Bach1 inhibits oxidative stress-induced cellular senescence by impeding p53 function on chromatin.

Abstract

Cellular senescence is one of the key strategies to suppress expansion of cells with mutations. Senescence is induced in response to genotoxic and oxidative stress. Here we show that the transcription factor Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1), which inhibits oxidative stress-inducible genes, is a crucial negative regulator of oxidative stress-induced cellular senescence. Bach1-deficient murine embryonic fibroblasts showed a propensity to undergo more rapid and profound p53-dependent premature senescence than control wild-type cells in response to oxidative stress. Bach1 formed a complex that contained p53, histone deacetylase 1 and nuclear co-repressor N-coR. Bach1 was recruited to a subset of p53 target genes and contributed to impeding p53 action by promoting histone deacetylation. Because Bach1 is regulated by oxidative stress and heme, our data show that Bach1 connects oxygen metabolism and cellular senescence as a negative regulator of p53.

Full text

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Bach1 inhibits oxidative stress–induced cellular
senescence by impeding p53 function on chromatin
Yoshihiro Dohi1,2,7, Tsuyoshi Ikura1, Yutaka Hoshikawa3, Yasutake Katoh1, Kazushige Ota1, Ayako Nakanome1,
Akihiko Muto1, Shinji Omura2, Tsutomu Ohta4, Akihiro Ito5, Minoru Yoshida5,6, Tetsuo Noda3&
Kazuhiko Igarashi1
Cellular senescence is one of the key strategies to suppress expansion of cells with mutations. Senescence is induced in response
to genotoxic and oxidative stress. Here we show that the transcription factor Bach1 (BTB and CNC homology 1, basic leucine
zipper transcription factor 1), which inhibits oxidative stress-inducible genes, is a crucial negative regulator of oxidative stress–
induced cellular senescence. Bach1-deficient murine embryonic fibroblasts showed a propensity to undergo more rapid and
profound p53-dependent premature senescence than control wild-type cells in response to oxidative stress. Bach1 formed a
complex that contained p53, histone deacetylase 1 and nuclear co-repressor N-coR. Bach1 was recruited to a subset of p53
target genes and contributed to impeding p53 action by promoting histone deacetylation. Because Bach1 is regulated by oxidative
stress and heme, our data show that Bach1 connects oxygen metabolism and cellular senescence as a negative regulator of p53.
Induction of premature cellular senescence is thought to be one of the
key strategies to preclude transformed cells from expansion in higher
eukaryotes1–4. It is often induced by the tumor-suppressor gene
pathways involving p53 and retinoblastoma (Rb) proteins5. p53
induces premature cellular senescence by inducing genes such as p21
(ref. 6) and Serpine1 (also known as PAI-1, encoding plasminogen
activator inhibitor-1)7. p53 is usually degraded through polyubiqui-
tination mediated by the E3 ubiquitin ligase Mdm2 (refs. 8,9), but it is
stabilized upon genotoxic stress, in part by phosphorylation of p53,
which negates the recognition of p53 by Mdm2 (ref. 10). DNA is
exposed to reactive oxygen species (ROS) that are generated endo-
genously as by-products of respiration11. When not properly mana-
ged, oxidative stress induces premature cellular senescence of murine
embryonic fibroblasts (MEFs), at least in part by causing DNA
damage5,12. Because genetic ablation of Trp53 (which encodes p53)
in MEFs abrogates cellular senescence13, the p53-mediated response,
but not the accumulation of DNA damage per se, is responsible for the
premature senescence. Thus, oxidative stress–induced cellular senes-
cence is a specific event governed by a genetically determined program
of transcription factor–network activities that includes p53. However,
little is known about the mechanisms that control the implementation
of the senescence program by p53 in response to oxidative stress.
Transcription factor Bach1 is a repressor of the oxidative stress
response in mice14,15. Bach1 forms a heterodimer with the small Maf
oncoproteins and binds to the Maf-recognition element (MARE) to
inhibit target genes, including that encoding heme oxygenase-1
(Hmox1, also known as HO-1), a critical protective gene against
oxidative stress15,16. Bach1–/–mice show reduced arteriosclerosis in
an injury model, and their smooth muscle cells show reduced
proliferation in vitro17, raising the possibility that Bach1 regulates
cell proliferation. To understand the molecular mechanisms under-
lying Bach1-mediated regulation of cell proliferation, we carried out a
detailed analysis of Bach1-deficient (Bach1–/–) MEFs. We found that
Bach1 was required to suppress the p53-dependent cellular senescence
of MEFs in response to oxidative stress. Purification and characteriza-
tion of the Bach1 complex from murine erythroleukemia (MEL) cells
revealed a biochemical interaction of Bach1 and p53. These genetic
and biochemical data suggest that Bach1 forms a complex with p53 on
a subset of its target genes to restrain the transcriptional activity of p53
by recruiting histone deacetylase 1 (Hdac1), thus inhibiting the
process of cellular senescence.
RESULTS
Suppression of cellular senescence by Bach1
To investigate the involvement of Bach1 in cell proliferation, we
prepared Bach1–/–MEFs. Bach1–/–MEFs showed a markedly reduced
proliferation rate when compared with control wild-type cells
(Fig. 1a). Cell staining with propidium iodide did not reveal major
Received 13 March; accepted 16 October; published online 16 November 2008; doi:10.1038/nsmb.1516
1Department of Biochemistry, Tohoku University Graduate School of Medicine, Seiryo-machi 2-1, Sendai 980-8575, Japan.2Department of Cardiovascular Physiology
and Medicine, Hiroshima University Graduate School of Biomedical Science, Kasumi 1-2-3, Hiroshima 734-8551, Japan.3Japanese Foundation for Cancer Research,
Cancer Institute, Ariake 3-10-6, Tokyo 135-8550, Japan.4Center for Medical Genomics, National Cancer Center Research Institute, 5-1-1 Tsukiji Chuo-ku, Tokyo
104-0045, Japan.5Chemical Genetics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.6Japan Science and Technology Corporation (JST), CREST Research
Project, Kawasaki, Saitama 332-0012, Japan.7Present address: Department of Cardiovascular Medicine, Hiroshima University Graduate School of Biomedical
Sciences, Hiroshima 734-8551, Japan. Correspondence should be addressed to K.I. (igarak@m.tains.tohoku.ac.jp).
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differences in the sub-G1 populations between Bach1–/–and control
MEFs (Supplementary Fig. 1 online), indicating that the reduced
proliferation was not due to an increase in apoptosis. To analyze the
effects of Bach1 deficiency on the cell cycle, we measured the
percentage of replicating cells after incubation with 5-bromodeoxy-
uridine (BrdU) for 30 min (Fig. 1b and Supplementary Fig. 2
online). Whereas 12.8% of control cells were positive for BrdU,
indicating replication, only 6.7% of Bach1–/–MEFs incorporated
BrdU (Fig. 1b and Supplementary Fig. 2). Accordingly, the percen-
tage of cells in G0-G1 phase of the cell cycle increased in the absence of
Bach1 (Supplementary Fig. 2). To investigate the effects of Bach1
ablation on cell-cycle progression, we synchronized control and
Bach1–/–MEFs by serum starvation (0.5% (v/v) FBS) for 48 h
(Fig. 1c). After serum stimulation, the percentage of cells that entered
S phase was lower in Bach1–/–MEFs. These results indicated that the
reduced proliferation of Bach1–/–MEFs was due to the lower replica-
tive capacity of these cells. To confirm that Bach1 directly affects cell
proliferation, we used RNA interference (RNAi) to reduce Bach1
expression in wild-type MEFs. We used sequence composition–
matched negative controls to verify silencing specificity and to control
for any effects related to delivery. As expected, knockdown of Bach1 in
MEFs resulted in reduced proliferation when compared with controls
(Fig. 1d,e). Efficient knockdown of Bach1 was observed after 5 d of
RNAi treatment, but began to weaken at 7 d (Fig. 1f). These results
suggest that the reduced proliferation of Bach1 knockout or knock-
down MEFs ensued from Bach1 deficiency.
Unexpectedly, Bach1–/–MEFs showed a senescence-like phenotype
more rapidly than control cells, as judged by enlarged cell morphology
(data not shown) and expression of senescence-associated b-galacto-
sidase (SA b-Gal) activity18(Fig. 2a,b). A major cause of senescence of
MEFs is oxidative stress–induced DNA damage, which can be alle-
viated by lowering the oxygen concentration12. As reported pre-
viously12, wild-type MEFs proliferated faster in 3% than in 20%
oxygen, the usual concentration for cell culture (Figs. 1a and 2c),
and showed no signs of senescence during the period examined here
(data not shown). Notably, Bach1–/–MEFs proliferated nearly as fast as
control cells (Fig. 2c) and did not show senescence-like morphology
(data not shown) or SA b-Gal activity (Fig. 2a,b) under 3% oxygen
conditions. When we examined cell-cycle progression after starvation
followed by serum stimulation, the kinetics and efficiency of S phase
entry were almost the same in control and Bach1–/–MEFs (Fig. 2d).
Thus, Bach1–/–MEFs became senescent in response to oxygen.
109
abcde
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048 1212 18
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β-actin
Time (d)
Bach1
Tubulin
D3D5D7
*
Co
Co
Co Co KD
KD
(kDa)
100
KD KD
7
12.8%
G0-G1 G2-M
S
6.7%
Figure 1 Bach1 deficiency resulted in reduced proliferation in MEFs. (a) Proliferation of Bach1–/–(gray triangles) and control (black circles) MEFs in 20%
oxygen. The averages of two independent cultures are shown. Similar results were obtained in five experiments using independently prepared MEFs. (b) Cell-
cycle analysis of MEFs from Bach1–/–(KO) and wild-type control mice (WT) at passage 2. (c) Percentages of Bach1–/–(gray triangles) and control (black
circles) cells that entered S phase after serum starvation and restimulation in 20% oxygen. The averages of three independent cultures with s.d. are shown.
(d) Proliferation of control (black) and Bach1 knockdown (gray) MEFs in 20% oxygen. The averages of three independent cultures with s.d. are shown.
(e) Immunoblotting analysis of Bach1 in MEFs expressing Stealth RNAi duplexes targeting Bach1 (KD) or control (Co). Tubulin was used as loading control.
* Indicates a nonspecific band. (f) Expression of Bach1 and b-actin mRNAs in wild-type MEFs expressing Stealth RNAi duplex targeting Bach1 (KD). RNAi
duplex with mismatches was used as a control (Co). Three-fold dilutions of samples from the indicated number of days after transfection were examined.
20%
3%
WT
abc
def
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40
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DCF fluorescence level
107
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20% late
WTKO
18126
30
30
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20
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20
10
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Time (d)
Time (h)
WTKO WTKO
0
Figure 2 Oxidative stress–induced premature
senescence in Bach1–/–(KO) MEFs. (a) SA b-Gal
staining at passage 8 in 20% or 3% oxygen. WT,
wild type. (b) Percentages of SA b-Gal–positive
cells were determined with or without NAC
treatment. (c) Proliferation of Bach1–/–(gray) and
control (black) MEFs in 3% oxygen. The averages
of two independent cultures are shown. Similar
results were obtained in three experiments using
independently prepared MEFs. (d) Percentages
of Bach1–/–(gray) and control (black) cells that
entered S phase after serum starvation and
stimulation in 3% oxygen. (e) ROS levels in
control (black) and Bach1–/–(gray) MEFs at
passage 0 or at passage 8 in 20% oxygen.
(f) Immunoblotting analysis of g-H2AX in
Bach1–/–(K) and control (W) MEFs under normal
condition (20% oxygen) or following treatment
with 50 mg ml–1bleomycin for 8 h. H2AX served
as a loading control. The averages of three
independent cultures are shown with s.d. in
b, d and e.
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Higher levels of oxygen result in the production of ROS, which
causes DNA damage, p53 activation and cellular senescence12. We thus
investigated whether Bach1–/–MEFs produced more ROS than control
cells in 20% oxygen. We found that the ROS levels increased during
culture in control MEFs but not in Bach1–/–MEFs (Fig. 2e), indicating
that Bach1–/–MEFs produced less ROS than control cells in 20%
oxygen. ROS produced in 20% oxygen can cause DNA damage in
MEFs12. To address the possibility that Bach1 deficiency caused
increased levels of DNA damage irrespective of ROS levels, we
examined levels of phosphorylated histone H2AX (g-H2AX), an
early marker of cell response to DNA damage19, in Bach1–/–and
control MEFs. When the cells were cultured in 20% oxygen, g-H2AX
was not detectable in both types of cells (Fig. 2f). g-H2AX levels
increased similarly in both cells when DNA damage was induced with
bleomycin (Fig. 2f). Thus, amounts and sensing of DNA damage and
subsequent signaling to g-H2AX were not altered in Bach1–/–MEFs.
These results indicate that the marked senescence of Bach1–/–-MEFs
was not due to exacerbated accumulation of ROS or DNA damage.
To further confirm the effect of oxidative stress on the reduced
proliferation we observed in Bach1–/–MEFs, we cultured MEFs in the
presence of the radical scavenger N-acetylcysteine (NAC). In the
presence of NAC, proliferation of Bach1–/–MEFs was partially rescued
(Supplementary Fig. 3 online) and the frequency of SA b-Gal–
positive cells was reduced (Fig. 2b). Unexpectedly, the frequency of
SA b-Gal–positive cells increased in wild-type MEFs with NAC. The
reason for this is not clear at present, but it may result from inhibition
of redox signaling, which regulates diverse cellular processes. These
results suggest that Bach1 deficiency causes an extravagant activation
of a senescence-like program in response to oxidative stress.
Involvement of p53 in Bach1-regulated cellular senescence
Senescence of MEFs relies predominantly on the p19Arfand p53
pathway5,20. To investigate the potential genetic interaction between
Bach1 and Trp53, we generated MEFs lacking both genes (Fig. 3a).
Bach1–/–Trp53–/–MEFs proliferated vigorously and did not show the
senescent phenotype observed in Bach1–/–MEFs (Fig. 3a). To further
investigate the genetic interaction, we reduced Bach1 expression using
RNAi in Trp53–/–MEFs. Knockdown of Bach1 reduced proliferation in
wild-type MEFs (Fig. 1e) but not in Trp53–/–MEFs (Fig. 3b). More-
over, when we reduced Trp53 expression using RNAi in senescent
wild-type and Bach1–/–MEFs, both cell types resumed growth
(Fig. 3c,d), and the senescence-like morphology completely disap-
peared (data not shown). This is consistent with the character of p53-
mediated cellular senescence, in that it is reversible upon subsequent
inactivation of p53 (refs. 21,22). These results suggest that Bach1 is a
negative regulator of the p53-mediated senescence program in
response to oxidative stress, although Bach1 could be either parallel
or upstream to p53 in this process.
If Bach1 is an inhibitor of p53-dependent cellular senescence, Bach1
should block Ras-induced cellular senescence in MEFs23. To explore
this possibility, we expressed the activated form of Ras (H-RasV12)
1013
1011
Cell number
Cell number (×106)
Cell number (×106)
Cell number (×106)
109
107
105
0
Trp53 RNAi / WT
Control RNAi / WT
Perp RNAi / WT
Trp53 RNAi / Bach1 KO
Control RNAi / Bach1 KO
Perp RNAi / Bach1 KO
Trp53 RNAi / WT
Control RNAi / WT
p21 RNAi / WT
Trp53 RNAi / Bach1 KO
Control RNAi / Bach1 KO
p21 RNAi / Bach1 KO
8 16
Time (d)Time (d)
24
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2
3
ab
cd
2
1
0
3
2
1
0
135
Time (d)
135
Time (d)
4
6
1357
Figure 3 p53-dependent senescence in Bach1–/–MEFs. (a) Proliferation of
MEFs from Bach1–/–Trp53+/+(gray triangles) and Bach1–/–Trp53–/–mice
(black circles) in 20% oxygen. The averages of three independent cultures
are shown. Error bars represent s.d. (b) Proliferation of Trp53–/–MEFs
expressing either Stealth RNAi duplexes of control (black circles) or
targeting Bach1 (gray triangles). The averages of three independent cultures
are shown. Error bars represent s.d. (c,d) Proliferation of senescent control
and Bach1–/–MEFs expressing either control Stealth RNAi duplex (control
RNAi) or targeting Trp53 (Trp53 RNAi). Proliferation of senescent Bach1–/–
MEFs expressing Stealth RNAi duplexes targeting Perp (Perp RNAi) or
Cdkn1a (p21) is also shown in c and d, respectively. Similar results were
obtained by using distinct RNAi duplexes targeting Trp53, Perp, or Cdkn1a
and MEFs from different embryos.
80
pBabe
abc
pBabeRas
1.1
1.0
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Perp/actin
Mock
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SA β-Gal (%)
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POZ-Bach1
40
0
1 2 3 4
MockBach1
pBabe
pBabeRas
MockBach1
1 2 3 41 2 3 4 1 2 3 4
Figure 4 Inhibition of Ras-induced cellular senescence in MEFs by Bach1. (a) Control or eBach1-overexpressing MEFs were infected with control or
H-RasV12(Ras) retroviruses. Cells were stained for SA b-Gal at the indicated time points. (b) Percentages of SA b-Gal–positive cells were determined.
The averages of three independent cultures are shown with s.d. (c) qPCR analysis of p53 target gene expression reveals that p21 and Perp mRNA were
downregulated in Bach1-expressing MEFs after retrovirus transduction of H-RasV12.
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using retroviral gene transfer. MEFs were first infected with control or
Bach1-expressing retroviruses. Mouse Bach1 was tagged with both
Flag and hemagglutinin epitopes at its C terminus (eBach1). MEFs
were then infected with the RasV12retrovirus. RasV12induced rapid
cellular senescence of MEFs, an effect that was substantially inhibited
by coexpression of Bach1 (Fig. 4a,b). As readout of the Bach1 effect at
a transcriptional level, we monitored expression of p53 target genes.
Expression of p21, which is implicated in cellular senescence6, was
inhibited by overexpression of Bach1, even in the presence of RasV12
(Fig. 4c). p53 apoptosis effector related to PMP22 (Perp)24, another
target gene of p53 (see below), was also inhibited by Bach1. These
observations indicate that Bach1 indeed inhibits the process of cellular
senescence, raising the possibility that levels of Bach1 could set a
threshold for cellular senescence.
Having established that Bach1 regulates the p53-dependent senes-
cence of MEFs, we examined whether Bach1 could regulate p53-
dependent apoptosis. Thymocytes undergo p53-dependent apoptosis
upon irradiation25. When thymocytes prepared from control or
Bach1–/–mice were irradiated, we did not detect any substantial
difference in apoptosis between control and Bach1–/–thymocytes
(Supplementary Fig. 4 online). In addition, when control and
Bach1–/–MEFs were irradiated, again we did not detect any appreci-
able difference in apoptosis (Supplementary Fig. 5 online). Thus, it
seems that Bach1 does not regulate the process of p53-dependent
apoptosis, although we cannot exclude the possibility that it does so in
other cell types or in response to other stimuli.
Bach1 represses a subset of p53 target genes
As Bach1 is a transcriptional repressor, we hypothesized that it may
modulate the transcriptional program of p53. We thus examined the
expression levels of several p53 target genes in control and Bach1–/–
MEFs. Using quantitative real-time PCR (qPCR), we observed that
several target genes such as Perp, p21, Noxa (also known as Pmaip1)
and Serpine1 were upregulated in Bach1–/–MEFs (Fig. 5a). In contrast,
expression of B-cell translocation gene 2 (Btg2) or Puma (also known
as Bbc3) was not affected by Bach1 deficiency (Fig. 5a). Because
Bach1–/–MEFs become senescent in response to oxidative stress, we
next examined the expression levels of these genes during the course of
culture in 20% oxygen. These four genes were induced by repeated
passage (data not shown). Among these genes, the expression levels of
Perp and p21 were much higher in Bach1–/–MEFs compared to levels
in control cells (Fig. 5b). In contrast, the expression levels of Puma
were indistinguishable between control and Bach1–/–MEFs in all
culture conditions (Fig. 5b). Notably, the effects of Bach1
deficiency upon Perp and p21 were lost when the cells were cultured
in 3% oxygen (Fig. 5b), and expression of Perp was suppressed
in 3% oxygen.
To further reveal a global view of the genes that are regulated by
Bach1 and p53 in response to oxidative stress, we carried out gene
expression profiling of control and Bach1–/–MEFs cultured in 20% or
3% oxygen (Fig. 5c). We compiled a list of p53 target genes on the
basis of previous reports7,26. Among the 94 genes on this list, 48 genes
were upregulated when control cells were maintained in 20% oxygen
(Fig. 5c and Supplementary Fig. 6 online). Expression of these genes
was similar in control and Bach1–/–MEFs at the start of culturing
(P0). However, 33 genes among the 48 upregulated genes were
expressed at higher levels in Bach1–/–MEFs at passage 3 or 5 (P3,
P5) including Perp, Serpine1, p21 and Noxa. Moreover, almost all of
these 33 genes were expressed at lower levels in 3% than in 20%
oxygen (Fig. 5c). In contrast to these genes, Puma and p53R2, a DNA
repair–related gene27, were not affected by Bach1 deficiency (Fig. 5c).
These results indicate that Bach1 might repress a subset of genes
regulated by p53 in response to oxidative stress.
We confirmed that acute knockdown of Bach1 in MEFs resulted in
higher expression of p21, Serpine1 and Perp (data not shown). p21 and
Serpine1 were previously reported to be involved in the process of
cellular senescence6,7. Perp may have a role in the induction of
senescence, because the closely related molecule PMP-22, also
known as growth arrest–specific 3 (Gas3), is upregulated in response
2
ac
d
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0
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GAPDH
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Figure 5 Bach1 repressed a subset of p53 target genes. (a) qPCR analysis
of several p53 target genes in control (black) and Bach1–/–(gray) MEFs at
passage 3 in vitro. The averages of three independent experiments with s.d.
are shown. (b) qPCR analysis of Perp, p21 and Puma in control (black) and
Bach1–/–(gray) MEFs under 20% or 3% oxygen at the indicated passage.
(c) Hierarchical clustering of p53 target genes in control and Bach1–/–
MEFs. Among 94 genes of p53 targets, 48 genes were induced by repeated
passage in 20% oxygen and are shown. Black bars indicated gene clusters
upregulated in Bach1–/–MEFs compared with control cells. The color bar
indicates relative expression levels. (d) Immunoblotting analysis of several
proteins in Bach1–/–(K) and control (W) MEFs at passages 0 and 4 in 20%
and 3% oxygen. (e) Total and acetylated (Ac) p53 in Bach1–/–and control
MEFs, with or without treatment of 50 mg ml–1bleomycin (Bleo).
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to serum starvation in NIH3T3 cells28. To investigate whether these
genes were involved in the rapid cellular senescence of Bach1–/–MEFs,
we carried out acute knockdown of Perp or p21 in Bach1–/–MEFs
(Fig. 3c,d); however, knockdown of these genes in Bach1–/–MEFs did
not bypass the exacerbated senescence (Fig. 3c,d). When taken
together with the phenotypic rescue of the senescence in Bach1–/–
MEFs by Trp53 ablation, these results indicate that Bach1 deficiency
causes upregulation of several p53 target genes in response to oxygen,
which may collaborate redundantly to induce premature cellular
senescence of MEFs.
To examine whether Bach1 affected the known regulation of p53
activity, we compared the levels of p53 in control and Bach1–/–MEFs.
Cells were cultured in 20% or 3% oxygen for the time periods
indicated in Figure 5d. The levels of p53 increased when the cells
were cultured in 20% oxygen. The initial and induced levels of p53
were similar between control and Bach1–/–MEFs in 20% oxygen
(Fig. 5d). Acetylation of p53 causes its activation29. There was no
difference in acetylated p53 levels between control and Bach1–/–MEFs
(Fig. 5d). When cells were cultured in 3% oxygen, p53 levels did not
increase irrespective of the genotypes. We observed no marked
difference in protein levels of p19Arfand p16Ink4a, which regulate
p53 and pRb, respectively, between control and Bach1–/–MEFs
(Fig. 5d). These results suggest that the two senescence pathways
were activated similarly in both cells. Moreover, when DNA
damage was induced with bleomycin (Fig. 5e), acetylation of p53
increased similarly in these cells. These observations demonstrate
that Bach1 did not affect accumulation or acetylation of p53 and
raise the possibility that Bach1 has a role in a step subsequent to
p53 activation.
Proteomic identification of Bach1 complexes
To investigate the molecular mechanism of Bach1 in suppression
of the p53-mediated transcriptional program of senescence, we
undertook a biochemical purification of Bach1 (ref. 30), using MEL
cells that express endogenous Bach1 at relatively high levels (data not
shown) and that can be cultured on a large scale. We generated stable
MEL cells expressing mouse eBach1 and subjected nuclear extracts
from these cells to sequential purification using anti-Flag and anti-
hemagglutinin antibody columns. As a control, we performed a mock
purification from nontransduced MEL cells. The purified eBach1
fraction contained several other proteins at varying stoichiometric
ratios (Fig. 6a), and we identified these proteins by MS. The presence
of small Maf (MafK and MafG)31and intracellular hyaluronic acid
binding protein (IHABP)32verified the purification procedure,
because they are known to interact with Bach1 (Supplementary
Fig. 7 online)15,32. In addition, there were at least 22 bands that
seemed to be specific, because they were present in several indepen-
dent purifications but absent in the mock purification. Whether these
proteins are important for Bach1 function awaits further study.
Whereas the small Maf proteins were present at a level that was
close to stoichiometric with Bach1, other bands were present at
substoichiometric levels, suggesting that only a fraction of Bach1
was associated with one or other of these proteins (Fig. 6a). Western
blotting analysis of the Bach1 complex revealed co-purification of p53
along with eBach1 (Fig. 6b). Immunoprecipitation of the Bach1
complex with p53 antibody brought down Bach1 (Fig. 6c), verifying
the specificity of their interaction. The fact that the amount of p53 in
the complex seemed to be substoichiometric indicated that only a
fraction of Bach1 interacted with p53 and vice versa.
To characterize the relationship between Bach1 and p53, we
fractionated Bach1-enriched material obtained by single anti-
Flag affinity purification using 10–35% (v/v) glycerol gradient
sedimentation (Fig. 6d). Whereas Bach1 and MafK/G formed peaks
corresponding to around 200 kDa, substantial portions of them
sedimented much faster, indicating the presence of high-molecular-
mass form(s). Bach1 may form several different complexes, or
(kDa)
Mock
Flag/HA
Flag/HA
eBach1
(kDa)
Flag
Sample 1
TopBottom
670 kDa 158 kDa
Sample 2
250
150
100
75
50
37
25
Bach1
p53
p53
MafG
IHABP
eBach1
N-CoR
Input
Flag
Input
Flag
S1S2
MafK
IP : anti-p53
–EtBr
Input
Anti-Bach1
Anti-p53
IB
IgG
p53
Input
IgG
p53
+EtBr
MafK
p53
N-CoR
HDAC1
af
g
bde
Bach1
IP :
IgG
Bach1comp.
anti-p53
MafK
p53
N-CoR
HDAC1
c
eBach1
Mock
eBach1
eBach1
IHABP
MafG
MafK
250
150
100
75
50
37
25
15
–++–++
+
MBP-MafK
Anti-Bach1
IB
Anti-MafK
His6-Bach1
Input
+
Pull-down
++
Pull-down
+
–
Input
+
GST-p53
Anti-Bach1
Anti-p53
IB
His6-Bach1
+
+
–+
–
+
+
+
–
––
Figure 6 Purification of the Bach1 complex. (a) Silver staining of Bach1-associated proteins. A mock purification was used as a control. Specific and
reproducible bands are indicated with dots. Protein bands identified by MS analysis are indicated. (b) Flag- and hemagglutinin (HA)–purified Bach1
complex was analyzed by immunoblotting analysis. (c) Flag- or HA-purified Bach1 complex was immunoprecipitated (IP) with control or anti-p53 antibody.
Precipitated materials were analyzed in immunoblotting with indicated antibodies. (d) Glycerol gradient analysis. Above, silver staining. Below,
immunoblotting analysis of Bach1 and p53. (e) Second affinity purification of pooled sample 1 (fractions 8–14) and sample 2 (fractions 15–22) in d
using an anti-Flag antibody column. Above, silver staining. Below, immunoblotting analysis of p53. Protein bands identified by MS analysis are indicated.
(f) Immunoprecipitation of endogenous p53 from wild-type MEL cells using anti-p53 or a control antibody in the absence or presence of 50 mg ml–1
ethidium bromide. Precipitated materials were analyzed in immunoblotting (IB) with the indicated antibodies. (g) Interaction of His6-tagged Bach1 with
maltose binding protein (MBP)-MafK or GST-p53. Proteins bound to nickel-agarose beads were detected using the indicated antibodies.
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associations of the complex components may be fragile. p53 was
found in the relatively faster-sedimenting fractions. To compare the
smaller and larger Bach1 complexes in detail, we pooled the respective
fractions (Fig. 6d) and carried out a second affinity purification using
an anti-Flag antibody column (Fig. 6e). p53 was found mainly in the
larger complex (Fig. 6e). MS analysis of the larger Bach1 complex
revealed the presence of the transcription co-repressor N-CoR (Fig. 6e
and Supplementary Fig. 7). N-CoR often represses gene expression by
recruiting histone deacetylase Hdac1 (refs. 33,34). We also confirmed
the presence of N-CoR and Hdac1 in the Bach1 and p53 complex by
immunoblotting analysis (Fig. 6b,c).
To address whether Bach1 and p53 also interact in wild-type MEL
cells with endogenous levels of Bach1, we performed reciprocal
immunoprecipitation of p53. The p53 antibodies brought down not
only p53 but also a portion of endogenous Bach1, indicating that p53
was indeed an authentic component of the Bach1 complex (Fig. 6f).
The presence of ethidium bromide in the immunoprecipitation
reaction did not affect the interaction of Bach1 and p53; thus, their
interaction is not mediated by DNA (Fig. 6f). To determine whether
Bach1 binds directly to p53, we performed an in vitro binding assay
using recombinant Bach1 and p53. Because Bach1 forms a hetero-
dimer with MafK, we also used recombinant MafK as a positive
control. Bach1 definitely bound to MafK in vitro but not to p53
(Fig. 6g). These results suggest that the interaction between Bach1 and
p53 is indirectly mediated by some other protein(s). Taken together,
these results raise the possibility that the biochemical interaction of
Bach1 with p53 constituted a key regulatory mechanism of cellular
senescence in response to oxidative stress.
Regulation of p53 target genes by Bach1
Considering that Bach1 deficiency did not affect the overall levels of
p53, we hypothesized that Bach1 may interfere with transactivation by
p53 through formation of a complex. To test this idea, we first
examined whether Bach1 was recruited along with p53 to its target
genes using chromatin immunoprecipitation (ChIP) assays. We
focused on Perp and p21 as models because they are directly or
indirectly regulated by Bach1. A previous report revealed the presence
of a p53 site (site D) in the intron of mouse Perp that was conserved in
the human Perp gene35. p21 is a well-characterized p53 target gene
in vivo36with a confirmed p53 binding site in its promoter region. We
also examined Bach1 binding to the HO-1 gene (Hmox1) enhancer E2,
which contains a MARE, as a positive control, and the pro-
moter region of Mcm5 (located 9 kb downstream of Hmox1) as a
negative control37.
Endogenous Bach1 in MEL cells was clearly recruited to the p53
binding sites of Perp and p21 together with p53 (Fig. 7a). The
observed Bach1 recruitment was specific, because we detected no
binding of Bach1 to the Mcm5 promoter. Under normal culture
conditions of 20% oxygen, both Bach1 and p53 endogenous to
MEFs were also recruited to the p53 sites of Perp and p21 (Fig. 7b).
Notably, Bach1 was not recruited to the p53 site of Btg2 (Fig. 7b),
whose expressionwas not affected by the Bach1 deficiency (Fig. 5a). In
contrast, Bach1 but not p53 bound to the Hmox1 E2 enhancer,
suggesting that there were at least two Bach1 complexes with or
without p53. This idea is consistent with the result of glycerol-gradient
analysis of the Bach1 complex (Fig. 6e).
Because MAREwas not found around these p53 sites, Bach1 may be
recruited to the gene by p53 and repress transcription. We tested this
idea using reporter assays. Consistent with Perp being a direct target of
p53 (ref. 24), the reporter was transactivated by p53 overexpression
(Fig. 7c). Bach1 repressed the p53-mediated activation of the Perp
promoter (Fig. 7c). When Bach1 was expressed alone, repression was
less marked (Fig. 7c). When targeted to a promoter using the GAL4
DNA binding domain, Bach1 efficiently repressed transcription, even
though the test thymidine kinase promoter lacked MARE (Fig. 7d),
indicating that the repression activity of Bach1 is separable from its
DNA binding activity. To confirm that Bach1 was recruited to the p53
binding sites in a p53-dependent manner, we performed ChIP assays
using Trp53–/–MEFs. We observed reduced recruitment of Bach1 to
the p53 sites of Perp and p21 in Trp53–/–MEFs compared with control
MEFs (Fig. 7e). These results suggest that Bach1 binds to a subset of
target genes of p53 by forming a complex with p53 while repressing
p53’s transcriptional activity.
The presence of N-CoR and Hdac1 in the Bach1 complex (Fig. 6)
raised the possibility that Bach1 facilitates repression of target genes
such as Perp or p21 by recruiting Hdac1. Consistent with this idea, we
observed a moderate but reproducible increase of H4 acetylation at the
p53 binding sites of Perp or p21 in Bach1–/–MEFs (Fig. 8a). In
contrast, we could not detect any difference in H4 acetylation at the
Hmox1 E2 MARE or the p53 binding site of p53R2. Inhibition of
HDAC activities with trichostatin A (TSA) increased Perp expression
in MEFs and HeLa cells (Fig. 8b and data not shown), suggesting that
Perp was repressed by histone deacetylation. Noxa showed a similar
response to TSA, whereas BCL2-associated X protein (Bax) and
Hmox1 did not show any response (Fig. 8b), indicating that the
regulatory mechanisms by p53 and/or Bach1 were different among
these genes. ChIP analysis of control MEFs revealed that Hdac1 was
recruited to Perp but not to p53R2 or Btg2 (Fig. 8c). By examining
Bach1–/–and Trp53–/–MEFs, we found that recruitment of Hdac1 to
Relative luciferase activity
8
6
4
2
0
p53
Bach1
Relative luciferase activity
0
Gal4/Bach1+
W K
Perpp21
W K
4
8
Relative recruitment of Bach1
0
2
4
12
16
++
+
+
++
+
+++ +++ +++
cde
Perp siteD
InputIgG
Bach1
p53
p21 promoter
Hmox1 E2 MARE
Mcm5
a
InputIgG
Bach1
p53
Perp siteD
p21 promoter
Hmox1 E2 MARE
Btg2
Mcm5
b
Figure 7 Recruitment of Bach1 to p53 target genes. (a,b) ChIP analysis
for the recruitment of Bach1 or p53 to the p53 sites of Perp, p21 or
Btg2 genes and the Hmox1 E2 enhancer in MEL cells (a) or MEFs (b).
(c,d) Reporter assays. The pPERPluc1 reporter plasmid was transfected
into Saos-2 cells with p53 and Bach1 expression plasmids in the indicated
combinations (c). The Gal4TKluc reporter plasmid was transfected into
NIH3T3 cells in the presence or absence of the Gal4-Bach1 expression
plasmid (d). The averages and s.d. of three independent experiments are
shown. (e) ChIP analysis for recruitment of Bach1 to the p53 binding
sites of Perp and p21 in wild-type (Black) and Trp53–/–(gray) MEFs.
Specifically immunoprecipitated DNA and input DNA were analyzed
quantitatively by qPCR. The amounts of immunoprecipitated DNA
were normalized to input DNA.
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the p53 binding sites of Perp was shown to be dependent on both
Bach1 and p53 (Fig. 8c). Moreover, consistent with the observations
that Bach1 did not affect the protein levels of p53 and its acetylation
(Fig. 5d,e), recruitment of p53 to the Perp target site was not affected
in the absence of Bach1 (Fig. 8d). These results suggest that Bach1
impedes p53-mediated transactivation by binding to a subset of target
genes along with p53 and by recruiting Hdac1.
DISCUSSION
To understand how Bach1 regulates cell proliferation, we carried out a
detailed analysis of Bach1-deficient MEFs. We found that Bach1-
deficient MEFs entered into the p53-dependent senescence state
more rapidly than the control MEFs. Notably, Bach1-deficient MEFs
did not enter senescence when cultured in 3% oxygen, a condition
that lowers the level of oxidative stress and activation of p53, thus
suppressing cellular senescence of MEFs12. We confirmed that p53
accumulation was mitigated in 3% oxygen. This may explain why
Bach1-deficient MEFs did not undergo senescence in the 3%-oxygen
culture condition. Our results strongly suggest that Bach1 is a negative
regulator of p53-dependent cellular senescence in response to oxida-
tive stress. We also analyzed the effect of acute knockdown of Bach1 in
MEFs to circumvent possible adaptation of MEFs to Bach1 deficiency
along the process of development. The acute knockdown of Bach1
resulted in slower proliferation of MEFs. In contrast, Trp53-deficient
MEFs were less sensitive to the Bach1 knockdown, corroborating the
idea that Bach1 impeded p53 function. We could not examine
senescence in these acute knockdown cells because the effect of
siRNA on Bach1 was reduced after 7 d (Fig. 1f). Nonetheless, the
results confirmed the genetic interaction between Bach1 and Trp53.
Consistent with the p53 dependency of the observed senescence in
Bach1-deficient MEFs, a subset of p53 target genes was found to be
upregulated in these cells. We obtained similar results using acute
knockdown of Bach1 (data not shown). Although it is difficult to
relate these changes as a whole to the enhanced senescence of Bach1-
deficient MEFs, some of the affected genes have been implicated in
cellular senescence. Specifically, the higher expression of p21 and
Serpine1 is interesting because these genes are known to be involved
in the process of cellular senescence6,7. In addition, we found that the
expression of Perp correlated well with the appearance of the senes-
cence phenotype and was highly induced under the 20% oxygen
condition where senescence was observed. Furthermore, the induction
level of Perp was higher in Bach1-deficient MEFs than in control cells;
in contrast, it remained low under the 3% oxygen condition, which
suppressed senescence. Although the molecular function of Perp in the
process of senescence needs to be investigated further, these observa-
tions suggest that p53 implements cellular senescence by inducing
multiple target genes, as shown previously6,7,38,39. In contrast to
senescence, we did not observe any increase in apoptosis in these
cells, although the pro-apoptotic gene Noxa was also upregulated in
Bach1-deficient MEFs. The reason for this is not clear at present, but it
is possible that the expression of other genes might modify the
function of these genes.
We found that Bach1 forms protein complexes with p53, N-CoR
and Hdac1. The results of ChIP experiments indicated recruitment of
Bach1 to the Perp and p21 promoters together with p53 and Hdac1. At
present, we envisage two molecular mechanisms that allow the
regulation of p53 target genes by Bach1. Bach1 may be recruited to
the p53 target genes via a protein-protein interaction independent of
its DNA binding activity. Alternatively, the protein complexes con-
taining both Bach1 and p53 may allow cooperative DNA binding of
these factors. This possibility is supported by a recent report that
binding sites of p63, a p53 family member40, are often accompanied
by nearby Bach1 binding sites41. Although the promoter regions of
Perp and p21 seem to lack a canonical Bach1 binding site or
MARE, binding of Bach1 to a degenerate site may be facilitated in
the presence of p53. Our two observations (that is, reduced recruit-
ment of Bach1 to the target genes in the absence of p53 and the
presence of a Bach1–p53 protein complex) support both models with
regard to the involvement of DNA recognition by Bach1. Although
further study is necessary to resolve this issue, our observations
suggest that a subset of p53 target genes is modulated at the
transcriptional level by Bach1 (Fig. 8e).
Bach1 stands in contrast with other known negative co-regulators
of p53 such as the apoptosis suppressor NIR (also known as Noc2l)42
in its ability to repress selectively the senescence-associated gene
program. Although p53 is the shared key component of apoptosis
8
WT
abce
d
Perp siteD
p21 promoter
HO-1 E2
p53R2 p53BS
KO
WTKO
2
1
0
W K
Perp
W K
p21
WT
Bach1 KO
p53 KO
DNA damage
Oxidative stress
p53
Apoptosis
Senescence
Bach1
X
Perp siteD
Perp siteD
Hmox1 E2
Input
HDAC1 HDAC1
Input
IgG
Input
Ac-H4
IgG
Input
Ac-H4
IgG
Input
p53
IgGInput
p53
IgG
IgG
HDAC1
Input
IgG
Btg2
p53R2 p53BS
6
4
2
0
Relative expression levels
Acetylation levels
of H4
Perp Noxa Bax Hmox1
Figure 8 Bach1 facilitates repression of target genes by recruiting Hdac1. (a) ChIP analysis for acetylation (Ac) levels of histone H4 at indicated sites
in Bach1–/–and control MEFs. Specifically immunoprecipitated DNA and input DNA were quantitatively analyzed at the p53 sites of Perp and p21 by
conventional PCR or qPCR. The amounts of immunoprecipitated DNA are normalized to input DNA. The average and s.d. of three independent cultures
were shown in the graph below. (b) Control MEFs were treated with (gray) or without (black) 2 mM TSA for 24 h. Expression levels of indicated genes were
determined with qPCR. (c) ChIP analysis for Hdac1 recruitment at indicated sites in control, Bach1–/–or p53–/–MEFs. (d) ChIP analysis for recruitment of
p53 at indicated sites in Bach1–/–and control MEFs. (e) Model of Bach1-mediated repression of oxidative stress–induced senescence signaling pathway.
Bach1 forms a protein complex with p53 on chromatin to inhibit the transcriptional activity of p53. Their interaction seems to involve a third unknown
factor. Their interaction inhibits a subset of p53 target genes, restricting cellular senescence in response to oxidative stress.
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and cellular senescence, outputs of the p53-dependent programs could
be regulated specifically, as suggested previously43. We have shown
here that Bach1 deficiency in MEFs caused enhanced cellular senes-
cence with no apparent effect on apoptosis. The suppression of cellular
senescence by Bach1 reflects its cross-talk with p53, which most likely
involves their protein complex formation and thus inhibition of a
subset of p53 target genes (Fig. 8e). Because Bach1 can be regulated by
redox44or heme15, their cross-talk can be viewed as an integral process
that sets the threshold of cellular senescence against oxidative stress. In
this model, oxidative stress induces cellular senescence of MEFs
when p53 activation and Bach1 inactivation take place simultaneously
to certain levels. Considering the result that Bach1 deficiency
entailed a propensity to undergo cellular senescence without increas-
ing ROS or DNA damage, Bach1 could be a molecular rheostat
placed between oxidative stress and senescence. In this regard,
cellular senescence could be induced without severe DNA damage
by modulating Bach1, pointing to a new potential therapeutic
approach against cancer.
METHODS
Isolation and culture of mouse embryonic fibroblasts. We derived MEFs
from 14.5-day-old embryos of various genotypes. Following removal of the
head and organs, embryos were rinsed with PBS, minced and digested with
trypsin (0.05% (v/v) solution containing 0.53 mM EDTA and 1.8 mg ml–1
DNase I in PBS (Gibco) and incubated for 60 min at 37 1C. Trypsin was
inactivated by addition of DMEM (Gibco) containing 10% (v/v) FBS (JRH
Bioscience) and 0.1 mM MEM nonessential amino acids (GIBCO), 55 mM
2-mercaptoethanol (Wako). Cells from a single embryo were plated into a
100-mm diameter culture dish and incubated at 37 1C. Cells (2–3 ? 105) were
cultured in 20% or 3% oxygen by subculturing in a 60-mm diameter culture
dish every 3–4 d, and cell number was determined at each passage.
Analysis of cell cycle and senescence. To measure BrdU incorporation, we
incubated control and Bach1–/–MEFs for 30 min with 10 mM BrdU (Roche).
After treatment, cells were fixed with 70% (v/v) ethanol. The cells were then
reacted with anti-BrdU antibody (Roche) followed by a secondary anti-mouse
IgG fluorescein isothiocyanate (FITC) conjugate (Sigma). The cells were
resuspended in 1 ml PBS with 160 mg ml–1RNase A and incubated at room
temperature (23–26 1C) for 10 min. After adding propidium iodide (final
concentration 5 mg ml–1), we used a FACSCalibur to analyze the cell suspension
for DNA content including sub-G1 (apoptotic cells) and for BrdU incorpora-
tion. We carried out cytometric analysis on a FACSCalibur with CellQuest
software (Becton Dickinson). SA b-Gal assays were carried out using a
Senescence Detection kit (BioVision).
Detection of intracellular ROS levels. We determined ROS levels using
dichlorodihydrofluorescein diacetate (DCF-DA, Sigma) as described pre-
viously43with FACSCalibur.
Antibodies. For immunoblotting analysis and ChIP analysis, we used the
following antibodies: anti-p53 (CM5p, Novocastra), anti-p16Ink4a(M-156,
Santa Cruz Biotech), anti-p19Arf(ab80, Abcam), anti-GAPDH (Santa Cruz
Biotech), anti-N–CoR (Affinity BioReagents) anti-Hdac1 (H3284, Sigma),
anti–acetylated histone H4 (Upstate). Anti-acetylated p53 antibody was
provided byT.P.Yao (DukeUniversity).
described previously16.
Anti-Bach1antibodywas
Immunoblotting analysis. We prepared whole-cell extracts from control
and Bach1–/–MEFs as described previously32. The extracts were separated by
SDS-PAGE. Following SDS-PAGE, the proteins were electrotransferred to
PVDF membrane (Millipore). The membranes were blocked for 1 h in
blocking buffer (1% (w/v) skimmed milk, 0.05% (v/v) Tween 20 in TBS),
and subsequently incubated with primary and secondary antibodies in
the blocking buffer for 1 h. To detect immunoreactive proteins, we used
ECL blotting reagents (Amersham).
RNA interference. Stealth RNAi duplexes were designed to target Bach1 and
p53 using the BLOCK-iT RNAi Designer (Invitrogen). For knockdown of
Bach1 and Trp53, we transfected 1.5 ? 106cells with 6 ml of stock Stealth RNAi
duplex (20 mM) using the basic nucleofection solution for MEFs (VPD-1004,
Amaxa). The transfected cells were cultured in a 60-mm diameter culture dish
by subculturing every 2 d. Whole-cell extracts were prepared from transfected
cells to monitor Bach1 knockdown at 48 h after transfection. We harvested total
RNA from transfected cells on the third and fifth days after transfection to
perform gene expression profiling. Sequence information for the Stealth RNAi
and corresponding Stealth controls used in this study are described in
Supplementary Methods online.
Ras-induced cellular senescence. MEFs were plated at approximately 1 ? 105
cells per well in 12-well plates and infected with control or eBach1-expressing
retroviruses. After MEFs were selected as previously described30, retrovirus
transduction of pBabe-puro-H-RasV12or control pBabe-puro (provided by
N. Tanaka (Nippon Medical School) and H. Ogawa (National Institute of Basic
Biology), respectively) was performed and infected cells were selected in
puromycin (0.7mg ml–1) for 4 d as previously described23. Cells were stained
with SA b-Gal–staining solution (BioVision). Cells were visualized in the
following days and quantified (minimum of 100 cells per trial) for the presence
or absence of staining.
Expression profiling, RNA amplification and quantitative real-time poly-
merase chain reaction. We prepared total RNAs from various cells using the
Total RNA Isolation minikit (Agilent Technologies). Agilent whole mouse
genome (4 ? 44K, G4122F) arrays were used for this study. RNA samples
were amplified and labeled with cyanine-3 (Cy3) dye. Agilent Low RNA Input
Fluorescent Linear Amplification Kits were used to amplify RNA samples
following the manufacturer’s protocol. After amplification and labeling, cRNA
yields and dye incorporation efficiencies were determined using a Nanodrop
spectrophotometer. For hybridization, 1.65 mg Cy3-labeled cRNA samples were
fragmentated and incubated with Agilent microarray slides for 17 h using an
Agilent Gene Expression Hybridization Kit. After hybridization, the array slides
were washed using Agilent Gene Expression Wash Buffer 1 and 2. The washed
slides were immediately scanned using an Agilent Scanner. We carried out
analysis and clustering of p53 target genes using Genespring software (Agilent
Technologies). qPCR experiments were performed with LightCycler (Roche)
using the primers described in the Supplementary Methods.
Bach1 complex purification and analysis. We purified the Bach1 complex
from nuclear extracts prepared from MEL cells stably expressing eBach1 and
analyzed them as described previously30. Endogenous p53 was immunopreci-
pitated from whole-cell extracts prepared from wild-type MEL cells with goat
anti-p53 (Sigma) and protein G beads (Pierce).
Chromatin immunoprecipitation assay. We carried out chromatin fixation
and purification as described previously16. In brief, suspensions of MEL cells or
MEFs (1–2 ? 107) were fixed by adding formaldehyde to 1% (w/v) final
concentration for 10 min at room temperature. Cells were then sonicated to
prepare a chromatin suspension of 200–500 bp DNA. Immunoprecipitations
were carried out using anti-Bach1, anti-p53, anti–acetylated histone H4 and
anti-Hdac1 antibodies as described previously37. PCR was performed using the
primers described in the Supplementary Methods.
In vitro binding assay. Recombinant Bach1 and MafK proteins were expressed
in Escherichia coli BL21 (DE3) and purified as described previously45,46.
Recombinant p53 proteins were described previously47. The indicated protein
mixtures were added to binding buffer (20 mM Tris-HCl, pH 8.0, 100 mM KCl,
10% (v/v) glycerol, 10 mM 2-mercaptoethanol) with 20 ml of slurry of nickel-
agarose beads, and incubated at 4 1C for 30 min. After incubation, the nickel-
agarose beads were washed three times with wash buffer (20 mM Tris-HCl, pH
8.0, 100 mM KCl, 10% (v/v) glycerol, 10 mM 2-mercaptoethanol, 20 mM
imidazole). Proteins were eluted by elution buffer (20 mM Tris-HCl, pH 8.0,
100 mM KCl, 10% (v/v) glycerol, 10 mM 2-mercaptoethanol, 200 mM
imidazole), and analyzed by immunoblotting analysis.
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Transfection reporter assays. The promoter and full-length Perp reporter
plasmids (pPERPluc1 and pPERPlucFL) were provided by T. Jacks35(Massa-
chusetts Institute of Technology), and the Gal4TKluc reporter plasmid, contain-
ing the Gal4 binding site preceding the minimal thymidine kinase promoter, was
provided by K. Umesono (Kyoto University). The expression plasmid for
human p53 was provided by K. Tanimoto (Hiroshima University). Mouse
Bach1 and Gal4 DNA binding domain–tagged Bach1 (Gal4-Bach1) were
reported previously14,16. We performed transfection reporter assays as described
previously16. In brief, cells were seeded in 12-well dishes 24 h before transfection.
The cells were transfected with the reporter plasmids together with the effector
plasmids using Genejuice (Novagen). Luciferase activities in cell lysates were
measured using Luciferase Assay System (Promega) with a Biolumat Lumin-
ometer (Berthold Technologies). Firefly luciferase activity was normalized to
transfection efficiency as determined from the control sea pansy luciferase
activity. The averages and s.d. of three independent experiments are shown.
Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
ACKNOWLEDGMENTS
We thank T. Jacks (Massachusetts Institute of Technology), T.P. Yao (Duke
University), K. Tanimoto (Hiroshima University), H. Ogawa (National Institute
of Basic Biology), N. Tanaka (Nippon Medical School) and K. Umesono for
providing plasmids and antibodies; Y. Ishikawa and K. Nakayama (Tohoku
University) for providing p53–/–MEFs; and M. Katsuki (National Institute of
Basic Biology) for providing Trp53–/–mice. We also thank M. Kobayashi and
Y. Taya for comments on the manuscript; S. Tashiro and T. Ide for valuable
advice to initiate the project; M. Ikura for help in cell culturing; and
M. Yoshizumi and N. Tanaka for advice. This work was supported by
Grants-in-aid and the Network Medicine Global-COE Program from the
Ministry of Education, Culture, Sports, Science and Technology of Japan,
and grants from the Uehara Foundation, the Takeda Foundation and the
Astellas Foundation for Research on Metabolic Disorders.
AUTHOR CONTRIBUTIONS
Y.D., T.I., Y.K., K.O., A.N., A.M., S.O., A.I. and M.Y. contributed to performing
and assisting with experiments; A.M. and T.O. contributed to performing gene
expression profiling; Y.H. and T.N. contributed to performing MS/MS analysis;
K.I. designed and conceptualized the study; Y.D., T.I., T.N. and K.I. interpreted
the data and Y.D., T.I. and K.I. wrote the manuscript. All authors made
comments on the manuscript.
Published online at http://www.nature.com/nsmb/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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