI). Expression is normalized to beta
Actin and the median wild type EtOH signal was set to 1 for
each panel. (mean + s.e.m.; 3 MEFs each genotype, N=8-12
(A) or one MEF each genotype, N=3 (B). P value shown is
from one-tailed t-test on FI-treated wild type and mutant MEFs.
C. 133 bases of the Hlf promoter, containing the CRE and
TSS were cloned into pGL3 Basic luciferase (vector). The
CRE was mutated (3rd through 5th bases of the CRE were
changed to Ts, confirmed by sequencing) and
control,
parental and mutant vectors were transfected into wild type
MEFs with SV40 Renilla as an internal control. MEFs were
treated for 6-8 hr with FI or EtOH. Data shown is average of
two experiments, each done in triplicate (mean + s.e.m.; N=6).
Note construct shows little FI-inducibility and that mutation of
the CRE has little effect on luciferase activity. D. WT MEFs
were infected with MSCV retrovirus expressing GFP alone or
the indicated FLAG-CREB constructs. ChIP was performed
using anti-FLAG antibody. ChIP signals were normalized to
input DNA and the percentage of GFP+ MEFs as determined
by flow cytometry. FLAG-CREB for each experiment set to
100. (mean + s.e.m., N=2-4).