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Nucleic Acids Research, 2007, 1–13
doi:10.1093/nar/gkm638
Heme oxygenase-1 induction by NRF2 requires
inactivation of the transcriptional repressor BACH1
John F. Reichard*, Gregory T. Motz and Alvaro Puga
Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati, Cincinnati,
OH 45267-0056, USA
Received May 21, 2007; Revised July 12, 2007; Accepted August 1, 2007
ABSTRACT
Oxidative stress activates the transcription factor
NRF2, which in turn binds cis-acting antioxidant
response element (ARE) enhancers and induces
expression of protective antioxidant genes. In con-
trast, the transcriptional repressor BACH1 binds
ARE-like enhancers in cells naı¨ve to oxidative stress
and antagonizes NRF2 binding until it becomes
inactivated by pro-oxidants. Here, we describe the
dynamic roles of BACH1 and NRF2 in the transcrip-
tion of the heme oxygenase-1 (HMOX1) gene.
HMOX1 induction, elicited by arsenite-mediated
oxidative stress, follows inactivation of BACH1 and
precedes activation of NRF2. BACH1 repression is
dominant over NRF2-mediated HMOX1 transcription
and inactivation of BACH1 is a prerequisite for
HMOX1 induction. In contrast, thioredoxin reduc-
tase 1 (TXNRD1) is regulated by NRF2 but not by
BACH1. By comparing the expression levels of
HMOX1 with TXNRD1, we show that nuclear accu-
mulation of NRF2 is not necessary for HMOX1
induction; rather, BACH1 inactivation permits NRF2
already present in the nucleus at low basal levels
to bind the HMOX1 promoter and elicit HMOX1
induction. Thus, BACH1 confers an additional level
of regulation to ARE-dependent genes that reveals
a new dimension to the oxidative stress response.
INTRODUCTION
Reactive oxygen species (ROS) pose a serious threat to all
aerobic organisms that maintain redox homeostasis in the
face of constant exposure to environmental oxidants. ROS
are harmful due to their reactivity with many cellular
macromolecules. To maintain cells in a state of redox
balance, biochemical antioxidants and a host of anti-
oxidant enzymatic reactions supply the needed reduction
potential. Antioxidant defenses exist in a balance with
endogenous oxidants, and it is the disruption of this
balance that characterizes the pathogenesis of many
human diseases and aging.
Arsenite, the trivalent form of inorganic arsenic, is an
environmental contaminant of major concern. Arsenic is
a potent electrophilic inducer of oxidative stress with
many of its effects attributable to its affinity for soft
nucleophiles, such as cysteine residues in glutathione
(GSH) and proteins (1). Arsenite exposure results in
rapid oxidation of glutathione (2) thereby disrupting
intracellular redox status (3). In response to the oxidative
stress mediated by arsenite, cells induce the antioxidant
battery of protective enzymes, of which heme oxygenase-1
(HMOX1) and thioredoxin reductase-1 (THXRD1) are
two well-recognized members.
Transactivation of HMOX1 and of other antioxidant
genes is regulated by binding of the transcription factor
NRF2 (Nuclear factor erythoid-derived 2 related factor 2)
to a cis-acting enhancer element known as the antioxidant
response element (ARE). Activation of NRF2 requires its
translocation to the nucleus, formation of a transcription-
ally active complex through dimerization with small MAF
(sMAF) proteins and binding to ARE enhancer motifs (4).
The ARE is one form of MAF response element (MARE)
having the core sequence RTGAYNNNGC (reverse
complement: GCNNNRTGAY) (5) and additional flank-
ing nucleotides that increase the specificity of NRF2
recognition (6,7). Some variations of the ARE motif can
be recognized by other regulatory factors, in addition to
NRF2, including NRF1 (8), NRF3 (9) and BACH1 (
BTB
and CNC homolog 1) (10). Thus, variation of ARE motif
sequences contribute to overlapping DNA binding by
factors that compete with NRF2 for ARE binding (11).
This arrangement appears to exist between NRF2 and
BACH1.
BACH1 is a transcriptional repressor (12) that is
conserved and ubiquitously expressed in tissues (13,14)
though its global activity in gene regulation is poorly
characterized. Like several other MAF-related trans-
cription factors, particularly NRF2, BACH1 hetero-
dimerizes with sMAF proteins in order to bind DNA.
In the basal state, BACH1/sMAF heterodimers interact
with the MARE-like enhancer sites recognized by NRF2
*To whom correspondence should be addressed. Tel: +1 513 558 0712; Fax: +1 513 558 0925; Email: john.reichard@childrens.harvard.edu
ß 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acids Research Advance Access published October 16, 2007
or NF-E2 to inhibit expression of the corresponding genes
(13,15). As a repressive transcription factor, BACH1
allows gene induction upon its release from enhancer
elements. Originally, BACH1 was described as a heme-
regulated repressor of b-globin genes (12) and HMOX1
(14–18). More recently, BACH1 has been suggested to
play a role as a sensor of oxidative stress. Human BACH1
is a thiol-rich protein possessing 34 interspersed cysteine
amino acids, of which two are responsible for BACH1
inactivation by oxidants (17). Therefore, it is possible that
heme and oxidants trigger gene induction simply by
relieving BACH1 repression (19). In this regard, it has
been demonstrated that BACH1 plays a role in redox
induction of HMOX1 (17) and NQO1 (10), though the
exact mechanism of this repression is not clear.
The relationship between BACH1 inactivation and
NRF2 activation during the induction of antioxidant
genes is unknown. NRF2 coordinates induction of genes
through its interaction with ARE enhancer motifs,
frequently located 5
0
to the transcriptional start site
(TSS) of several well-characterized antioxidant genes (5).
In the absence of oxidative stress, the cytosolic protein
KEAP1 (Kelch-like ECH Associated Protein 1) directs E3
ligase-dependent proteasomal degradation of newly
synthesized NRF2. As a result of this continuous
degradation, NRF2 is effectively maintained at very low
cellular levels. KEAP1, like BACH1, is a thiol-rich
protein, and oxidation of a few of KEAP1’s cysteines
blocks NRF2 degradation (20–22). Consequently, activa-
tion of NRF2 is dependent on nuclear accumulation of
de novo synthesized protein for subsequent binding of
ARE motifs (23). At a few known genes, including
HMOX1, BACH1 binds ARE motifs to the exclusion
of NRF2.
Here, we have tested whether oxidative stress induced
by arsenite exposure can trigger gene induction by simply
releasing repressive BACH1 from ARE motifs. We find
that NRF2 and BACH1 bind to two distal ARE enhancer
sites far upstream of the HMOX1 TSS and that BACH1
removal is necessary for NRF2-mediated HMOX1 gene
induction. In contrast to HMOX1, TXNRD1, which has
a single ARE motif located 9 bp upstream of the TSS,
is regulated by NRF2 but not by BACH1. Comparison of
TXNRD1 expression with that of HMOX1 demonstrates
that BACH1 repression is dominant over NRF2-mediated
transcription. The dynamic interplay between BACH1
and NRF-2 can produce distinct regulatory expression
patterns at different oxidative stress-induced genes.
MATERIALS AND METHODS
Cells and chemical treatments
HaCaT (24) human keratinocytes were grown in DMEM
(Mediatech, Herndon, VA, USA) supplemented with 5%
fetal bovine serum (Sigma Aldrich, St Louis, MO, USA)
and 1% penicillin–streptomycin solution (Invitrogen,
Carlsbad, CA, USA). Cells were incubated at 378C with
5% CO
2
and grown to 90% confluence before treat-
ment. Aqueous solutions of NaAsO
2
(hereafter referred
to as arsenite) (Sigma Aldrich) were prepared from a
1000 stock in ddH
2
O.
Whole cell extracts, nuclear extracts and immunoblotting
For preparation of whole cell extracts, cells were washed
and harvested in PBS containing 1 Complete Protease
Inhibitor
Õ
(Roche Diagnostics Corporation, Indianapolis,
IN) and lyzed by sonication (Sonic Dismembrator 60,
Fisher Scientific) on ice 3 10 s in 300 ml NETN buffer
(100 mM NaCl, 20 mM Tris pH 8.0, 1 mM EDTA, 0.5%
NP-40, 1 Complete Protease Inhibitor). Nuclear and
cytosolic extracts were prepared using a nuclear extraction
kit from Panomics (Fremont, CA, USA) according to
the manufacturer’s protocol. Protein concentrations were
measured using the Bradford assay and concentrations
adjusted to 1 mg/ml. Proteins were separated by SDS–
PAGE, transferred to PVDF and probed for NRF2
(H300; Santa Cruz Biotechnology), b-actin (Sigma),
HMOX1 (C20; Santa Cruz Biotechnology), in blocking
buffer containing 3% NFDM in PBST (0.1 M PBS
with 0.2% Tween 20). Blots were probed for BACH1
(C20; Santa Cruz Biotechnology) in blocking buffer
containing 1% BSA in PBST. After washing, the blots
were incubated with species-appropriate HRP-conjugated
secondary antibody (Santa Cruz), incubated with chemi-
luminescent reagent (PicoWest Super Signal, Pierce
Rickford, IL, USA) and visualized by exposing to film
(GE Healthcare, Piscataway, NJ, USA).
RNA isolation and real-time RT–PCR
Total RNA was extracted using NucleoSpin RNA II
columns (Macherey-Nage, Bethlehem, PA) according to the
manufacturer’s protocol. c DNA was synthesized by reverse
transcription of 1 mg total RNA in a total volume of 20 ml
containing 1 reverse transcriptase buffer (Invitrogen,
Carlsbad, CA, USA), 25 mg/ml oligo (dT)
12–18
(Invitrogen),
0.5 mM dNTP mix (GeneChoice, Frederick, MD), 10 mM
dithiothreitol (Invitrogen), 20 U of RNase inhibitor
(RNasin
Õ
; Promega, Madison WI) and 100 U of
SuperScript
II reverse transcriptase (Invitrogen). Resulting
cDNA products were diluted in a final volume of 200 mlanda
2 ml aliquot was used as template for subsequent quantifica-
tion by real-time PCR amplification. Quantitative real-time
PCR was performed in a 25 ml reaction mixture containing
1 Clonetech QTaq polymerase reaction mix (Mountain
View,CA),1 SybrGreen (Invitrogen) as a marker of
amplification, 0.1 mMofeachprimerand2mloftemplate
cDNA. Products were amplified with human HMOX1
primers (forward: 5
0
-CTCAAACCTCCAAAAGCC-3
0
and
reverse: 5
0
-TCAAAAACCACCCCAACCC-3
0
), TXNRD1
primers (forward: 5
0
-CTTTTTCATTCCTGCTACTC-
TACC-3
0
and reverse: 5
0
-CTCTCTCCTTTTCCC
TTTTCC-3
0
), BACH1 primers (forward: 5
0
-TGCGATGT-
CACCATCTTTGT-3
0
and reverse: 5
0
-CCTGGCCTAC-
GATTCTTGAG-3
0
), NRF2 primers (forward: 5
0
-GAGA
GCCCAGTCTTCATTGC-3
0
and reverse: 5
0
-TGCT
CAATGTCCTGTTGCAT-3
0
). Amplifications were per-
formed using an Opticon 2 Real-Time PCR Detection
System (MJ research). Cycle threshold (C
t
)ofeachsample
was automatically determined to be the first cycle at which a
2
Nucleic Acids Research, 2007
significant increase in optical signal above an arbitrary
baseline was detected. Amplification of b-actin cDNA in the
same samples was used as an internal control for all PCR
amplification reactions. Relative mRNA expression was
quantified using the comparative C
t
(C
t
) method and
expressed as 2
C
t
. Each assay was done in triplicate.
Chromatin immunoprecipitation (ChIP) analysis
Nuclei were isolated from formaldehyde (1% final) fixed
cells by lysing cells in buffer containing 5 mM PIPES
(pH 8.0), 85 mM KCl, 0.5% NP-40 and 1 Complete
Protease Inhibitor
Õ
. DNA was fragmented by sonication
(Diagenode Bioruptor) in buffer containing 50 mM Tris–
HCl pH 8.1, 10 mM EDTA, 1% SDS, 1 Complete
Protease Inhibitor
Õ
. DNA-cross-linked proteins were
immunoprecipitated from precleared samples with 2–5 mg
of each specific antibodies, including anti-NRF2, anti-
BACH1 or rabbit IgG control (Millipore), or for use as
total input chromatin. Antibodies were pulled down with
protein A beads at 48C overnight. Recovered beads were
re-suspended in 1 dialysis buffer [2 mM EDTA, 50 mM
Tris–HCl (pH 8.0), 0.2% Sarkosyl and 1 Complete
Protease Inhibitor
Õ
] and washed twice with dialysis buffer,
following which pellets were washed four times with IP
wash buffer [100 mM Tris–HCl (pH 9.0), 500 mM LiCl,
1% NP-40, 1% deoxycholic acid and 1 Complete
Protease Inhibitor
Õ
]. Antibody/protein/DNA complexes
were eluted in IP elution buffer (50 mM NaHCO
3
,1%
SDS) with vigorous shaking. Immunoprecipitated DNA
and total input chromatin were diluted to 120 ml with
water, brought up to 0.3 M NaCl. Cross-linking was
reversed at 658C overnight in the presence of RNase A
(10 mg), followed by the addition of RNase A followed by
protease K digestion at 458C for 2 h. DNA was purified
using a Qiaquick PCR purification kit per manufacturer
protocols. Samples were evaluated for enrichment by
quantitative real-time PCR (qRT–PCR) in a 25 ml reaction
mixture containing 1 BD QTaq polymerase reaction mix
(BD Biosciences), 1 SybrGreen (Invitrogen) as a marker
of DNA amplification, and 0.1 mM of each primer
(Supplementary Table 1). ChIP enrichment was evaluated
using primers to regions upstream of the human HMOX1
or TXNRD1 genes (Table S1). Relative efficiency of each
PCR primer was determined using input DNA and
adjusted accordingly. The DNA in each ChIPed sample
was normalized to the corresponding input chromatin
(C
t
) and enrichment was defined as the change in C
t
in
treated samples relative to untreated controls (C
t
),
relative to the IgG negative control. Exponential C
t
values were converted to linear values (2
C
t
) for
graphical presentation as either as fold change or percent
change, where indicated.
RESULTS
BACH1 inactivation precedes NRF2 activation
To characterize activation of the antioxidant response
following arsenite exposure, we first characterized
cellular and subcellular changes in NRF2 and
BACH1 disposition. In control cells, NRF2 is present at
very low levels in whole cell extracts attributable to
ongoing proteasomal degradation mediated by KEAP1
(25). After treatment with 25 mM arsenite, NRF2 accu-
mulates to high levels in whole cell extracts consistent with
oxidative inactivation of KEAP1 (23) (Figure 1A and B).
Importantly, NRF2 accumulation is negligible during the
first hour after arsenite treatment, and does not reach
maximum levels until 3 h after treatment. This initial lag
period represents the time necessary for translational
synthesis of new NRF2 protein (26), which precedes
nuclear translocation and subsequent ARE-mediated gene
induction. In comparison to NRF2, BACH1 gradually
accumulates in whole cell extracts over 5 h after treatment.
The extent to which BACH1 accumulation is attributable
to protein stabilization is unknown; however, no change
in BACH1 mRNA expression was observed (data not
shown).
Nuclear localization of both NRF2 and BACH1 is
essential for their transcriptional activity and can be used
as an indicator of their activation. To investigate their
temporal activation following arsenite treatment, changes
in nuclear and cytosolic distribution were examined
(Figure 1C and D). Nuclear NRF2 accumulation parallels
that observed in whole cell extracts. Prior to treatment,
very low levels of NRF2 are present in nuclear fractions.
Consistent with NRF2 levels in whole cell extracts,
nuclear NRF2 accumulation is negligible during the first
hour after arsenite treatment but becomes highly elevated
by 3 h after treatment. Throughout the time course, and
despite the accumulation of NRF2 in whole cell and
nuclear fractions, there is little or no NRF2 accumulation
in the cytoplasmic fraction. Rather, whenever detected,
NRF2 is always associated with nuclear fractions,
consistent with the rapid transport of NRF2 into the
nucleus after its synthesis. Detection of NRF2 in nuclear
fractions of control cells shows that even in the absence
of oxidant exposure a low level of NRF2 resides in the
nucleus.
In contrast to NRF2, BACH1 is rapidly exported from
the nucleus following arsenite treatment, reaching minimal
nuclear levels by 30 min after treatment, corresponding
with an increase in cytosolic levels. BACH1 levels in the
cytosol increase by 2-fold during the first hour after
arsenite treatment and reach maximum observed levels
by 3 h after treatment (Figure 1C and D).
Oxidative stress and arsenite are known to strongly
induce HMOX1. Time-course analyses following 25 mM
arsenite treatment shows a rapid induction of HMOX1
mRNA following an initial 1-h lag period (Figure 1F),
coinciding with the time required for nuclear localiza-
tion of de novo synthesized NRF2 protein. This induction
continues in a linear fashion through 8 h after treat-
ment without achieving a steady-state plateau. Together,
these results show that HMOX1 transcription is
preceded by BACH1 inactivation and occurs in parallel
with, rather than following, NRF2 activation, suggest-
ing that BACH1 inactivation is the antecedent
event corresponding with transcriptional initiation of
HMOX1.
Nucleic Acids Research, 2007 3
Bach1
Nuclear
Actin
Nrf2
Cytosol
Hours
Whole cell lysate
Hours 0.5 1 2 3 4 50
0 0.5 1 3 0 0.5 1 3 N0
0.5 1 2 3 4 50
Bach1
Nrf2
Actin
A
% Maximum
0
20
40
60
80
100
120
NRF2
BACH1
Whole cell lysate
Time (Hours)
B
C
D
BACH1
Time (Hours)
0 0.5 1 3 0 0.5 1 3
0
20
40
60
80
100
120
Nuclear
Cytosol
NRF2
% Maximum
Nuclear
Hours
02 46 8
Fold Control
0
100
200
300
400
500
F
Arsenite (µM)
Fold Control
1 5 25 50
0
100
200
300
400
E
Figure 1. Activation of antioxidant response and induction of HMOX1. (A) Representative immunoblots of total cellular proteins (20 mg) illustrating
the effect of 25 mM arsenite on NRF2 and BACH1 protein levels. (B) Graphical representation of three separate experiments showing changes in
expression of NRF2 and BACH1 proteins normalized to b-actin levels. (C) Representative immunoblots of nuclear (10 mg) and cytosolic proteins
(20 mg) illustrating changes in subcellular localization of NRF2 and BACH1 following arsenite treatment (25 mM). (D) Graphical representation of
three separate experiments showing changes in nuclear and cytosolic NRF2 and BACH1 proteins levels normalized to b-actin levels. The presence
of NRF2 in nuclear extract at 0 h (N0) is provided as a positive immunoblot control for cytosolic NRF2. Quantification represents the mean SEM
of three independent experiments. (E) Dose response of HMOX1 mRNA expression in HaCaT cells following a 6-h continuous treatment with
the indicated concentration of arsenite. (F) Time course of HMOX1 mRNA expression in HaCaT cells following treatment with 25 mM arsenite.
HMOX1 expression was determined using quantitative real-time PCR (qRT–PCR). HMOX1 mRNA concentrations were normalized to
b-actin mRNA and expressed as fold change relative to untreated controls. Values represent at least three independent experiments quantified
in triplicate.
4 Nucleic Acids Research, 2007
BACH1 and NRF2 bind to the same ARE motifs
in the HMOX1 promoter
To identify the maximum possible BACH1- and NRF2-
binding sites, we searched 10 kb upstream of the HMOX1
TSS for core ARE motifs conforming to the sequence
RTGAYNNNGC or its reverse complement (5). Twelve
consensus elements were identified (Table 1) and each
of these sites were investigated for NRF2 and BACH1
interactions by ChIP analysis (Figure 2A). Of the 12 ARE
motifs, NRF2 and BACH1 interact with the same two
sites containing multiple ARE motifs; one, a more
proximal site located at 3928 bp upstream of the TSS
(E1) and the other a more distal site at 8979 bp upstream
(E2). NRF2 binds both of these sites after arsenite
treatment (Figure 2B) while BACH1 binds both of them
in arsenic naı
¨
ve cells (Figure 2C). The proximal E1
element is composed of two slightly different ARE core
motifs having the sequences GCtgcGTCAT and GCtga
GTCAC and separated by 54 nt. The more distal E2 site
consists of four identical repeats of the core ARE motif
having the sequence GCtraGTCAC, each separated by
19 nt. An additional single motif is located at 9491 bp,
415 bp further upstream from the end of this ARE tetrad
for a total of five elements in this DNA region. In control
cells, BACH1 binding at the E2 site is 5-fold greater than
at E1. Following arsenite treatment, NRF2 binding at the
distal E2 tetrad is greater than at the E1 site by 2.5-fold.
None of the other five remaining ARE motifs were bound
by either NRF2 or BACH1. These data show that BACH1
and NRF2 undergo reciprocal binding at the E1 and E2
enhancers following oxidative stress initiated by arsenite
treatment.
Differential regulation of BACH1 and NRF2 activity
To investigate the relative contributions of BACH1 and
NRF2 to HMOX1 expression, we differentially regulated
their activities by treating cells with the proteasome
inhibitor MG132 or with hemin. MG132 indirectly
causes NRF2 activation by inhibiting KEAP1-dependent
proteasomal degradation (26). Hemin inactivates BACH1
through interaction with its multiple heme-binding motifs
leading to a conformational change and nuclear export
(27,28). Treatment of HaCaT cells with MG132 for 3 h
induces extensive NRF2 accumulation in whole cell lysates
relative to untreated control cells (Figure 3A). This
increase in NRF2 is almost entirely localized to the
nuclear fraction (Figure 3B) with negligible levels detected
in the cytosolic fraction (Figure 3C). In contrast to NRF2,
BACH1 levels are only slightly affected by MG132
treatment in whole cell lysates or nuclear distribution
(Figure 3). Hemin treatment contrasts with MG132
treatment in that it prominently triggers BACH1 inactiva-
tion, which manifests as a significant decrease in whole cell
BACH1 protein levels (Figure 3A) and as its disappear-
ance from the nuclear fraction (Figure 3B). Interestingly,
this effect is not associated so much with nuclear efflux to
the cytosolic compartment as occurs following arsenite
treatment, but rather is associated with a net loss of total
BACH1 (Figure 3A). Co-treatment of MG132 and hemin
blocks this net loss of BACH1 (Figure 3A) and results in
its cytoplasmic accumulation due to efflux from the
nucleus (Figure 3C). Thus, inactivation of BACH1 by
hemin triggers both its nuclear export and subsequent
proteasomal degradation. In contrast to hemin, arsenite
only mediates subcellular redistribution of BACH1,
as indicated by nuclear efflux, without notable protein
loss in whole cell lysates (Figure 3). Importantly, hemin
treatment has only minimal effects on NRF2 activation,
as indicated by the relative absence of NRF2 accumula-
tion in either whole cell extracts or nuclear fractions.
Co-treatment with hemin plus MG132 produces a
combined pattern of NRF2 nuclear translocation and
BACH1 efflux similar to that elicited by arsenite. Consis-
tent with these findings, the expression of HMOX1 protein
(Figure 3A) is associated only with treatments that result
in BACH1 efflux from the nucleus, but not with NRF2
activation alone.
The elimination rate of NRF2 exceeds its rate of
synthesis (26); hence, NRF2 activation requires both
inhibition of proteasomal degradation and continuous
NRF2 synthesis. To characterize the roles of NRF2 in
basal and inducible expression of HMOX1 and TXHRD1,
NRF2 activation was prevented by pretreating cells
for 30 min with cycloheximide (CHX) to block protein
synthesis prior to treatment with MG132. Blocking
ongoing protein synthesis prior to inhibition of NRF2
degradation by MG132 prevents accumulation of de novo
synthesized NRF2 while eliciting depletion of basal
nuclear NRF2. The right half of Figure 3A shows that
pretreatment of HaCaT cells with 5 mM CHX blocks
synthesis of new NRF2, thereby preventing its activation
when followed by MG132 or arsenite treatment. The fact
that CHX pretreatment results in the elimination of NRF2
Table 1. DNA sequences consistent with the consensus ARE motifs
located within 10 Kb upstream of the HMOX1 TSS and 1 Kb upstream
of TXNRD1 TSS
Postulated antioxidant response elements of the HMOX1
and TXNRD1 genes
PCR primer location
for ChIP (bp)
Motif sequence Motif location
(bp)
Consensus motif:
GCnnnRTCAY or
CGnnnYAGTR
HMOX1
9069 GTGACagaGC 9491
GCtgaGTCAC 9066
GCtaaGTCAC 9037
GCtgaGTCAC 9008
GCtgaGTCAC 8979
7232 GCcttGTCAC 7104
6060 GCagaATCAT 6008
GCtgaATCAT 5967
3928 GCtgcGTCAT 3992
GCtgaGTCAC 3928
1319 GCgtgGTCAC 107
148 GCaaaATCAC 200
TXNRD1
91 & 36 GCtttGTCAT 19
PCR primer and motif positions correspond to the most distant 5
0
nucleotide from the TSS on the forward strand. Sequences were
considered candidate ARE motifs regardless of orientation or strand.
Nucleic Acids Research, 2007 5
(Figure 3) establishes that under these conditions NRF2
cannot be the mediator of gene transcription. In parallel
treatments, CHX has no effect on hemin- or arsenite-
induced export of BACH1 from the nucleus, though total
BACH1 levels increase in the presence of MG132 relative
to untreated and CHX-treated controls. These data
support a role for proteasomal degradation in the turn-
over of BACH1 and indicate that BACH1 has a much
longer half-life than NRF2 in cells naı
¨
ve to oxidative
stress or hemin.
The effect of differentially modulating BACH1 or
NRF2 activities was examined in relation to HMOX1
expression. HMOX1 is expressed in control cells at nearly
undetectable levels and its induction coincides with
the disappearance of nuclear BACH1 but not nuclear
translocation of NRF2. Treatment with MG132 triggers
prominent nuclear accumulation of NRF2 but fails to
induce HMOX1 expression (Figure 3A). In contrast,
treatment with either arsenite or hemin leads to nuclear
export of BACH1 and to a prominent increase in HMOX1
expression. Combined treatment with MG132 and hemin
augments HMOX1 protein levels, suggesting that activa-
tion of NRF2 contributes to HMOX1 expression in a
manner that is at best additive to the effect of BACH1.
These data suggest that BACH1 inactivation plays a larger
role in the regulation of antioxidant gene expression
than NRF2 activation.
Temporal induction of HMOX1 transcription depends
on BACH1 removal from the enhancers
The observation that expression of HMOX1 is associated
with nuclear efflux of BACH1 rather than with NRF2
activation, suggests that BACH1 removal is the dominant
event regulating HMOX1 induction. To test this hypo-
thesis we followed the temporal dynamics of HMOX1
Relative DNA Enrichment
Relative DNA Enrichment
B
NRF2 Interactions
Kbp −10 −1−2−30−4−5−6−7−8−9
1 211
21
4
1
2
A
C
BACH1 Interactions
Contro
l
As
+3
&
&
HMOX1 Promoter Position
−10 −9 −8 −7 −6 −5 −4 −3 −2 −1
0
−10 −9 −8 −7 −6 −5 −4 −3 −2 −1
0
0
1000
2000
3000
4000
5000
HMOX1 Promoter Position
−9076
−7114
−6018
−5206
−3928
−3033
−2518
−1734
−1304
−148
0
100
200
300
400
500
600
−9076
−7114
−6018
−5206
−3928
−3033
−2518
−1734
−1304
−148
E2
E1 Pr
Figure 2. Identification of NRF2 and BACH1 interactions with core ARE motifs of HMOX1.(A) The position of all 12 putative ARE motifs,
relative to the HMOX1 transcription start site as annotated by the NCBI Homo sapiens Genome Map Viewer, Build 36.2. Open boxes = motif
conforming to the consensus ARE sequence, shaded boxes = imperfect ARE motif. Boxed numerals indicate the number of motif repeats in that
region. Arrows indicate the plus-strand orientation of each ARE motif relative to the consensus ARE sequence (RTGAYnnnGC). Relative DNA
enrichment associated with NRF2 (B) and BACH1 (C) ChIP at each of the HMOX1 ARE-containing sites. Immunoprecipitated DNA was analyzed
for enrichment by qRT–PCR using primers flanking ARE motifs at the positions indicated. Vertical bars and circle symbols graphically depict the
relative magnitude of DNA enrichment at each position. Vertical bars indicate enrichment at positions 3928 bp (E1) and 9069 bp (E2) while
circles correspond with regions where negligible DNA enrichment was observed. Values oriented vertically along the abscissa indicate the position of
each DNA region amplified by qRT–PCR. Amplification was expressed in terms of C
t
as calculated by normalizing the C
t
for each primer in
chromatin immunoprecipitated samples to the C
t
obtained from the respective input DNA and expressed as a percentage relative to the PCR primer
having the maximum enrichment (–9069 bp). Values represent the mean SEM of at least three independent experiments performed in triplicate.
6 Nucleic Acids Research, 2007
transcriptional initiation by ChIP analysis. The time
course of BACH1 and NRF2 binding to the E1
(Figure 4) and E2 (data not shown) enhancer regions of
HMOX1, as well as RNA pol II binding at the proximal
promoter (148), were quantified after treatments with
arsenite, hemin or MG132. These treatments induced very
similar interactions of NRF2 and BACH1 at both of
these enhancer regions, of which the observed interactions
at E1 are representative. As anticipated, arsenite treat-
ment produces a significant loss of BACH1 binding
that is clearly detected 30 min after treatment and reaches
minimal level by 60 min (Figure 4A). Conversely, arsenite
A
HMOX1
DMSO
CHX
Whole Cell Lysates
As
+3
MG132
Hemin
B
Nuclear Lysate
DMSO CHX
b-actin
b-actin
Bach1
Bach1
Nrf2
Nrf2
As
+3
MG132
Hemin
DMSO CHX
Bach1
Nrf2
Cytosolic Lysate
b-actin
C
––––+----+
–+–+--+-+-
–++---++--
––––+----+
–+–+--+-+-
–++---++--
––––+----+
–+–+--+-+-
–++---++--
As
+3
MG132
Hemin
Figure 3. Differential regulation of NRF2 and BACH1 activation.
Immunoblots illustrating differential expression of NRF2 and BACH1
protein following treatment of HaCaT cells with 25 mM arsenite, 5 mM
MG132, 25 mM hemin or MG132 + hemin combined. HaCaT cells were
treated as indicated for 3 h or co-treated with 5 m M cycloheximide
(CHX) following a 30-min CHX pretreatment. (A) Total cellular
proteins (20 mg) from whole cell lysates. (B) Proteins extracts (10 mg)
from nuclear lysates. (C) Proteins extracts (20 mg) from cytosolic
lysates. Blots are representative of 2–3 separate experiments.
Fold change
Nrf2
0
5
10
15
20
25
A
Bach1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Arsenite
Hemin
Mg132
C
RNA pol II
Minutes
0 30 60 90 120 150 180
0
2
4
6
8
10
12
Fold changeFold change
B
Figure 4. Association of NRF2 and BACH1 DNA binding with
transcriptional activation. Time course of DNA binding by BACH1
(A) NRF2 (B) and RNA polymerase II (C) following treatment with
25 mM arsenite, 5 mM MG132 or 25 mM hemin. ChIP-enriched DNA
was quantified using qRT–PCR with primers flanking the HMOX1
ARE motifs at positions 3992 (NRF2 and BACH1) and 148 (RNA
Pol II) and is expressed as the value for each treatment normalized
to its corresponding input and expressed as fold enrichment relative to
untreated control. Figures represent the results of two independent
experiments performed in triplicate SEM.
Nucleic Acids Research, 2007 7
triggers a prominent increase in NRF2 binding
(Figure 4B), but this increase is delayed by at least
60 min relative to the BACH1 decrease, and is maximal
at 150 min after treatment. Arsenite also produces a
large increase in RNA pol II binding (Figure 4C) that
temporally follows the loss of BACH1 but precedes NRF2
binding by at least 60 min and this coincides with the
relatively low level of NRF2 binding during the period
between 60 and 120 min.
To investigate the individual contributions of BACH1
and NRF2 to HMOX1 induction, cells were treated
with either hemin or MG132 in parallel with arsenite.
Hemin triggers a rapid decrease in BACH1 DNA binding,
mirroring the effect of arsenite and producing
nearly complete BACH1 loss from the promoter 60 min
after treatment (Figure 4A). In comparison to arsenite-
mediated NRF2 binding, hemin treatment is associated
with relatively weak NRF2 binding at time points earlier
than 90 min (Figure 4B) and significantly less than that
observed with arsenite after 120 min. Interestingly, hemin
induces RNA pol II binding that proceeds in a manner
similar in magnitude and time course to that associated
with arsenite treatment (Figure 4C).
In stark contrast to both arsenite and hemin, MG132
has little effect on promoter binding by BACH1
(Figure 4A) and limited binding of NRF2, even after
120 min of treatment (Figure 4B). The level of NRF2
binding elicited by MG132 is of the order of that observed
with hemin and approximately one-half of the level found
with arsenite. Furthermore, MG132 elicits weak RNA pol
II DNA binding relative to the levels associated with
hemin and arsenite treatments, implying much lower levels
of transcription initiation (Figure 4C). Together, these
results strongly suggest that loss of BACH1 from the
HMOX1 promoter is associated with a high level of RNA
polymerase II binding, while NRF2 activation in the
absence of BACH1 removal is associated with weak levels
of gene induction, as inferred from the extent of RNA
pol II binding. Consistent with whole cell lysate
and nuclear extract immunoblots, it is evident that
RNA pol II HMOX1 binding precedes NRF2 activation,
further supporting the conclusion that loss of BACH1-
mediated transcriptional repression is more important
than nuclear accumulation of NRF2 for HMOX1
induction.
To confirm that RNA pol II binding is associated
with gene induction by hemin but not by MG132,
relative steady-state levels of HMOX1 mRNA accumula-
tion were measured by qRT–PCR over the time course
of HMOX1 protein induction. Induction of HMOX1
mRNA expression was differentially regulated by treat-
ment with either hemin or MG132. Despite the absence
of NRF2 activation, hemin triggers induction of
much greater levels of HMOX1 mRNA than MG132
(Figure 5). Given that NRF2 activation is generally
considered a critical event for antioxidant gene
induction, it is surprising that inactivation of BACH1
by hemin produced 5-fold greater levels of HMOX1
induction than were elicited by MG132-mediated NRF2
activation.
Differential gene induction regulated by NRF2 and BACH1
Although NRF2 and BACH1 interact with ARE motifs,
several lines of evidence support that these interactions
with DNA are not identical, thereby suggesting that genes
could be identified that are regulated by one factor but not
by the other (12,29,30). TXNRD1 was identified as one
such gene. ChIP analysis was used to test for NRF2 and
BACH1 binding to the single ARE motif located 9 bp
upstream of the TSS in the TXNRD1 5
0
-flanking region.
As illustrated in the upper panel of Figure 6A, NRF2
binds the ARE motif of the TXNRD1 in ‘control cells’
with an affinity significantly greater than either at
HMOX1 E1 or E2. After arsenite-induced oxidative
stress, this site becomes more strongly bound by NRF2,
reaching a level that is intermediate between NRF2
binding at the HMOX1 E1 and E2 sites after activation,
and 6.5-fold greater than control levels. In contrast,
the lower panel of Figure 6A shows negligible binding
of BACH1 to the TXNRD1 ARE in either control or
arsenite-treated cells. As a consequence of differential
transcription factor binding, we anticipated that MG132,
but not hemin, would elicit NRF2-dependent TXNRD1
induction. To test this prediction, we treated HaCaT cells
with MG132 or hemin and quantified mRNA levels
of HMOX1 and TXNRD1 by qRT–PCR. In untreated
control cells, basal TXNRD1 mRNA levels are 35-fold
greater than the levels of HMOX1 mRNA. Inhibition of
NRF2 activation with CHX significantly inhibited basal
TXNRD1 expression but left HMOX1 expression
unchanged (Figure 6B and C). This pattern of expression
is consistent with functionally active NRF2 residing
basally in the nucleus and rules out the possibility that
this resident nuclear NRF2 contributes to basal HMOX1
expression. The role of activated NRF2 is demonstrated
HMOX1 Expression
Minutes
0 60 120 180
HMOX1 Expression
(% Maximum)
0
20
40
60
80
100
120
Hemin
Mg132
Figure 5. Time course of HMOX1 expression elicited by hemin and
MG132 treatment. The time course of HMOX1 mRNA expression
following treatment with 25 mM hemin or 5 mM MG132. HMOX1
mRNA was determined by quantitative real-time PCR (qRT–PCR),
normalized to b-actin mRNA and expressed as percent maximum
expression. Values represent at least three independent experiments
quantified in triplicate SEM.
8 Nucleic Acids Research, 2007
by the MG132 treatments that trigger maximal TXNRD1
induction but mediate only weak HMOX1 expression
(Figure 6B and C). Pretreatment with CHX prior to
MG132 blocks NRF2 synthesis, returning HMOX1
mRNA to control levels and reducing TXNRD1 mRNA
to the levels of CHX-treated control cells. These expres-
sion data reflect the primary role of NRF2 in basal
and inducible expression of TXNRD1 but not HMOX1.
Hemin treatment, which inactivates BACH1, has no effect
on TXNRD1 expression (Figure 6C), reflecting the fact
that TXNRD1 is regulated independently of BACH1.
In contrast, hemin elicits a significant and nearly maximal
increase in HMOX1 expression (Figure 6B). CHX pre-
treatment predominantly blocks this induction, demon-
strating a requisite role for resident nuclear NRF2 in
HMOX1 induction without necessitating nuclear accumu-
lation of activated NRF2. Combined treatment with
hemin plus MG132 results in induction of TXNRD1
to levels similar to those in cells treated with MG132
alone while the level of HMOX1 induction is similar to
that elicited by hemin (Figure 6B and C). These results
show that TXNRD1 induction is attributable solely
Nrf2
% Enrichment
(relative to E2)
0
20
40
60
80
100
Bach1
Promoter
HMOX1
E2
HMOX1
E1
TXNRD1
0
20
40
60
80
100
Control
Arsenite
A
Treatment
0
20
40
60
80
100
120
Control
Mg132
Hemin
Hemin +
MG132
TXNRD1 Expression
(% Maximum)
C
DMSO
Cycloheximide
Treatment
Control
Mg132
Hemin
Hemin +
MG132
HMOX1 Expression
(% Maximum)
0
20
40
60
80
100
120
B
Figure 6. Differential regulation of HMOX1 and TXNRD1 by BACH1 and NRF2. (A) ChIP analysis of NRF2 and BACH1 interactions with ARE
sites of HMOX1 and TXNRD1 before and after 25 mM arsenite treatment for 3 h. Binding of NRF2 (top panel) and BACH1 (bottom panel)
to HMOX1 E1 and TXNRD1 is expressed relative to binding at the HMOX1 E2 enhancer element. Expression of HMOX1 (B) and TXNRD1
(C) mRNA was measured in HaCaT cells following treatment with 25 mM hemin, 5 mM MG132 or both in the presence (shaded bars) or absence
(filled bars) of 5 m M CHX. Relative mRNA was determined by quantitative real-time PCR (qRT–PCR), normalized to b-actin mRNA and expressed
as percent maximum expression. Values represent at least three independent experiments quantified in triplicate SEM.
Nucleic Acids Research, 2007 9
to activation of NRF2 by proteasome inhibition but that
HMOX1 induction requires inactivation of BACH1.
Thus, BACH1 inactivation is predominantly responsible
for HMOX1 induction and that NRF2 activation contrib-
utes nominally to this process. Interestingly, the combina-
tion of MG132 plus hemin also shows that, when preceded
by CHX treatment, HMOX1 expression remains signifi-
cantly induced despite the lack of NRF2 and hints at the
possible role for BACH1 in repressing the activity
of transcriptional activators in addition to NRF2. These
data establish that inactivation of BACH1 is
more important than activation of NRF2 for induction
of HMOX1 expression and that not all NRF2-regulated
genes respond in this manner, as is the case of TXNRD1.
DISCUSSION
In the present article, we report for the first time the
dynamics associated with inactivation of BACH1 and
NRF2 activation underlying the initiation of endogenous
gene targets. BACH1, which is predominantly localized to
the nucleus of control cells, is exported to the cytosol
within 30 min following arsenite or hemin treatment.
Temporally, inactivation of BACH1 precedes NRF2
activation by at least 30 min, correlating with the period
necessary for de novo synthesis of NRF2, as supported by
the absence of immunoreactive NRF2 when CHX
treatment precedes proteasome inhibition. Consequently,
biological coupling of these transcriptional regulators
insures that the response to stressors is rapid and not
dependent on the delay required for synthesis of high
NRF2 levels.
Of the 12 putative HMOX1 ARE motifs, only two sites,
at 3928 bp (E1) and 8979 bp (E2), are reciprocally
bound by BACH1 and NRF2. Both sites contain multiple
ARE motifs and have been previously recognized as
HMOX1 transcriptional regulators (15). Our data confirm
that these are the only two HMOX1 elements recognized
by NRF2 and BACH1 in vivo. This pattern of binding
contrasts markedly with binding at the TXNRD1 promo-
ter, where NRF2 is capable of significantly binding the
ARE motif but BACH1 is not. Distinct recognition of
ARE motifs by BACH1, compared to NRF2, conveys an
additional level of transcriptional regulation to HMOX1
that is lacking in the regulation of TXNRD1. The fact that
BACH1 and NRF2 differently recognize ARE motifs
presents the possibility that BACH1 regulates an over-
lapping but separate gene battery. NRF2 and BACH1
differ in their affinities for ARE motifs that otherwise
appear to conform equally well to the consensus ARE core
motif; however, this short 10 bp sequence is not the sole
determinant of factor binding. Nucleotides flanking the
ARE core as well as motif multiplicity also contribute
to differential motif recognition by BACH1 and NRF2.
Several reports indicate that extended sequences flanking
the ARE core are vital for recognition by NRF2 (5,31–33),
though the precise sequence requirements of the extended
ARE motif are not well defined leaving a great deal of
uncertainty when attempting to predict high-affinity
NRF2-binding elements. Similar difficulties exist in
predicting the sequence requirements for high-affinity
BACH1-binding sites. Unlike NRF2 however, detailed
description of BACH1-binding elements is hampered by
the fact that only a few genes are known to be under its
regulatory control (13,34), including NQO1 (10) and
HMOX1 (14). The sequences that BACH1 interacts with
at these genes seems to conform to the core ARE motif
recognized by NRF2; however, only one report has
experimentally examined sequence requirements for
BACH1 binding, the results of which generally support
motif homology with the ARE (29). Binding element
multiplicity may also contribute to differences in DNA-
binding affinity between NRF2 and BACH1. While Nrf2
is capable of binding an individual ARE motif, BACH1
appears to bind poorly to single motifs, preferring to
interact at sites containing multiple motifs. The BTB/POZ
domain of BACH1 is thought to contribute to DNA
binding through oligomerization with neighboring
BACH1 heterodimers to stabilize their interactions with
DNA (12). In this respect, both HMOX1 enhancers
recognized by BACH1 consist of multiple elements while
the TXNRD1 element is a single consensus motif. Thus,
the multiple ARE motifs of HMOX1 might permit
BACH1 oligomerization while TXNRD1, with its single
motif, would not allow formation of stable BACH1
oligomers. Together, nucleotide sequence and motif
multiplicity may account for the observation that the
TXNRD1 promoter is poorly recognized by BACH1.
In control cells, we have noted that the cytosol is
essentially devoid of detectable NRF2 consistent with
reports that NRF2 activity is governed by regulation of
its stability (23,35,36) rather than by being sequestered
in the cytosol (25,37). In the absence of oxidative stress,
KEAP1 mediates Cul3-dependent ubiquitylation of NRF2
that targets it for rapid proteasomal destruction (36).
During oxidative stress several sensitive KEAP1 cysteines
are oxidized resulting a loss of KEAP1-directed protea-
somal degradation of NRF2 and increased proteasomal
degradation of KEAP1 itself (21,38). In the absence of
ongoing KEAP1-directed degradation of NRF2, the
half-life of newly synthesized protein is prolonged permit-
ting rapid nuclear translocation and accumulation with
subsequent antioxidant gene induction (35). Consistent
with a short residence time of NRF2 in the cytosol, we
observe that when NRF2 escapes proteasomal degrada-
tion it accumulates in the nucleus while remaining nearly
undetectable in the cytoplasm. In this respect, we have
observed the persistence of nuclear NRF2 in untreated
control cells indicating that even in the absence of
oxidative stress some NRF2 escapes KEAP1-mediated
degradation and is capable of binding ARE elements and
eliciting gene induction. We propose that the persistence
of resident nuclear NRF2 plays a crucial role in both the
induction of HMOX1 as well as basal TXNRD1 expres-
sion. BACH1 inactivation elicits maximal HMOX1
expression without requiring concurrent NRF2 activation
by permitting resident nuclear NRF2 to bind HMOX1
ARE motifs. In contrast, BACH1 does not inhibit basal
nuclear NRF2 binding to the TXNRD1 promoter and
therefore basal TXNRD1 expression is 20% of maximal
expression, and 35-fold higher than basal HMOX1
10
Nucleic Acids Research, 2007
ARE Motif Position (Kb)
BACH1
BACH1
NRF2 NRF2
NRF2
NRF2
NRF2
BACH1
NRF2
−8−9Kb-10 −7 −6 −5 −4 −3 −2 −10
−8−9Kb-10 −7 −6 −5 −4 −3 −2 −10
−8−9Kb-10 −7 −6 −5 −4 −3 −2 −10
−8−9Kb-10 −7 −6 −5 −4 −3 −2 −10
NRF2
BACH1
BACH1
NRF2
Nucleus
K
off
Naïve
A
B
C
D
Hemin
MG132
Arsenite
or
Hemin+
MG132
PolII
PolII
K
on
K
off
K
off
K
on
Nucleus
Nucleus
BACH1
K
off
K
on
K
off
K
on
K
off
K
on
K
off
K
on
K
off
K
on
K
on
NRF2
NRF2
NRF2
NRF2 NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
NRF2
BACH1
BACH1
NRF2
Cytosol
Cytosol
Nucleus
Cytosol
C
y
tosol
Figure 7. Induction of HMOX1 through the interplay of BACH1 and NRF2 with enhancer elements. (A) Binding of BACH1 (red ovals) at the
E1 and E2 enhancer elements of HMOX1 in untreated control cells blocks NRF2 (blue ovals) binding and HMOX1 induction. Competition
Nucleic Acids Research, 2007 11
expression. Loss of basal nuclear NRF2 following CHX
treatment precipitously decreases both hemin-mediated
HMOX1 induction and basal TXNRD1. It is noteworthy
that some HMOX1 expression elicited by hemin inactiva-
tion of BACH1 persists despite the absence of NRF2
protein, suggesting BACH1 may also play a role in the
repression of factor(s) in addition to NRF2. In this regard,
HMOX1 expression is even more pronounced when CHX
treatment precedes co-treatment with hemin plus MG132,
suggesting that proteasomal inhibition may stabilize other
putative transcriptional activator(s).
Without prior inactivation of BACH1, activated NRF2
binds inefficiently to HMOX1 enhancers compared to the
binding elicited by arsenite. Although the level of NRF2
binding associated with MG132 treatment is similar
in extent to that initiated by hemin treatment, NRF2
activation in the absence of BACH1 inactivation results in
significantly lower levels of HMOX1 transcription. This
contrasts profoundly with TXNRD1 expression, where
MG132 strongly stimulates induction and hemin treat-
ment has no effect. Since transcription factor binding to
cognate sites is a stochastic event (39), the massive increase
in nuclear NRF2 concentration associated with its
activation could increase competition with BACH1 for
ARE motifs or sMAF-binding partners. By mass action,
NRF2 activation could establish a new equilibrium
favoring DNA-bound NRF2/sMAF, thereby increasing
the stoichiometric DNA residence of NRF2. Since
BACH1 appears to bind at sites containing multiple
AREs, NRF2 may not displace a large enough fraction of
DNA-bound BACH1 to overcome transcriptional repres-
sion. CHX treatment decreased hemin-mediated HMOX1
induction and basal TXNRD1 expression. When inter-
preted in the light of our ChIP enhancer binding data,
these findings support the conclusion that inactivation of
BACH1 by hemin decreases its DNA-binding affinity
resulting in its generalized removal from ARE motifs and
concomitant loss of transcriptional repression, leaving the
AREs available for binding by NRF2 and possibly other
transcription that mediate gene induction.
Based on the dynamic exchange of BACH1 and NRF2
we propose that in cells naı
¨
ve to oxidative stress, BACH1
is bound to the ARE enhancer motifs preventing
NRF2 from binding and thereby repressing transcription
(Figure 7A). Hemin treatment triggers removal of BACH1
from HMOX1 enhancers thereby allowing NRF2 that is
already present in the nucleus to interact with ARE motifs
and elicit gene induction (Figure 7B). On the other hand,
MG132 triggers NRF2 translocation to the nucleus but
DNA-bound BACH1 blocks NRF2–ARE interactions to
prevent gene induction (Figure 7C). The vast increase
in the presence of nuclear NRF2 permits some increase
in NRF2 binding to HMOX1 AREs but the presence of
BACH1 maintains gene repression. Treatment with either
arsenite or hemin plus MG132 triggers both the removal
of BACH1 and the activation of NRF2, which can now
freely bind vacant enhancer motifs (Figure 7D).
Though not all ARE-containing genes are regulated
by BACH1, whenever BACH1 interacts with ARE motifs
it contributes an important regulatory dimension that
provides a more complex response to environmental
stress. Reciprocal ARE binding of BACH1 for NRF2
imparts an increased level of complexity to the ARE-
regulated genes producing distinct patterns of gene
expression patterns, as shown here for HMOX1 and
THXRD1. The differential interaction of BACH1 and
NRF2 with various forms of the ARE motif suggests
the possibility that BACH1 may regulate a distinct but
overlapping battery of genes. In this case, genes specifi-
cally repressed by BACH1 could be globally induced in
response to redox stress.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank Dr N. Fusenig (Division of Differentiation
and Carcinogenesis in Vitro, German Cancer Research
Center, Heidelberg, Germany) for a gift of HaCaT
keratinocytes. This research was supported by NIEHS
grants R01 ES10807, The NIEHS Center for Environ-
mental Genetics grant P30 ES06096 and the NIEHS
Superfund Basic Research Program grant P42 ES04908.
J.F.R. is a Postdoctoral Trainee partly supported by
NIEHS T32 ES07250, Environmental Carcinogenesis and
Mutagenesis Training Grant. Funding to pay the Open
Access publication charges for this article was provided
by NIEHS grants RO1 ES10807.
Conflict of interest statement. None declared.
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