(Received for publication, March 19, 1997, and in revised form, March 31, 1997)
From the Laboratory of Biochemistry, Transcription factor PEBP2/CBF consists of a DNA
binding subunit, Polyoma virus enhancer-binding protein 2 (PEBP2)1 (1), also called core binding
factor (2), is a heterodimeric transcription factor composed of two
different subunits, In mammals, PEBP2 has been implicated in the transcriptional regulation
of lymphoid cell-specific genes such as T-cell receptors, CD3, and
myeloperoxidase, neutrophil elastase, granulocyte/macrophage colony-stimulating factor, macrophage colony-stimulating factor receptors, interleukin-3, and granzyme B (for review, see Ref. 10).
Chromosomal translocations involving the human AML1 gene, such as t(8;21), t(3;21), and t(12;21), lead to various types of
leukemia including acute myeloid leukemia, the blast crisis of chronic
myeloid leukemia, and B-lineage acute lymphoblastic leukemia,
respectively (for review, see Refs. 10 and 11). Moreover, an inversion
in human chromosome 16 that gives rise to a fusion product,
PEBP2 Our previous functional characterization of PEBP2 (6) has revealed that
the DNA binding activity of the Runt domain is subject to regulation by
a reduction/oxidation-dependent mechanism (redox
regulation). Since the finding of human thioredoxin/adult T-cell
leukemia-derived factor (15), the importance of the thiol-mediated redox regulation has been well recognized in various biological responses (16, 17). Particularly notable are accumulating examples of
redox-responsive transcription factors, such as the AP-1 family (18,
19), the Rel family (20-22), Myb (23), Ets-1 (24), and p53 (25). In
these transcription factors, the reduced state of cysteine in the
DNA-binding domain is essential for their DNA binding. Coincidentally,
the Runt domain in all three mammalian submembers of the Plasmid pQE-RD (6) encoding
the Runt domain of PEBP2
His-tagged
derivatives of the Runt domain and the A DNA probe
containing the wild type PEBP2 binding site was prepared as described
(6). The DNA binding reaction was routinely carried out for 10 min at
25 °C in 10 µl of the EMSA buffer containing 20 mM
HEPES-KOH (pH 8.0), 4% (w/v) Ficoll 400, 2 mM EDTA, 100 mM KCl, 0.1 µg of poly(dI-dC), 6% glycerol, 0.2 mg/ml
bovine serum albumin, 0.04% bromphenol blue, 10 fmol of
32P-labeled probe, 5 ng of purified His-tagged Runt domain,
and the The two conserved cysteine residues in the Runt domain of
PEBP2 We then used the three serine mutants together with the wild type to
evaluate the susceptibilities of the two cysteine residues to a thiol
oxidizing reagent, diamide (Fig. 2A). Fig.
2B shows changes in the relative DNA binding activity of
these constructs after their treatment with increasing concentrations
of diamide relative to the respective mock-treated controls. In the
absence of the
In Fig. 2B are also indicated changes in the overall -fold
stimulation of DNA binding by the We further addressed the question of what cellular component(s) could
serve for reductive activation of the Runt domain. Three known factors
were tested as candidates: glutathione (GSH), Trx/ADF, and Ref-1.
Trx/ADF is known to activate transcription factor NF-
The present mutational study has demonstrated that both of the two
cysteine residues within the Runt domain are responsible for redox
regulation in their own ways, which are unique in comparison with known
other redox-responsive transcription factors. Cys-124 apparently
resembles the redox-responsive cysteine identified in Jun and Fos (18)
in its high oxidizability due to flanking basic amino acids (Fig.
1A; see also Ref. 30) and augmented DNA binding by its
substitution to serine. In Jun and Fos, conversion of cysteine to
oxidation products of acidic nature is supposed to confer negative
effects on DNA binding. A similar scenario would likely apply to
Cys-124 in the Runt domain. Conversely, the lesser redox sensitivity of
Cys-115 is likely attributable to the absence of any basic amino acid
in its vicinity (Fig. 1A). Nevertheless, the reduced DNA
binding activity caused by its substitution to serine as well as
aspartate suggests that the free sulfhydryl group per se
might be functionally essential. Similar mutational effects have
previously been reported for NF- In extension of our previous observation (6), the results of this study
also highlighted the potential significance of the The finding that Trx and Ref-1 can efficiently activate the Runt domain
in a manner synergistic with each other and enforceable by the While this work was in progress, in vivo evidence
underscoring the functional importance of cysteine residues in the Runt domain protein was provided by the report (35) that the transactivation and cell-transforming potentials of AML1/PEBP2 Worth noting finally is the pattern of evolutionary conservation of
cysteine in the Runt domain proteins. This protein family has been
identified in a wide range of animal species from Caenorhabditis elegans (deduced from the C. elegans genomic data
base)2 to mammals, and shown thus far to
contain at least three subclasses in mammals (8-10) and two in
Drosophila (Runt (3) and Lozenge (36)). As indicated in Fig.
4, all the members identified in species classified to
deuterostomia including sea urchin (37), chicken (38), and
mammals (8-10) share the two conserved cysteine residues at positions
115 and 124. On the other hand, those found in Drosophila
and C. elegans, which belong to protostomia, have one or both of these cysteines altered to serine or tyrosine. Discernible also is an opposite change from cysteine to serine at
position 157 in an apparent association with the advent of vertebrate.
It thus seems that there were a number of possible ways in utilizing
cysteine to control DNA binding by the Runt domain and nature had
shifted its choices among them at major steps of evolutionary
divergence in the animal kingdom. Further comparative studies of the
Runt domain proteins are awaited to explore the potential evolutionary
implications of its redox regulation in full scope.
We thank Dr. Thomas Bürglin (Basel
University) for providing the deduced sequence of C. elegans
Runt homolog.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
, and a regulatory subunit,
. The
subunit
has an evolutionarily conserved 128-amino acid region termed "Runt
domain" that is responsible for both DNA binding and
heterodimerization with the
subunit. The Runt domain in all
mammalian submembers of the
subunit contains two conserved cysteine
residues, and its DNA binding activity undergoes redox regulation. To
investigate the mechanism of this redox regulation, we performed
site-directed mutagenesis of the two conserved cysteines in the Runt
domain of the mouse PEBP2
A homolog. Substitution of Cys-115 to
serine resulted in a partially impaired DNA binding, which remained
highly sensitive to a thiol-oxidizing reagent, diamide. Conversely, the
corresponding substitution of Cys-124 caused an increased DNA binding
concomitant with an increased resistance to diamide. In contrast,
substitution of either cysteine to aspartate was destructive to DNA
binding to marked extents. These results have revealed that both
Cys-115 and Cys-124 are responsible for the redox regulation in their
own ways with low and high oxidizabilities, respectively. We have also
found that two cellular thiol-reactive proteins, thioredoxin and Ref-1,
work effectively and synergistically for activation of the Runt domain. Interestingly, the
subunit further enhanced the activation by these
proteins and reciprocally prevented the oxidative inactivation by
diamide. These findings collectively suggest the possibility that the
Runt domain's function in vivo could be dynamically
regulated by the redox mechanism with Trx, Ref-1, and the
subunit
as key modulators.
and
. The
subunit binds directly to a
specific DNA sequence, RACCRCA, while the
subunit does not by
itself contact with DNA but facilitates the DNA binding activity of the
subunit through allosteric interactions (1, 2). The
subunit
shares a 128-amino acid region of high homology with the
Drosophila segmentation gene runt (3) and the
human AML1 gene (4). The conserved region, termed "Runt domain," is responsible for both DNA binding and heterodimerization with the
subunit (5, 6). After the identification of the first
member of the
subunit, PEBP2
A (1), two additional members of the
Runt family have subsequently been isolated in mice and humans:
PEBP2
B (mouse homolog of AML1) (7), and PEBP2
C (8) or AML2 (9).
-SMMHC (smooth muscle myosin heavy chain), was also found to
cause a M4Eo subtype acute myeloid leukemia (12). Recent gene
disruption studies in mice of AML1 (13) as well as
PEBP2/CBF
(14) have confirmed that these genes are essential for
definitive hematopoiesis.
subunit
contain two cysteine residues that are perfectly conserved among them
(8). Our recent random mutagenesis study with the Runt domain of
PEBP2
A (26) has suggested that one target for its redox regulation
should be Cys-124, because substitution of this residue to serine
resulted in an enhanced DNA binding concomitant with decreased
redox-dependence. However, the possibility remains that the other
cysteine residue, Cys-115, could also be redox-responsive. To further
define the potential roles of the two conserved cysteine residues in
the redox regulation of PEBP2, we performed their site-directed
mutagenesis and examined the DNA binding ability of resulting mutants
under various redox conditions in vitro.
Mutagenesis of the Runt Domain
A with an N-terminal hexahistidine tag (for
its structure, see Fig. 1A) was used as a vector for
construction and overexpression of Runt domain mutants. Each or both of
Cys-115 and Cys-124 were mutagenized to serine or aspartate by
polymerase chain reactions using Vent DNA polymerase (New England
Biolabs) as described (27). The substitutions of the targeted residues
were confirmed by sequencing.
Fig. 1.
DNA binding activities of the wild type and
its six cysteine substitution mutants. A, structures of
PEBP2A (top) and its hexahistidine-tagged Runt domain
derivative (bottom). The two conserved cysteines (Cys-115
and Cys-124) and their flanking amino acids are shown in the
box representing the Runt domain. The cysteines were changed
to serine (S) or aspartate (D) as indicated. The
arrows indicate the minimum regions responsible for DNA
binding and heterodimerization with the
subunit. B, EMSA
patterns of the Runt domain derivatives in the presence and absence of
the
subunit as indicated. See "Results" for the notation of
each construct. The bands were visualized and quantitated by a
phosphorimager. Open, shaded, and solid
arrows indicate the free DNA probe, the Runt domain-DNA complex,
and the heterodimer-DNA complex, respectively. C, comparison
of the DNA binding activities (% DNA bound) as measured above by EMSA.
Open and solid bars stand for the activities
obtained in the absence and presence of the
subunit, respectively.
Striped bars indicate -fold stimulation by
subunit.
[View Larger Version of this Image (28K GIF file)]
subunit were expressed in
Escherichia coli and purified on a nickel-nitrilotriacetic acid resin (QIAGEN) as described (6). Trx was purchased from Ajinomoto.
TrxR was purified as described (28). Ref-1 was purified as described
(29).
subunit (50 ng) where indicated. In some experiments, the Runt domain was pretreated with redox reagents such as diamide, DTT,
Trx, and Ref-1. The reaction mixture was loaded on a 10% nondenaturing
polyacrylamide gel (acrylamide:bisacrylamide, 39:1) in 0.25 × Tris borate-EDTA buffer and electrophoresed at room temperature. Gels
were dried and visualized by a phosphorimager (Fujifilm BAS 2000) or
autoradiography with x-ray films.
A to redox regulation were substituted to serine or aspartate, either separately or simultaneously (Fig.
1A). In the following, the resultant mutants
are denoted by abbreviations in the form XY, in which the
first and second letters represent amino acids in one-letter codes that
replace Cys-115 and Cys-124, respectively. All the serine mutants
showed readily detectable DNA binding, although those with Cys-115
substituted to serine (SC and SS) were severalfold less active than the
wild type (CC) in the absence of the
subunit (Fig. 1B,
left panel; see also Fig. 1C for quantitative comparison). In confirmation of our previous observation (26), mutant
CS displayed stronger than normal DNA binding. On the other hand, the
aspartate mutants invariably showed drastically impaired DNA binding,
which was virtually undetectable in DC and DD, and barely recognizable
in CD even after a prolonged exposure for autoradiography (Fig.
1B, right panel). When the
subunit was present, the wild type and all the mutants with any recognizable DNA
binding activity gave supershifted DNA bands, whose intensities, except
for CS, were prominently increased over those observed without the
subunit. This indicates that substitution of either cysteine residue to
serine or aspartate is tolerable for the heterodimerization between the
Runt domain and the
subunit as well as their allosteric regulatory
interaction.
subunit (open circles), SC remained
nearly as sensitive as the wild type to diamide, showing more than 75%
inhibition at 1 mM. In contrast, CS was only weakly
affected (25% inhibition) at 1 mM. However, the activity
of this mutant was progressively and extensively decreased with further
increments of diamide up to 100 mM. Thus we conclude that
Cys-115 is also responsive to redox regulation, although being much
less sensitive than Cys-124. When the
subunit was present, all
variants having one or both cysteine residues showed decreased
sensitivities to diamide. CC and SC remained 90% active at 1 mM, although they became drastically inactivated at higher
concentrations; CS was only moderately inhibited even at 100 mM. Thus, the
subunit appeared to render both Cys-115 and Cys-124 tolerable to proportionately increased dosages of diamide
by one order of magnitude or more. No such
subunit-dependent protection was observed with the
cysteine-less mutant, SS, as expected.
Fig. 2.
Effects of varying concentrations of diamide
on the DNA binding activities of the wild type and serine-substituted
Runt domain mutants. A, EMSA patterns of the respective
proteins pretreated with indicated concentrations of diamide in the
presence (right) and absence (left) of the subunit at 25 °C for 10 min. Only the region covering shifted bands
is shown. Notations and symbols are the same as in Fig. 1B.
B, relative diamide sensitivities of the respective
variants. The DNA binding activities of each construct measured above
in the absence (open circles) and presence (solid
circles) of the
subunit are replotted after normalization to
the respective control values obtained with no added diamide. Crosses on dotted lines show -fold stimulation by
the
subunit.
[View Larger Version of this Image (44K GIF file)]
subunit with the diamide
concentration for each Runt domain construct (crosses on
dotted lines). Note first that SS showed moderate
stimulation (severalfold), which is almost invariant with the diamide
concentration and hence supposed to represent the redox-independent
regulatory action of the
subunit in augmenting the intrinsic
affinity of the Runt domain for DNA as previously defined by Kagoshima
et al. (6). By contrast, CC and SC exhibited much greater
stimulation than SS, attaining a peak of 30-fold or more at 1 mM diamide. This enormous stimulation over that observed
with SS is taken to reflect the above-noted anti-oxidation effect of
the
subunit on Cys-124 as target. Although the -fold stimulation of
CS was comparatively very low, it still showed a gradual rise with
increasing concentrations of diamide, eventually surpassing that of SS
at 100 mM. Evidently, therefore, Cys-115 is also subject to
the protective action of the
subunit.
B under the
cooperation with TrxR and NADPH (15, 16, 28). Ref-1 (19) has been
identified as a nuclear factor that stimulates the DNA binding activity
of transcription factor AP-1. Ref-1, in turn, can be readily reduced by
Trx (19). As shown in Fig. 3A, GSH was
virtually inert in activating the Runt domain in the absence of the
subunit. When the
subunit was present, GSH showed moderate
activation at low concentrations (1-10 µM: lanes 4 and 6), but it became inhibitory at 0.1 mM or above. In contrast, Trx consistently showed
substantial activation at micromolar levels (Fig. 3B). Ref-1
was even more potent than Trx with still lesser concentrations (0.5-50
nM, Fig. 3C). Interestingly, the
subunit tended to stimulate the activation of the Runt domain by these proteins, particularly Ref-1 (Fig. 3C, even-numbered
lanes). Possible mechanistic and regulatory implications of this
effect will be given in "Discussion." Furthermore, Trx and Ref-1
were found to act synergistically with each other. When preoxidized CC
was incubated with either of them alone supplemented with a minimum
amount of DTT to quench the residual diamide, no or only marginal DNA
binding was observed. In combination, however, they restored the DNA
binding as effectively as 100 mM DTT (compare lanes
2 and 5). The effect of Trx was also improved
considerably by its co-incubation with TrxR and NADPH (lane
6). In the presence of this coupled reducing system, the
supplementary DTT could be omitted with only a moderate reduction in
DNA binding (lane 7), which was again fully restored by the
addition of Ref-1 (lane 8).
Fig. 3.
Effects of various cellular thiol-reducing
factors on the DNA binding activity of the Runt domain. In
A-C, the wild type Runt domain was subjected to EMSA after
co-incubation with increasing concentrations of indicated factors in
the presence (even-numbered lanes) and absence
(odd-numbered lanes) of the subunit. A (GSH),
lanes 1 and 2, none; lanes 3 and
4, 1 µM; lanes 5 and 6,
10 µM; lanes 7 and 8, 0.1 mM; lanes 9 and 10, 1 mM.
B (Trx/ADF), lanes 1 and 2, none;
lanes 3 and 4, 0.2 µM; lanes 5 and 6, 2 µM; lanes 7 and
8, 20 µM. C (Ref-1), lanes
1 and 2, none; lanes 3 and 4, 0.5 nM; lanes 5 and 6, 5 nM;
lanes 7 and 8, 50 nM. D,
CC was preoxidized with 1 mM diamide, treated with indicated thiol-reducing reagents either separately or simultaneously, and then subjected to EMSA: lane 1, 0.4 mM DTT;
lane 2, 100 mM DTT; lane 3, 0.4 mM DTT and 20 µM Trx; lane 4, 0.4 mM DTT and 1 µM Ref-1; lane 5, 0.4 mM DTT, 20 µM Trx, and 1 µM
Ref-1; lane 6, 0.4 mM DTT and Trx/TrxR system
(20 µM Trx together with 5 × 104 units
of TrxR and 1 mM NADPH); lane 7, Trx/TrxR system
with no DTT; lane 8, Trx/TrxR system and 1 µM
Ref-1. The EMSA pattern was visualized by autoradiography with x-ray
films in A-C or by a phosphorimager in D.
Shaded and solid arrows on the left
indicate shifted bands due to the Runt domain alone and the Runt
domain-
subunit heterodimer, respectively.
[View Larger Version of this Image (56K GIF file)]
B (21), Myb (23), and Ets-1 (24).
Indeed, the x-ray structural analysis of NF-
B has demonstrated
direct contacts of the free sulfhydryl group of cysteine with its
target DNA (31). A priori, the two cysteines in the Runt
domain might form a reversible disulfide bridge, as reported for p53
(25). However, their differential redox susceptibilities as observed
rather favor the view that the two cysteines undergo redox reactions
independently from each other. Such a two-pronged mechanism would
enable more flexible regulation of DNA binding in response to diverse
cellular redox signals than otherwise.
subunit as a
modulator of redox sensitivity of the Runt domain, in addition to its
role in allosterically enhancing the intrinsic DNA binding affinity of
the Runt domain. The
subunit acted to protect both cysteines from
oxidation by diamide. Puzzlingly, however, the
subunit did not
hinder the access of the Runt domain by much larger molecules, Trx and
Ref-1. How then was the
subunit able to block the action of
diamide? We infer that the intrinsic oxidizability of these cysteine
residues could be modulated by
subunit-induced alterations in the
local environment around them in terms of their spatial alignment with
neighboring charged amino acids or their involvement in intra- or
inter-molecular hydrogen bonding, rather than a simple steric
hindrance.
subunit raises the question of where and how these protein factors
interact with each other within the cell. While the
subunit is a
nuclear protein, the
subunit by itself is localized in the
cytoplasm and transported to the nucleus through association with the
subunit (32). Available evidence (6, 32) has suggested that the
heteromeric interaction of the
subunit with the intact
subunit
is somewhat restricted both in vitro and in vivo
by a putative intramolecular interference between the Runt domain and
its flanking regions and that a specific mechanism would exist to
promote this heterodimerization. Likewise, Ref-1 is a nuclear protein
(19), whereas Trx is normally localized in the cytoplasm but is
mobilized into the nucleus upon exposure of cells to various chemical
and physical stimuli such as phorbol ester and ultraviolet (UV)
irradiation (33). The expression of Trx also can be strongly induced by
various oxidative stresses (34). In the light of these observations, it
is tempting to speculate that the DNA binding activity of PEBP2 would
be regulated by dynamic nuclear trafficking of the
subunit and Trx
in response to oxidative stresses.
B were markedly weakened by substitution of serine for Cys-72 (corresponding to Cys-115
in PEBP2
A). To further delineate the dynamics and significance of
redox regulation of PEBP2 to their full extents, it should be
informative to extend such functional studies using a wider variety of
mutants as constructed here in combination with various conditions
affecting the cellular redox state.
Fig. 4.
Phylogenetic comparison of the pattern of
distribution of cysteine residues in the Runt domain. The amino
acid sequences of Runt homologs identified to date in the indicated
metazoan phyla were aligned together, and their amino acids are listed up for the positions within the Runt domain (numbered after PEBP2A) at which cysteine was found in at least one of the homologs. See "Discussion" for the source of each sequence.
[View Larger Version of this Image (14K GIF file)]
*
This work was supported in part by Research Grant RG-357/94
(to K. S.) from the Human Frontier Science Program Organization (Strasbourg) and by Research Project 08NP0601 (to J. Y.) under the
"New Program System" of the Ministry of Education, Science, Culture
and Sports of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a research fellowship from the Japan Society for the
Promotion of Science for Young Scientists. Present address: Dept. of
Molecular Biology, Massachusetts General Hospital, 50 Blossom St.,
Boston, MA 02114.
§
The first two authors contributed to this work equally.
Present address: Biozentrum, der Universität Basel,
Abteilung Zellbiologie, Klingelbergstr. 70, CH-4056 Basel,
Switzerland.
**
To whom correspondence should be addressed. Tel.: 81-75-751-4019;
Fax: 81-75-751-3992; E-mail: kshigesa{at}virus.kyoto-u.ac.jp.
1
The abbreviations used are: PEBP, polyoma virus
enhancer-binding protein; Trx, thioredoxin; TrxR, thioredoxin
reductase; EMSA, electrophoretic mobility shift assay; DTT,
dithiothreitol.
2
H. Kagoshima and T. Bürglin, personal
communication.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.