(Received for publication, July 13, 1995; and in revised form, October 6, 1995)
From the
We show here that the OxyR response element (ORE) in the
bacterial oxyR promoter can also function as a redox-dependent
enhancer in mammalian cells. Fusion of ORE to an SV40 basal promoter
driving chloramphenicol acetyltransferase (CAT) expression confers
HO
inducibility to expression of the cat gene in mouse Hepa-1 hepatoma cells. Nuclear extracts from these
cells contain DNA-binding proteins that specifically interact with ORE
DNA, cannot be competed by cognate oligonucleotides to AP-1 or
NF
B, and are constitutively expressed, since treatment with
H
O
causes no detectable changes in binding
activity or DNA-protein interaction. Recombinant cDNA clones that
express ORE-binding proteins were isolated from a mouse hepatoma
expression library and found to be representatives of two different
members of the murine Y-box family of transcription factors. Canonical
Y-box and ORE oligonucleotides compete with each other for binding to
Y-box proteins in gel shift assays and antibodies to FRGY2, a Xenopus Y-box protein, supershift both Y-box and ORE
DNA-protein complexes. In addition, antisense oligonucleotides to mouse
YB-1 mRNA abolish induction of ORE-mediated cat expression by
H
O
, and luciferase reporter constructs
containing ORE, or the Y-box from the human MHC class II HLA-DQ gene,
exhibit identical dose-dependent H
O
inducibilities, which can be abolished by addition of
2-mercaptoethanol to the culture medium. These results suggest that the
Y-box proteins may be an integral component of a eukaryotic redox
signaling pathway.
Reactive oxygen intermediates, including superoxide anion, hydrogen peroxide, and hydroxyl radical, are generated during normal aerobic metabolism by the incomplete reduction of oxygen to water and are crucial for many physiologic processes, such as the respiratory burst of phagocytic cells. Reactive oxygen intermediates also pose a continuous risk of cell injury to aerobic organisms, a risk that is greatly elevated by exposure to environmental oxidants such as ionizing radiation, heavy metals, redox active chemicals, and hyperoxia, that increase their production(1, 2, 3, 4) . Reactive oxygen intermediates react with DNA, proteins, lipids, and cellular membranes, causing behavioral, cytotoxic, and mutagenic damage(5, 6, 7, 8) . With an increased understanding of the pathways leading to oxidant injury and of the protective role of antioxidant compounds, it is apparent that endogenous antioxidant mechanisms exist in a balance with each other and with endogenous oxidants and that perturbation of this balance contributes to the pathogenesis of many human diseases, including pulmonary oxygen injury, cancer, aging, and degenerative diseases(1, 2, 3, 4) .
Much of our current understanding of genetically determined enzymatic antioxidant defenses derives from work in enteric bacteria, where approximately 80 different proteins controlled by two different regulons are induced by oxidative stress(9) . One of these regulons is stimulated by superoxide radicals and is controlled by the products of the soxR and soxS genes(10, 11, 12, 13) . The other is stimulated by hydroxyl radicals and is controlled by the product of the oxyR gene(14, 15, 16, 17) . The OxyR protein is an autoregulatory, DNA-binding, redox-sensitive transcriptional activator of genes coding for peroxide-inactivating enzymes such as catalase (katG), NADPH-dependent alkylhydroperoxidase (ahpFC), and many others(18, 19, 20) . The promoters of the genes regulated by OxyR show little sequence relatedness, and consequently there is a poor definition of consensus sequence elements for OxyR-binding DNA motifs (21, 22, 23, 24) .
Our
understanding of the regulation of antioxidant defenses in eukaryotic
systems is far less defined than in prokaryotes. Many genes are induced
by reactive oxygen
species(25, 26, 27, 28) , including
transcription factors like those in the AP-1 (29, 30, 31, 32) and NF-B (33, 34, 35) families, but regulons
resembling those controlled by soxRS and oxyR have
not been described, although their existence has long been suspected.
We report here that the bacterial OxyR-binding motif functions as a
redox-dependent transcriptional enhancer in murine cells. This function
results from the interaction of the ORE (
)motif with a
member of the Y-box family of DNA- and RNA-binding proteins.
For analysis of their oxidant status,
cells were grown on microscope coverslips at 37 °C in the absence
or presence of 25 µM 2-ME. When the cells were at 60%
confluence, one-half of the coverslips were treated with 300
µM HO
in PBS for 10 min, and the
other half was treated only with PBS. Following treatment, all
coverslips were washed with fresh PBS and incubated in complete medium
for an additional 1 h. Thereafter coverslips were washed in PBS,
incubated for 10 min at 37 °C with 2`,7`-dichlorofluorescein
diacetate, washed again with PBS, and examined by fluorescent
microscopy. The excitation and emission wavelengths for fluorescence
measurements were 495 and 535 nm, respectively.
For transient expression of ORE constructs in the presence of anti-Y-box antisense oligonucleotides, the following oxi843 (musybox, see Fig. 4) antisense oligonucleotides were used as phosphorothioated derivatives to block mRNA translation in vivo(41) : Y1, (12) CTCGCTGCTCATGGTTGAGGT(-9); Y2, (8) CTGCTCATGGTTGAGGTGGAT(-13); Y3: (3) CATGGTTGAGGTGATGGTGAT(-18). The numbers in parentheses indicate the coordinates relative to the ATG initiation codon (or its complement, CAT, underlined above). Yc, GCATTTTCTACGAGGGTGTTG, was a control generated by random rearrangement of the sequences between coordinates -18 and +12. Cells were grown for three generations in the presence of the antisense oligonucleotides, which were kept in the culture medium throughout transfection and the subsequent growth period prior to assay of the reporter genes.
Figure 4: Amino acid sequence comparison of human and murine Y-box- and ORE-binding proteins. Alignment of sequences was obtained with the multiple sequence alignment algorithms. GenBank(TM) accession numbers of the sequences used are, M60419 and X57621 (Musybox, the murine YB-1 protein); M24069 (Humdbpa2, the human YB-2 protein); M24070 (Humdbpb2, the human YB-2 protein). The two Y-box sequences determined in the present studies are oxi843 and oxi17. The cold-shock domain spans the 80 amino acids between residues 84 and 164 (bold and over- and underlined) and is almost identical for the five sequences shown.
For methylation interference analysis, approximately 1 µg (60
pmol) of each single-stranded oligonucleotide in the double-stranded
ORE was labeled with T4 polynucleotide kinase in the presence of
[-
P]ATP to a specific activity of 2
10
dpm/pmol. Each labeled oligonucleotide was partially
methylated with dimethyl sulfate as described (45) and annealed
to an equal amount of its unlabeled complement. Binding reactions were
scaled up to contain 150 µg of protein and 10 ng (1.2
10
dpm) of probe. Bound probe was separated from free probe
by electrophoresis in nondenaturing 4% polyacrylamide gels and
identified by exposing the wet gel to x-ray film. The complex A bands
were excised from the gel and electroblotted onto NA 45 paper
(Schleicher and Schuell). DNA was eluted from the paper by incubation
in 1 M NaCl, 10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA at 56° for 1 h, extracted with an equal volume of
phenol:chloroform (1:1) and ethanol precipitated. The DNA was dissolved
in water, cleaved with 1 M piperidine at 90 °C for 30 min,
and analyzed in denaturing 15% polyacrylamide-urea gels.
For
Southwestern analyses, nuclear and cytosolic extracts from
HO
-treated and untreated cells were prepared as
described(43, 44) , and proteins from coupled in
vitro transcription-translation assays were synthesized with the
TNT kit from Promega, using 1 µg of recombinant plasmid DNA as the
transcription template and following the manufacturer's
instructions. Protein samples (30-50 µg of cell extract or 5
µl of a 50-µl in vitro translation reaction) were
separated in 10% or 12.5% SDS-acrylamide gels and transferred to an
Immobilon membrane (Millipore). After transfer, membranes were washed
in buffer B (20 mM Tris-HCl, pH 7.8, 60 mM KCl, 1
mM MgCl
, 1 mM DTT), and proteins were
denatured by immersing the membranes in 6 M guanidine
hydrochloride in buffer B twice for 10 min at room temperature. Graded
protein renaturation was accomplished by sequentially washing the
membranes for 5 min in four successive 2-fold serial dilutions of the
denaturing solution in buffer B, followed by two 5-min washes in buffer
B. In some experiments, denaturation in 6 M guanidine
hydrochloride and graded renaturation were omitted. Nonspecific binding
sites were blocked by incubation for 1 h at room temperature in 5%
non-fat milk in buffer B. Probes were prepared by labeling each strand
of a double-stranded oligonucleotide separately at the 5` end with T4
polynucleotide kinase in the presence of
[
-
P]ATP and allowing the strands to anneal.
For maximum binding efficiency, probes were catenated by ligation with
T4 DNA ligase. Binding of end-labeled, catenated DNA probes took place
for 2 h at room temperature in 0.25% non-fat milk in a high ionic
strength buffer B, containing 120 mM KCl and 2
10
dpm/ml probe. After binding, the membranes were washed
three times in a buffer of the same composition, blotted, and exposed
to x-ray film.
For Western blots, proteins were transferred to Immobilon, and the blot was blocked with 2% goat serum, 2% non-fat milk in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1% Tween 20 (TBS-T) for 1 h at room temperature. A rabbit anti-Xenopus FRGY2 antibody (a kind gift of Dr. Alan Wolffe) was used at a 1:4,000 dilution. Detection was by the ECL chemiluminescence detection kit of Amersham Corp.
For UV cross-linking, 50 pmol of ORE noncoding
strand was annealed to an equimolar amount of a 10-mer oligonucleotide
complementary to the 3` end of the template. The BrdUrd-substituted
coding strand was synthesized by incubation with the Klenow fragment of E. coli DNA polymerase in the presence of 50 µM each Br-dUTP, dATP, dGTP and 50 µCi of
[-
P]dCTP (3,000 Ci/mmol) for 1 h at 37
°C. Approximately 10 ng of probe (1
10
dpm)
were incubated with 20 µg of nuclear extract protein from untreated
cells. Binding mixtures were exposed to 12 J/m
of 254-nm UV
light or left unexposed. Complexes were digested with 2 units of DNase
I in the presence of 5 mM MgCl
for 20 min at room
temperature; one-third volume of 3
SDS-polyacrylamide gel
electrophoresis buffer was added, the samples were heated to 100 °C
for 5 min and analyzed in 10% SDS-polyacrylamide gels.
The actual
extent of peroxide-induced oxidative stress in the Hepa-1 cells used in
the rest of these experiments was estimated by measuring the uptake and
oxidation of the fluorescent probe 2`,7`-dichlorofluorescein diacetate
(DA-DCFH). DA-DCFH is a reliable indicator of intracellular oxidant
state(50, 51, 52, 53, 54) ,
since it is taken up by cells and deacetylated by esterases, releasing
free dichlorofluorescein (DCFH). DCFH is readily oxidized by reactive
oxygen intermediates to form the fluorescent product
2`,7`-dichlorofluorescein
(DCF)(54, 55, 56, 57) . Cells grown
under reducing conditions, i.e. Dulbecco's modified
Eagle's medium supplemented with 25 µM 2-mercaptoethanol, displayed a mean DCF fluorescence per cell
significantly lower than that of cells grown in unsupplemented medium.
In both instances, fluorescence was nearly doubled after a 10-min
treatment with 300 µM HO
(Table 2), indicating that H
O
causes a significant change in the level of intracellular
oxidants.
Figure 1:
Characterization of
ORE-binding proteins in Hepa-1 cells. A, gel retardation
analyses of ORE-binding nuclear proteins. Nuclear extracts (30 µg)
used were from Hepa-1 cells grown in the absence (-2ME)
or in the presence (+2ME) of 2-mercaptoethanol. In both
cases, one-half of each culture was treated with 300 µM
HO
(+) in PBS, and the other half
was mock-treated with PBS (-). In addition, nuclear
extracts were prepared in the absence (-) or presence (+) of 20 mM DTT. The positions of the five
complexes with ORE probes are indicated at the left. B, competition of ORE binding by different oligonucleotides.
Binding reactions were prepared in the presence of 1 mM DTT
with extracts from untreated cells grown in the absence of 2-ME. Prior
to addition of the labeled ORE probe, extracts were preincubated for 5
min with a 300-fold excess of the indicated oligonucleotides (see the
``Experimental Procedures'' for the complete sequences): AhpC, the 46-base pair-long OxyR binding sequence in the
bacterial ahpFC promoter; KatG, the 51-base pair long
OxyR binding sequence in the bacterial katG gene promoter; NF
B, the consensus NF
B binding motif; AP-1,
the consensus AP-1 binding site; and oxyR, the same 51-mer oxyR promoter sequence present in the
probe.
The
specificity of the DNA-protein interactions detected was examined in
greater detail by using an excess of various unlabeled oligonucleotides
as binding competitors in gel shift assays. Unlabeled ORE DNA was able
to compete for its own binding effectively, while an excess of
oligonucleotides containing the canonical binding sites for AP-1 or
NFB had no effect on ORE binding (Fig. 1B).
Remarkably, binding to ORE was readily competed by the OxyR binding
sites in the bacterial katG and ahpFC promoters (Fig. 1B), indicating that the binding characteristics
and specificity of the mammalian protein resembled very closely those
of bacterial OxyR.
Figure 2:
Methylation interference analyses. A, methylation interference. To maximize any possible
differences in binding characteristics between oxidative and reducing
environments, DNA binding reactions were prepared with extracts from
cells grown under the most divergent conditions. The nuclear extract
from untreated cells (U) was from cells grown in the presence
of 2-ME and prepared with 20 mM DTT; the nuclear extract from
HO
-treated cells (H) was from cells
grown without 2-ME and prepared without DTT. In addition, binding
reactions were run in the presence (U) or absence (H)
of 20 mM DTT. Results of both coding and noncoding strands are
shown. C, control with free probe. The solid arrows point to the three G residues in the coding strand whose
methylation interferes strongly with binding; the open arrow points to the G residue in the noncoding strand that shows less
pronounced methylation interference. B, partial sequence of
the binding motif in the oxyR promoter. The sequence shows the three G
residues in the coding strand (closed stars) and the single G
residue in the noncoding strand (open star) that form contacts
with the binding protein.
The size of the murine
ORE-binding protein was determined by Southwestern blot analyses of
cytosolic and nuclear protein extracts from
HO
-treated or untreated cells. A protein with
an apparent molecular mass of 46 kDa was detected in cytosolic extracts
by interaction with a catenated ORE probe; in addition, two other minor
species with apparent sizes of 33 and 55 kDa, respectively, were
observed (Fig. 3A). In the nuclear fraction, proteins
with masses of 33-35, 46, 50, and 55 kDa were all very prominent (Fig. 3A). As with the gel retardation experiments,
there were no differences between binding activities in
H
O
-treated or untreated cells. The binding
specificity between fractionated proteins and the wild-type ORE probe
was confirmed by probing with a mutant ORE, which did not bind under
the conditions used to any of the proteins detected with the wild type (Fig. 3A).
Figure 3:
Molecular size determination of
ORE-binding proteins. A, Southwestern analyses. Nuclear (N) and cytosolic (C) extracts from
HO
-treated (+) and untreated (-) cells were prepared, and protein samples
(30-50 µg) were separated in 10% SDS-acrylamide gels and
transferred to an Immobilon membrane (Millipore). The probes used were
the wild-type (oxyR) and the mutant (mOxyR) ORE
oligonucleotides. B, UV cross-linking. Nuclear extracts were
allowed to interact with the ORE probe in the absence (-) or presence of competing unlabeled NF
B (
B) or OxyR (Ox) oligonucleotides. The sizes
indicated in kDa were determined by comparison with protein standards
run in the same gels.
UV cross-linking analyses with a
BrdUrd-substituted probe verified the physical interaction between the
ORE and a DNA-binding protein. A major DNA-protein complex was detected
that included a protein species with an apparent size of 46 kDa (Fig. 3B). UV cross-linking of this complex could be
almost abrogated by competition with an excess of unlabeled ORE
oligonucleotide, but not by an excess of an oligonucleotide containing
the NFB consensus binding site (Fig. 3B) or a
mutant ORE or an AP-1 binding site (data not shown). No cross-linking
was observed in the absence of UV irradiation (Fig. 3B)
or when a mutant ORE was used as the probe (not shown). The intensity
of the cross-linked band was considerably lower when the noncoding
strand was substituted with BrdUrd, suggesting that this strand is less
likely to make stable contact with the protein (data not shown). Since
only the 46-kDa polypeptide can be cross-linked to ORE, we conclude
that, of the six polypeptides detected by Southwestern analysis, the
one at 46-kDa is the one more likely to form a stable interaction with
ORE.
Figure 5: A Y-box protein recognizes the ORE. A, competition gel retardation. Prior to incubation with the ORE probe (OxyR) or the Y-box motif probe (Yb) nuclear extracts (5 µg) were incubated in the presence of increasing amounts (25-, 100-, and 300-fold excess) of competitor oligonucleotides with the wild-type ORE motif (OxyR), the mutant ORE sequence (mOxyR), or the Y-box motif (Yb). B, antibody supershift. Nuclear extracts were incubated with the indicated probes for 10 min and 1 µl of rabbit preimmune (PI) or immune (Im) serum was added and incubation continued for another 10 min. The double-headed arrow indicates the position of the supershifted band. C, Western and Southwestern analyses. oxi843 plasmid DNA was transcribed and translated in vitro; of the product was separated in 12.5% SDS-acrylamide gels (O), along with 30-50 µg of nuclear (N) and cytosolic (C) extracts from untreated Hepa-1 cells. For chemiluminescence detection, immune rabbit anti-Xenopus FRGY2 serum was used at a 1:4,000 dilution (lanes 1-3). For Southwestern analyses, the ORE probe was used as described in the legend to Fig. 3A. Lanes 4-6 were denatured in 6 M guanidine hydrochloride and subjected to graded renaturation; lanes 7-9 were the same ones used for the Western blot and were analyzed for ORE binding without denaturation and renaturation treatments.
To confirm that the ORE-binding protein in the nuclear extracts was indeed a Y-box family member, anti-Y-box antibodies were used to bind and displace the mobility of DNA-protein complexes in gel retardation assays. Immune rabbit anti-Xenopus FRGY2 antiserum supershifted both Y-box and ORE complexes, whereas the same amount of preimmune serum had no effect on the mobility of these complexes (Fig. 5B).
Western and Southwestern analyses were used to analyze the relationship between the protein encoded by the Oxi843 clone and the proteins in the DNA-protein complexes detected in extracts from Hepa-1 cells (Fig. 3A). In Western blots, immune anti-Xenopus antiserum clearly detected a major protein at approximately 46 kDa from nuclear and cytosolic Hepa-1 extracts as well as from the in vitro translation product of oxi843 (Fig. 5C, lanes 1-3). In a Southwestern blot of the same proteins, the ORE probe showed strong binding to the same 46-kDa protein (Fig. 5C, lanes 4-6), and, in addition, to the nuclear proteins in the 33- to 55-kDa size range observed previously. When the blot used for the Western analysis (Fig. 5C, lanes 1-3) was probed directly for ORE binding, omitting the denaturation and graded renaturation steps, only the 46- and the 33-kDa proteins gave strong signals, while the other additional proteins did not bind the probe (Fig. 5C, lanes 7-9). These results strongly suggest that oxi843 codes for the 46-kDa protein detected in nuclear and cytosolic cell extracts and that this protein is the one more likely to form stable complexes with ORE.
Figure 6:
Transient expression of ORE constructs in
the presence of anti-Y-box mRNA antisense oligonucleotides. Cells were
grown for at least three generations (72 h) in medium containing
oligonucleotides at the indicated concentrations. Thereafter, they were
seeded, transfected with pOxyRCAT and control pCMVgal plasmids,
and analyzed for transient expression. Y1, Y2, Y3, and Yc are described under ``Experimental Procedures.'' Ym refers to an equimolar mix of Y1, Y2, and Y3. Antisense
oligonucleotides were kept in the medium throughout transfection and
subsequent growth period prior to the assay. The values shown are the
mean ± S.E. of two experiments done in
duplicate.
The effect of the Y-box
protein on HO
-induced CAT expression might be
the result of an idiosyncratic phenomenon affecting the OxyR response
element and having no biological relevance in eukaryotic systems. To
address this question, the effect of peroxide treatment on the
transient expression of a luciferase reporter plasmid containing the
Y-box sequences from the human MHC class II HLA DQ
-2 gene was
determined. Expression of his reporter plasmid, termed pYbLuc, was
compared with expression directed by a similar pOxyRLuc reporter
plasmid carrying the OxyR response element instead of the Y-box. As
controls we used reporter constructs with mutated Y-box and ORE,
respectively. Luciferase induction after H
O
treatment showed identical dose-response curves, reaching a
plateau by 150 µM H
O
, for the two
wild-type constructs, whereas there was no effect on expression
directed by the mutated response elements (Fig. 7A).
The effect of H
O
was observed only if, after
treatment, cells were grown in unsupplemented culture medium; for both
constructs, inclusion of 140 µM 2-ME in the medium
abolished induction by peroxide, also decreasing by more than 10-fold
the basal expression level (Fig. 7B). These results
strongly suggest a role for the Y-box and Y-box proteins in eukaryotic
redox signaling.
Figure 7:
Redox effects on the transient expression
of Y-box reporter plasmids. A, HO
dose
response. Relative luciferase activity, normalized to the value in
untreated cells, is shown as a function of H
O
concentration for reporter plasmids containing Y-box (pYbLuc) and ORE (pOxyRLuc) motifs and their
corresponding mutant controls. Although the relative values can almost
be superimposed, the absolute values for pOxyRLuc were always
higher by a factor of 3-4-fold than the corresponding values for pYbLuc. B, 2-ME block. After treatment with 50 µM
H
O
, transfected cells were cultured in
unsupplemented medium (stippled) or in medium containing 140
µM 2-ME (filled). Luciferase activity values are
shown relative to those obtained in cells that were not treated with
either H
O
or 2-ME.
Our results show that the promoter of the bacterial oxyR gene functions in eukaryotic cells as an oxidative stress-responsive enhancer. At least one of the murine trans-acting factors that recognize this promoter has been cloned and is a member of the Y-box family of transcriptional activators. We show that the Y-box motif and Y-box proteins participate in redox-dependent gene expression based on three different criteria. First, peroxide-dependent activation of the reporter gene is blocked by antisense oligonucleotides to YB-1 mRNA. Second, a reporter gene carrying the Y-box motif responds to peroxide treatment as effectively as the reporter with the bacterial OxyR promoter. Third, the reducing agent 2-ME added to the culture medium abolishes the effect of peroxide.
The Y-box transcription
factors comprise a family of DNA- and RNA-binding proteins of molecular
sizes ranging from 36 to 56 kDa, conserved throughout evolution, from E. coli to humans (58, 59, 60, 62, 63, 64) .
Their DNA binding domain spans an 80-amino acid region near the
NH terminus, with a high degree of relatedness to the
prokaryotic cold shock response proteins CSPA and
CSPB(65, 66, 67) . It could be argued that
the Y-box transcription factors are not sequence-specific DNA-binding
proteins, yet the evidence to the contrary is overwhelming. In
eukaryotes, the cold shock domain plays an essential role in
sequence-specific DNA binding. Its recognition element is the Y-box, an
inverted CCAAT motif contained within the sequence GATTGGC common to
the promoter of many genes (Fig. 8), including all the MHC class
II genes, HSP70, histone H2B, c-jun, PCNA, herpes simplex
virus thymidine kinase, and DNA polymerase
, as well as DNA
elements in which the CCAAT motif is absent (see (60) and (63) for a review). More recently, a transcriptionally
functional Y-box has been found in the promoter of the cystic fibrosis
CFTR gene(68) . In the case of the ORE used in our experiments,
an imperfect Y-box is present in two motifs, ATAGGT and ATTGCT,
adjacent to the three G residues involved in methylation interference.
One of these motifs is identical to the sequence ATAGxt, and the other
matches it in 5-out-of-6 residues. This sequence has recently been
suggested as a potential consensus motif for oxidized OxyR
contacts(23, 24) . Similar imperfect Y-box motifs can
be found in the ahpFC and katG promoters (Fig. 8). In gel retardation experiments, canonical Y-box
oligonucleotides compete ORE binding and vice versa, and anti-FRGY2
antibodies supershift the ORE complexes, indicating that binding is
likely to involve these imperfect Y-box elements.
Figure 8: Alignment of Y-box elements and OxyR-binding promoters. References for the different Y-box sequences are given in the text. HSV tk* refers to a linker-scanning mutant of the HSV-1 tk gene promoter Y-box that has greater transcriptional activation activity than the wild type(71) .
The cold shock
domain also mediates the binding of Y-box proteins to mRNA molecules in
ribonucleoprotein storage
particles(60, 61, 63, 69) ; no clear
evidence exists of other physiologic functions for these proteins. Our
results strongly suggest that one likely role of Y-box proteins
involves redox-dependent gene activation. In this context, the
experiments with 2-ME-treated cells are particularly revealing.
Addition of 2-ME to the culture medium not only eliminates almost
completely the peroxide effect but also decreases expression to levels
well below those found in untreated Hepa-1 cells (Fig. 7B), suggesting that even in the absence of
HO
treatment, these cells are under
considerable oxidative stress, which is relieved by the presence of
2-ME. We would predict that the activity of the Y-box proteins might
depend on the oxidant status of the cell, and by extension, on cell
lineage and density, culture conditions, medium, and many other
factors. Preliminary experiments using other cell lines suggest that
this may in fact be the case. (
)
Little is known as to the activation mechanisms that regulate Y-box protein activity. Many recent studies indicate that redox cycling at cysteine residues regulates in part the DNA binding activity of several mammalian transcription factors(29, 30, 31, 32, 33, 34, 35, 70) . Although other amino acids may be capable of redox cycling, the lack of cysteine residues in Y-box proteins ( Fig. 4and (58, 59, 60, 61, 62, 63) ) is further indication that the actual oxidative stress sensor might be a different, as yet undetected, protein. If this were the case, Y-box proteins could be constitutively bound to their response element regardless of the prooxidant status of the cell, serving as a scaffold for the assembly of other factors, including the putative redox-sensing protein. Such a scaffolding role has been proposed for the region of alternating acidic and basic amino acid modules in the carboxyl-terminal third of the protein(60, 63) . This hypothesis would explain the absence of apparent changes in methylation interference patterns of treated and untreated extracts. Alternatively, methylation interference may be inadequate to detect subtle changes in DNA-protein contacts which might activate transcription.
In prokaryotes, the OxyR protein functions as a sensor, as well as a transcriptional activator/silencer that binds DNA and responds to hydroxyl radicals. In eukaryotes, the redox-sensing mechanism, transcriptional activation, and DNA binding may be functions performed by different proteins. There is little sequence relatedness between OxyR and Y-box proteins, indicating that they are unlikely to be evolutionarily related. It is striking, however, that some of the elements of the regulatory pathway that governs the response to oxidative stress appear to be shared by prokaryotic and eukaryotic organisms.