Supplementary Information
Materials and methods
Cells
MEFs were prepared from p53-/- or p53-/-Atf-2-/- embryos as described previously
(Maekawa et al., 2007), and cultivated using 3T3 protocol. Spontaneously immortalized
cells after at least 20 passages were isolated and used.
Luciferase reporter assays
A mixture of 1 g of reporter plasmid pGADD45-Luc that contains the human
GADD45 promoter region, 1 g of the plasmid pact-ATF-2 that expresses ATF-2, 2 g
of the plasmid pCMV-BRCA1 that expresses BRCA1, and 0.05 g of internal control
plasmid pRL-SV40 was transfected into p53-/- or p53-/-Atf-2-/- cells using
Lipofectamine (Invitrogen), and luciferase assays were performed. The total amount of
plasmid DNA was adjusted to 4.05 g by adding control plasmid lacking BRCA1.
The DNA fragment containing the human Maspin promoter (nucleotides -764 to
+91) was amplified by PCR and cloned into the luciferase reporter pGL3-basic vector
(Promega). A mixture of 8 g of reporter plasmid, 1 g of pSV40-puro and 1 g of the
internal control plasmid pRL-SV40 was cotransfected into p53--/- cells or p53-/-Atf-2-/cells by using the CaPO4 method. More than 300 puromycin-resistant colonies were
then pooled and used for luciferase assays.
Measurement of luciferase mRNA level using real-time RT-PCR
To determine the critical region in the GADD45 promoter for BRCA1-dependent
activation, a mixture of 1 g of reporter plasmid pGADD45-Luc that contained the
human GADD45 promoter region (-107 to +144 or -62 to +144), 2 g of
pCMV-BRCA1, and 1 g of pRL-SV40 was transfected into p53-/- cells. The amount of
luciferase mRNA was determined by real-time RT-PCR using the ABI 7500 Real-Time
PCR Instrument and the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit
(invitrogen), according to the manufacture’s instructions. Renila luciferase was used as
an internal control. The PCR conditions were 50°C for 15min, 90°C for 10 min, and 40
cycles of 95°C for 15 s and 60 °C for 1 min and 40°C for 1 min. The oligonucleotide
primers used were as follows: luciferase 5’-CAACTGCATAAGGCTATGAAGAGA-3’
and
5’-
ATTTGTATTCAGCCCATATCGTTT-3’,
GAGCATCAAGATAAGATCAAAGCA-3’
Renila
and
luciferase
5’5’-
CTTCACCTTTCTCTTTGAATGGTT-3’
RNase protection assay
Mixtures containing 6 g of pGADD45-Luc that harbor either the wild-type or mutant
GADD45 promoter (nucleotides –812 to +289) and 3 g of pRL-SV40 or 6 g of
pRL-TK as an internal control were transfected into p53-/- or p53-/-Atf-2-/- MEFs by
using Lipofectamine Plus (Invitrogen). The transfected cells were treated with AM (2.5
g/ml) for 4 or 12 h before being harvested. Twenty-four hours after transfection, the
cells were harvested and their poly(A)+ RNAs were isolated by using an mRNA
purification kit (Amersham Pharmacia). Ten g of poly(A)+ RNA was hybridized with
the 32P-labeled RNA probe at 42°C for 16 h according to the manufacturer's protocol
for the RPAII Kit (Ambion). After digestion with a mixture of RNaseA and RNaseT1,
the protected bands were separated on 5% polyacrylamide-8M urea gels. The RNA
probe for GADD45 contained the promoter region from nucleotides –84 to +218,
while the TK probe contained nucleotides –83 to +284 (including the 137-nucleotide
intron), and the SV40 probe contained nucleotides –59 to +306 from the most upstream
transcription initiation site and included the 137-nucleotide intron.
Coimmunoprecipitation
In coimmunoprecipitation assays of endogenous proteins, MCF7 cells were lysed by
mild sonication in NET-NB buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5%
NP-40, 150 mM NaCl) and immunoprecipitation was performed using anti-ATF-2 (N96,
Santa Cruz Biotech.), anti-BRCA1 (Ab-3, Oncogene Research), or anti-Oct-1 (C-21,
Santa Cruz Biotech.) antibodies. Immuno-complexes were resolved on 10%
SDS-polyacrylamide gels, transferred to a PVDF membrane (Immobilon, Millipore),
and probed with anti-Oct-1 or anti-NF-I antibodies (H300, Santa Cruz). Western blots
were developed by using ECL detection reagents (Amersham Pharmacia). To detect the
interaction with NF-I, LSLD buffer (50 mM Hepes-HCl, pH 7.4, 50 mM NaCl, 0.1%
Tween 20, 10% glycerol) was used.
DNA precipitation assay
MCF-7 cells were lysed by mild sonication in NET-NB buffer (20 mM Tris-HCl, pH
8.0, 1 mM EDTA, 0.5% NP-40, 150 mM NaCl, 0.5% 2-mercaptoethanol). 32P-labelled
GADD45 DNA probes that correspond to nucleotides -112 to +287 of the GADD45
promoter region and 1 g of poly(dI-dC) were mixed together with the cell extract.
After an incubation for 1 h on ice, anti-ATF-2 (C19, Santa Cruz), anti-BRCA1 (Ab-3,
Oncogene Research), or anti-Oct1 (12F11, Santa Cruz) antibodies were added and the
lysates were further incubated for 3 h. The protein-DNA complexes were precipitated
with Protein G-Sepharose and washed three times with NET-NB buffer. The recovered
DNA was concentrated by ethanol precipitations and separated on a 2% agarose gel.
In vitro binding assay
The GST pull-down assays using various mutants of GST-BRCA1, GST-Oct-1 or
GST-NF-I and in vitro-translated ATF-2, Oct-1 or NF-I were performed essentially as
described previously (Dai et al. 1996). The GST fusion proteins were expressed in
Escherichia coli and bacterial lysates containing 5 g of each GST fusion protein were
rocked for 2-3 h at 4°C with 100 l of glutathione-sepharose beads (Amersham
Pharmacia). The beads were washed twice with PBS containing 0.6 M NaCl, four times
with PBS containing 0.05% NP-40, and then once with binding buffer (20 mM Hepes,
pH 7.7, 150 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2 , 1% skim milk, 1 mM DTT,
0.05% NP40). The 35S-methionine-labeled ATF-2, Oct-1 and NF-I proteins were
synthesized by using an in vitro transcription/translation kit according to the procedures
described
by
the
supplier
(Promega).
GST-fusion
proteins
bound
to
the
glutathione-Sepharose beads were mixed with the in vitro translated proteins in various
combinations. The mixture were rocked overnight at 4°C, after which the resin was
washed with binding buffer five times, resuspended in SDS sample buffer, and boiled to
release the bound proteins. The proteins were then analyzed by SDS-PAGE followed by
autoradiography.
RT-PCR
Total RNA was isolated from MEFs, mouse tumor tissues, and normal mammary glands
by using Isogen (Nippon Gene) as described previously (Maekawa et al., 2007).
RT-PCR of Atf-2, Gadd45, Maspin, and p53 was performed using 0.5 g of total RNA
and the Superscript One-Step RT-PCR System (Invitrogen) as described previously
(Maekawa et al.,2007). Mouse Maspin mRNA levels were determined by real-time
RT-PCR using the ABI 9700 Real-Time PCR Instrument and the QuantiTect Probe
RT-PCR Kit (Qiagen), according to the manufacturers' instructions. The PCR conditions
were 50°C for 30 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1
min.
The
oligonucleotide
primers
and
probe
used
were
as
follows:
5’-GGAAGTACCATTGGCACAAACTG-3’
and
5’-GAGCAGCACATTGGGAACCT-3’,
and
5’FAM-AGCCCACATCAGCTATCGTTCGTCCA-TAMRA3’.
Gel mobility-shift assay
The gel shift assay was performed essentially as described previously (Maekawa et al.,
1989). Briefly, purified GST-ATF-2 protein spanning amino acids 254-505 was
incubated for 15 min at 25°C with a 32P-labeled oligonucleotide in a solution containing
10 mM Tris-HCl (pH 7.9), 50 mM KCl, 1 mM DTT, 0.04% NP-40, 1 g of poly(dI-dC),
and 5% glycerol. The reaction mixture was separated by electrophoresis through a 4%
polyacrylamide gel in 0.25 x TBE buffer (22 mM Tris-borate, 22 mM boric acid and 0.5
mM EDTA), followed by autoradiography. The oligonucleotides probes used were:
5`-GGGCGTGGCGGTGCTGCCCAGGTGAGCCACCGCTGCTTCTAAG -3` (wild
type) and 5`-GGGCGTGGCGGTGCTGCCTTTTAAAAAAACCGCTGCTTCTAAG
-3` (mutant).
Mice and histological analyses
All p53+/-Atf-2+/- and control p53+/- mice analyzed had a 75% C57BL/6J and 25%
CBA genetic background. Histological analysis of tumors was performed as described
previously (Maekawa et al., 2007).
References
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Maekawa T, Shinagawa T, Sano Y, Sakuma T, Nomura S, Nagasaki K et al. (2007).
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