From the
The DNA binding activity of the thyroid transcription factor-1
(TTF-1), a homeodomain-containing protein implicated in the control of
thyroid- and lung-specific transcription, is controlled, in
vitro, by the redox potential. Oxidation decreases TTF-1 DNA
binding activity, which is fully restored upon exposure to reducing
agents. The decrease in DNA binding activity is due to the formation of
disulfide bond(s), formed between two specific cysteine residues
located outside the TTF-1 homeodomain; hence, oxidation does not appear
to directly hinder TTF-1/DNA contacts. Disulfide bond formation seems
to stabilize preexisting, loosely associated, TTF-1 dimers, which, upon
oxidation, proceed in the formation of specific, higher order
oligomers.
Gene-specific transcriptional regulation can be achieved by
modulating either the amount or the activity of specific transcription
factors. Mechanisms capable of regulating the transcriptional potential
of transcription factors, with no changes in their intracellular
concentration, rely largely on post-translational modification, either
of the factors themselves or of associated proteins that affect their
activity. Post-translational modification, such as phosphorylation, can
affect either the activation or the DNA-binding domain of transcription
factors. Recently, redox potential has been proposed as a regulatory
mechanism capable of modifying the DNA binding activity of
transcription factors
(1, 2) . In the case of the
bacterial OxyR protein, oxidation produces a change in DNA binding
specificity
(3) , while in the case of all other proteins
studied, it is the overall DNA binding activity that is influenced by
the redox potential of the
medium
(4, 5, 6, 7, 8, 9, 10, 11) .
The redox-mediated regulation of DNA binding activity follows, in
several instances, a quite straightforward mechanism since the residues
affected by oxidation are cysteines located in the DNA-binding domain
of the protein. In these cases it is very likely that oxidation
directly influences DNA-protein contacts. In only one report, it has
clearly been shown that oxidation of cysteines located outside the
DNA-binding domain can also influence the DNA binding activity of the
transcription factor
(7) .
Homeodomain-containing proteins
(12) can also be regulated by redox, either directly via
oxidation of cysteines in their homeodomain
(8) or indirectly,
as for example in the case of HoxB5 where oxidation of extra
homeodomain cysteines affects the ability of the protein to bind
cooperatively to DNA
(5) . TTF-1 is a homeodomain-containing
protein (13) for which several indirect studies suggest that its
ability to activate transcription is subjected to regulation. Indeed,
TTF-1 has been implicated in the activation of both
thyroid-
(14, 15) and lung- (16) specific promoters, in
their respective cell type. Furthermore, during thyroid development,
TTF-1 protein appears 5 days before the onset of transcription of its
target genes
(17) and also in a Ha-ras-transformed cell
type the presence of inactive TTF-1 as been reported
(18) .
Here we show that the DNA binding potential of TTF-1 is redox
sensitive. This regulation appears to be indirect, since no cysteines
are present in the TTF-1 homeodomain and, moreover, replacement of two
cysteines located outside the homeodomain with serines transforms TTF-1
into an oxidation-insensitive DNA-binding protein. In the course of
these studies, we have also discovered that TTF-1 is able to dimerize.
Dimerization is not a common feature among homeodomain-containing
proteins and might have a role in specifying the different functions
that TTF-1 is able to perform.
Purified, recombinant, TTF-1 HD
was prepared as described previously (21).
The molecular state of TTF-1 was analyzed
also under native conditions. To this end, rate-zonal centrifugation
through preformed glycerol gradients was employed. The distribution of
the protein in such gradients was revealed by assaying the DNA binding
activity of each fraction under fully reducing conditions. Since the
diamide-induced loss of DNA binding activity is fully reversible
(Fig. 1), we assume that the measured binding activity reflects
the distribution of TTF-1 throughout the gradient. A typical
sedimentation profile obtained in the presence of 5 mM DTT or
5 mM diamide is shown in Fig. 5A. The majority
of TTF-1 protein, under fully reducing conditions, sediments as a
monomer. In the presence of diamide, two peaks can be observed: one
co-migrating with the fully reduced monomer and a shoulder
corresponding to a dimeric form. The total binding activity recovered
from the gradient under oxidizing conditions is almost half of that
obtained under reducing conditions. The formation of high molecular
weight aggregates could account for this reduction. In fact, in the
presence of diamide, a large amount of protein was found at the bottom
of the tube, while no protein pellet was found under reducing
conditions (Fig. 5B). However, no other species were
observed in the intermediate molecular mass range (i.e. between 100 and 600 kDa), in contrast to what seen by SDS-PAGE,
where both tetrameric and hexameric forms of TTF-1 were detected in the
presence of diamide (Fig. 4, lane1). We
suggest that the oxidation-induced, covalent TTF-1 multimers (see
Fig. 4
, lane1) establish noncovalent
interactions to form higher molecular weight aggregates. Such
aggregates are resolved by SDS and hence are not visible in the
experiment shown in Fig. 4.
Aggregates,
although to a lower extent, are also observed under reducing
conditions, as demonstrated by the presence of a broad peak in the
sedimentation profile shown in Fig. 5A. These
noncovalent aggregates can be resolved by the addition of nonionic
detergents, such as Nonidet P-40. Under these conditions, only the
monomeric form was observed (Fig. 5D). Furthermore, in
the presence of Nonidet P-40, oxidizing reagents fail to induce the
formation of covalent multimers (compare the profiles shown in
Fig. 5
, A and D), thus indicating that the
occurrence of non-covalent associations is necessary for the formation
of disulfide bridges.
Redox regulation
is a mechanism of transcriptional control that in prokaryotic systems
operates primarily in response to oxidative stress (1-3), while
it has been proposed as a more general eukaryotic mechanism for
transcription regulation by a rapid and reversible post-translational
modification of DNA-binding proteins
(7) . Our data show that
TTF-1 binding activity is sensitive in vitro to the redox
environment. However, we do not know at present if the redox regulation
observed in vitro could also play a role in vivo.
The data reported in this paper also show that TTF-1 is capable of
forming dimers in solution, both under reducing and oxidizing
conditions. The existence of dimers is not unique among homeodomain
(HD)-containing proteins. In fact, although the 60-amino acid
homeodomain of several transcription factors has been demonstrated to
bind to DNA as a
monomer
(28, 29, 30, 31) , several
HD-containing proteins have been shown to form dimers. Among them, two
major groups can be distinguished: (i) those homeoproteins that exist
in solution as dimers and (ii) those that are monomers in solution but
exhibit cooperative dimerization on the DNA. To the first group belong
the HNF-1 protein (32, 33), the yeast a1 and
The
behavior shown by TTF-1 falls in between the above-cited situations. In
fact, even though TTF-1 may exist in solution both as monomer and as
dimer, its oligomeric state strongly depends on the redox potential of
the environment, but it is not influenced by the presence of its
binding site, to which TTF-1 has been shown to bind as a
monomer
(13) . The ability of TTF-1 to dimerize raises the
question of whether dimerization per se has an important role
for its activity. In two thyroid-specific promoters, namely the
thyroglobulin and thyroperoxidase promoters, the existence of three
TTF-1-binding sites, 40-50 bp apart, has been demonstrated by
DNase I footprinting experiments
(14, 15) . However, in
the thyroglobulin proximal site, a palindromic motif can be observed,
with each half-site having at least 60% identity with the TTF-1
consensus sequence
(42) (Fig. 7). Interestingly, these
half-sites are separated by 10 bp (i.e. approximately one
helical turn of DNA) and may determine a functional apposition of TTF-1
molecules on the same face of the DNA helix (Fig. 7). Such an
arrangement of sites is not found in the proximal site of the
thyroperoxidase promoter (Fig. 7), from which transcription is
indeed only weakly stimulated by TTF-1
(43) . Furthermore, TTF-1
has been found to activate transcription from the promoter of the
lung-specific surfactant protein B gene
(16) . This promoter also
contains two closely spaced TTF-1-binding sites (Fig. 7). Hence,
it seems conceivable that dimerization of TTF-1 could play an important
role in transactivation from the thyroglobulin and surfactant protein B
promoters; alternatively, different dimeric forms of TTF-1, for which
we have provided evidences in this paper, could modulate transcription
from specific promoters in a cell type-dependent fashion. We have not
addressed in this study the issue of whether and where TTF-1 oligomers
occur in vivo and if they are covalently linked. Disulfide
stabilized dimers have been observed also in other systems, as in the
cases of human HoxB5 (5), and in the C/EBP-related family
(44) .
Dimer stabilization can also be mediated by cofactors, as shown
recently for some homeodomain-containing
proteins
(45, 46) . In this respect, the recent finding
that the E2A protein can form disulfide stabilized homodimers that are
detected (when the assay is performed at physiological temperature)
only in B cells is of great interest. Reduction of the disulfide bond
in E2A is achieved by an as yet not characterized, albeit specific,
enzymatic activity, which is probably limiting in B cells
(47) .
These observation stress the notion that redox control of transcription
factor could be involved in specific and quite complex regulation, such
as the cell type-specific selection of target promoters. It is then
conceivable that a specific redox activity or a specific cofactor may
be responsible for the stabilization of alternative TTF-1 oligomers,
with a consequent modulation of its target preference perhaps depending
on a specific cellular environment. Further investigations will focus
on this opportunity and on whether these covalent dimeric species are
required for specific transactivation by TTF-1.
We thank V. Cubellis and A. Papavassiliou for critical
reading of the manuscript, M. De Felice for helpful discussions, and H.
Stunnenberg for advice with vaccinia virus-related work. We also thank
G. Theil for a gift of purified, recombinant TTF-1 HD and G. de
Martinoff for a gift of pMS53/56 recombinant vector.
Purification of TTF-1 Protein and of Recombinant TTF-1
Homeodomain
HeLa spinner cells (about 5 10
cells/ml) were infected with a vaccinia virus containing the
entire open reading frame of TTF-1
(13) , using the method
described
(19) . In brief, the 1.2-kilobase cDNA coding for rat
TTF-1 protein was fused downstream to the 11K vaccinia late promoter,
in the recombinant vector pMS53/56 (kindly provided by G. de
Martinoff). The plasmid was then used to prepare a recombinant virus as
described previously
(20) . The nuclear extract from 5 liters of
infected cells was prepared as indicated
(14) . The extract (17
ml) was loaded on a blue Sepharose (Pharmacia Biotech Inc.) column (1
20 cm) previously equilibrated in buffer D (20 mM
Hepes, pH 7.9, 0.3 M KCl, 10% glycerol, 0.1 mM EDTA,
0.5 mM DTT
(
)
); the column was washed
with 4 volumes of the same buffer and eluted with buffer F (20
mM Tris-HCl, pH 8.5, 10% glycerol, 0.1 mM EDTA, 0.5
mM phenylmethylsulfonyl fluoride, 0.5 mM DTT, 0.02%
Nonidet P-40) containing 1 M KCl. Active fractions (as
assessed by gel shift assay) were pooled, poly(dI
dC) was added to
a final concentration of 10 µg/ml, and the sample was loaded on
oligonucleotide C
(14) affinity column (about 8 ml of resin,
obtained coupling a DNA concatenamer containing multiple copies of the
oligonucleotide C with CNBr-activated Sepharose) previously
equilibrated in buffer F with 0.2 M KCl. The column was washed
first with 2 volumes of equilibration buffer containing 2.5 µg/ml
poly(dI
dC) and then with 2 volumes of same buffer without
poly(dI
dC); proteins were eluted with a 0.2-1.5 M
linear KCl gradient in buffer F. The active fractions were pooled,
poly(dI
dC) was added to a final concentration of 2.5 µg/ml,
and the affinity step was repeated.
Mutant Preparation
In TTF-1 SCCC, CSCC, CCSC, and
CCCS mutants, the serine residue was substituted for cysteine by the
polymerase chain reaction overlap extension method
(22) , using
Start (5`-CCCGGGAAGCTTCTCCACTCAAGCCAATTAAGGCGG) and End
(5`-GCGCGCTCTAGAGAGCAGCGGGCGAATGGTGG) oligonucleotides as flanking
primers on a TTF-1 expression vector
(18) used as template. The
amplified fragments were digested with HindIII and
XbaI, subcloned into the same vector
(18) , and checked
by sequencing. TTF-1 CSSC was obtained as above using TTF-1 CSCC as
template. TTF-1 SCCS was constructed by EcoO109I digestion of
TTF-1 CCCS and fragment ligation to TTF-1 SCCC. TTF-1 SSSC and CSSS
were constructed by BstXI-BglII digestion of TTF-1
CSSC and fragment ligation to TTF-1 SCCC and CCCS, respectively. TTF-1
SSSS was obtained subcloning the fragment BstXI-XbaI
from TTF-1-CCCS into TTF-1 SSSC. Fidelity of the constructs was
verified by restriction mapping and sequencing of the junctions. The
proteins were expressed in HeLa cells by transfecting 20 µg of each
plasmid to approximately 10 cells in 100-mm tissue culture
dishes. Cell culture and transfection procedures are
described
(23) . Nuclear extracts were prepared as indicated
below for FRTL-5 cells
(24) .
Nuclear Extract Preparation and Gel Shift
Assays
FRTL-5 cells were cultured as described previously.
Nuclear extracts were prepared as described previously
(14) except for the absence of DTT, which was added only where
indicated. Gel retardation assays were performed with the indicated
amount of the purified proteins or of FRTL-5 and HeLa nuclear extracts
using the oligonucleotide C probe described previously
(14) .
Binding reactions were assembled in 20 mM Tris-HCl, pH 7.5,
200 mM KCl, 10% glycerol containing 20 µg bovine serum
albumin, and 0.3 µg poly(dIdC) (unless otherwise indicated),
in a final volume of 20 µl DTT, reduced or oxidized glutathione,
N-ethylmaleimide, and diamide were added at the final
concentration indicated. After 10 min of incubation, binding reaction
was started by the addition of 10-20 fmol of labeled
double-stranded oligonucleotide. After 30 min at room temperature,
samples were loaded on a native 8% polyacrylamide gel in 0.5
TBE (2 mM EDTA, 90 mM boric acid, 90 mM
Tris-HCl, pH 8.0) and electrophoresed for 3 h at 250 V. The gel was
dried and exposed to an x-ray film with an intensifying screen at
-80 °C overnight.
Glycerol Gradient Sedimentation
Sedimentation was
performed through linear 10-35% glycerol gradients containing 20
mM Hepes, pH 7.9, 200 mM KCl, and either 5
mM DTT or 5 mM diamide. Samples, in 0.1 ml of buffer
F containing 0.2 M KCl, were layered onto the preformed
gradients and sedimented at 400,000 g in a SW60Ti
rotor for 10 h at 4 °C in a L8-M ultracentrifuge equipped with a
slow acceleration accessory. The gradients were fractionated into 25
fractions of 0.2 ml by pumping from the bottom of the centrifuge tube.
The amount of TTF-1 protein in each fraction was determined by gel
shift assay and Western blotting analysis. Standard proteins were
sedimented, each in a different tube, and fractionated as above. The
standards used were as follows: thyroglobulin, dimer, M
(subunit) = 334,500; catalase, tetramer, M
(subunit) = 55,700; cytosolic aspartate aminotransferase,
dimer, M
(subunit) = 45,000; bovine serum
albumin, monomer, M
= 68,000.
RESULTS
Oxidation Reversibly Inactivates TTF-1 Binding
Activity
The binding of TTF-1 to a high affinity site
(oligonucleotide C)
(14) , as assessed by gel mobility shift
assay, was markedly reduced after the addition of oxidizing agents such
as 5 mM oxidized glutathione (GSSG) or 5 mM diamide
(1,1`-azobis(N-dimetilformamide)), a reagent that catalyzes
the formation of disulfide bonds between cysteines
(25) (Fig. 1Alane2versuslane1 and lane5versuslane4). The reduction in DNA binding activity
was accompanied by the appearance of a faster migrating protein-DNA
complex on gel shift assay (Fig. 1). These effects were observed
at a concentration as low as 0.5 mM diamide and, in all cases,
binding activity could be fully restored after oxidation by the
addition of dithiothreitol (Fig. 1A, lanes3 and 6).
Figure 1:
Oxidation of sulfhydryl groups
reversibly decreases TTF-1 DNA binding activity. Shown is a gel shift
DNA binding assay with purified TTF-1 produced in HeLa cells, 1 ng (He
TTF-1, panel A) and FRTL-5 cell nuclear extracts, 5 µg
(FRTL-5 NE, panels B and C) using a
P-labeled double-stranded 24-mer oligonucleotide
containing the sequence of the proximal TTF-1 binding site of the
thyroglobulin promoter (5`-CACTGCCCAGTCAAGTGTTCTTGA-3` (oligonucleotide
C, Ref. 14). Where indicated reduced (GSH) or oxidized
(GSSG) glutathione, DTT, or diamide were added to 5
mM and incubated at room temperature for 10 min before the
addition of labeled DNA. To test reversibility, DTT was subsequently
added to 50 mM after a 10-min incubation with the oxidizing
reagent (panel A, lanes3 and 6 and
panel B, lane3). In panel C,
lane3, the ratio [GSH]/[GSSG]
was 10 (4 mMversus 0.4
mM).
The same behavior was exhibited by
TTF-1 in FRTL-5 crude nuclear extracts (Fig. 1B).
Interestingly, when these extracts were prepared in the absence of DTT,
a greatly decreased DNA binding activity (which could be restored by
the addition of DTT) was observed (data not shown). These data imply
that TTF-1 is either partially oxidized in the cell or that it readily
oxidizes during the preparation of the extract, thus suggesting that
the redox potential of the reactive cysteine residues of TTF-1 is very
low. In support of this view, loss of TTF-1 DNA binding activity could
also be observed in a buffer system in which the ratio of reduced to
oxidized glutathione ((GSH)/(GSSG)) is very similar to that operating
in the cytosol
(26) (Fig. 1C, compare lane3 with lane1). Other transcription
factors, which have been shown to be regulated in a redox-dependent
manner, do not appear as sensitive to the absence of DTT during the
extraction procedure
(8, 9) .
The Targets of the Redox Effect are the Free Sulfhydryl
Groups of Cysteines
To demonstrate that oxidation decreases
TTF-1 DNA binding activity via the cysteine sulfhydryl groups, purified
TTF-1, previously reduced by DTT, was incubated with increasing amount
of N-ethylmaleimide, a reagent that irreversibly alkylates
free sulfhydryl groups. The resulting alkylated protein was assayed for
its DNA binding activity by gel shift assay in the presence of a molar
excess of either DTT or diamide. N-Ethylmaleimide modification
of TTF-1 results in a protein resistant to oxidation
(Fig. 2A, compare lanes1-3 with
4-6), thus demonstrating that the thiol groups are the
actual targets of this regulation. The same results were obtained using
iodoacetamide as alkylating reagent (data not shown). The fact that
alkylation of TTF-1 does not affect its DNA binding activity but rather
protects it against oxidative damage strongly suggests that the
inactivation of the protein by oxidation is not due to direct
interference with TTF-1-DNA contacts but, rather, to the formation of
inter- or intrachain disulfide bonds. In agreement with this view, the
isolated, recombinant TTF-1 homeodomain, which does not contain
cysteine residues, is insensitive to modifications by both oxidizing
and alkylating reagents (Fig. 2B).
Figure 2:
Alkylation of sulfhydryls does not
decrease TTF-1 DNA binding activity, but protects it against oxidative
damage. Panel A, gel shift assay performed with 1 ng of
purified TTF-1 (previously reduced in a 1-h incubation at room
temperature with a large molar excess of DTT) using oligo(C) probe (see
Fig. 1). N-Ethylmaleimide was added to the binding reaction
mixture and incubated at room temperature (lanes2-6). Where indicated DTT (lane5) or diamide (lane6) was added after
a 20-min incubation with N-ethylmaleimide. The DNA probe was
always added last after a 30-min incubation with the sulfhydryl
modifying reagents. Panel B, gel shift assay performed with
0.2 ng of purified, recombinant, TTF-1 HD (21), in the absence of
poly(dIdC). Incubations with the indicated regents were carried
out at room temperature for 10 min before addition of labeled oligo(C)
probe.
Both Cysteine 87 and Cysteine 363 Are Required for
Redox-dependent DNA1 Binding Activity
The amino acid sequence of
TTF-1 contains four cysteine residues at positions 87, 245, 271, and
363. To determine the residue(s) responsible for the sensitivity of the
DNA binding activity of TTF-1 to oxidizing reagents, mutant DNAs
encoding proteins in which each cysteine residue was replaced by a
serine residue were constructed (Fig. 3A). The binding
activity of nuclear extracts prepared from HeLa cells transfected with
expression vectors for each of these mutants was measured in the
presence of DTT or diamide. All mutated proteins were still able to
bind DNA in the presence of reducing agents, but none of the single Cys
Ser changes gave rise to a protein whose DNA binding activity
was insensitive to inactivation by diamide (Fig. 3B).
The analysis of three mutants that contain combinations of these Cys
Ser substitutions identified cysteine 87 and cysteine 363 as the
residues responsible for the inactivation by oxidizing reagents. In
fact, the combination of these two substitutions almost abolished the
inhibition by diamide (compare in Fig. 3B the mutant
TTF-1 SCCS to TTF-1 CCCC), whereas the substitution of the two inner
cysteine residues (TTF-1 CSSC) did not change the diamide sensitivity
of TTF-1 (Fig. 3B, compare lanes11,
12, 15, and 16). The tetramutant, TTF-1
SSSS, is constitutively active (see Fig. 3B, lanes13 and 14) and binds to DNA with the same
affinity as the wild-type protein (data not shown).
Figure 3:
Identification of the cysteine residue(s)
responsible for redox regulation. The cysteine residues at positions
87, 245, 271, and 363 were changed to serine in different combinations.
Panel A, schematic representation of the mutant proteins. The
mutated protein synthesized are referred to as TTF-1 SCCC, indicating
that the first cysteine residue (position 87) was changed to serine;
TTF-1 CSCC; the second cysteine residue (position 245), was changed to
serine; and so on. TTF-1 CCCC is the recombinant wild-type protein.
Panel B, gel shift analysis of the expressed proteins. Equal
amounts of expressed proteins (about 1 ng, as determined by Western
blot analysis) in nuclear extract of HeLa cells were used to shift the
oligo(C) probe. Where indicated, DTT or diamide were added to 5
mM and incubated at room temperature for 10 min before
addition of labeled DNA.
TTF-1 Is Able to Form Aggregates under Both Reducing and
Oxidizing Conditions
Since the DNA binding activity of TTF-1 is
affected by oxidation, we asked whether this was due to the formation
of inter- or intramolecular disulfide bonds. The molecular state of
both the fully reduced and the fully oxidized forms of TTF-1 was
analyzed by SDS-PAGE under nonreducing conditions. As shown in
Fig. 4
, fully reduced TTF-1 migrates as a monomer with an
apparent molecular mass of 38 kDa. After exposure to diamide, two
monomeric forms and a ladder of multimeric species are observed, even
though the absence of 2n + 1 oligomers and the presence
of two discrete species in the dimer population suggest a certain
specificity in the oligomerization process. The oligomerization of
TTF-1 as multimers of a dimeric form can also be induced by
cross-linking under reducing conditions, after exposure to diverse
cross-linking reagents (Fig. 4, compare lanes1, 3, and 4). Taken together, these
data indicate that oligomerization of TTF-1 has specific structural
requirements, as it has been observed in the case of other
transcription factors
(7) .
Figure 4:
TTF-1 is able to form covalently linked
dimers, as well as oligomers, both in oxidizing and reducing
conditions. SDS-PAGE analysis of purified TTF-1 (2 ng) in different
conditions. Samples were incubated in the presence of 4 mM
diamide (lane1), 4 mM DTT (lane2), 1 mM disuccinimidylsuberate, (DSS,
lane3), or 1 mM bis(maleimido)-methyl ether
(BMME, lane4) for 15 min at room
temperature. Samples were heated 5 min at 95 °C in the presence of
4% SDS (lanes1 and 2) or 4% SDS plus 10%
-mercaptoethanol (lanes3 and 4) and
than run on gel using Fast System (Pharmacia). Proteins were visualized
by means of Western blotting with a TTF-1
antiserum.
After diamide treatment, two
monomeric forms are observed. The slower migrating form appears to
co-migrate with the fully reduced form obtained in the presence of DTT
(see Fig. 4, lane2). The faster migrating
species probably represents a more compact form as a consequence of the
formation of an intramolecular disulfide bridge. This situation is
reminiscent of that observed in gel mobility-shift assay where, in the
presence of oxidizing reagents, a faster migrating complex is also
detected (see for example in Fig. 1A, lanes2 or 5). Since it has been demonstrated that
TTF-1 binds to the oligonucleotide C as a monomer
(13) , it seems
conceivable that the residual binding activity, which is observed as a
faster migrating complex under oxidizing conditions, is due to this
intramolecularly cross-linked form. Furthermore, this complex is
observed in the presence of diamide with the mutant TTF-1 CSSC but not
with the mutant TTF-1 SCCS (see Fig. 3B), thus
indicating that both Cys-87 and Cys-363 are responsible for this
internal disulfide bridge.
Figure 5:
TTF-1 can form aggregates in both reducing
and oxidizing conditions. Panel A, sedimentation profiles of
purified TTF-1 (100 ng) through glycerol gradients containing 5
mM DTT () or 5 mM diamide (
), in the
presence of 10 µg of bovine serum albumin. The amount of TTF-1
present in each fraction was evaluated by gel shift assay performed as
described (see ``Materials and Methods'') always in the
presence of 5 mM DTT and 1% Nonidet P-40. Panel B, Western blot analysis of the proteins sedimented at the bottom of
the tube for the gradients shown in A, performed in the
presence of DTT (5 mM) or diamide (5 mM). Panel
C, sedimentation profiles of nuclear extracts from HeLa cells
expressing mutant TTF-1 SCCC. About 100 ng of mutant protein, as
evaluated by Western blotting analysis, corresponding to 50 µg of
total nuclear proteins were subjected to centrifugation and analyzed in
the presence of DTT (
) or diamide (
) as indicated in
A. Similar results were obtained with the mutant TTF-1 CCCS.
Panel D, as A, except the gradient buffer that
contains 1% Nonidet P-40. Arrows in A, C,
and D indicate the position of the sedimentation peaks as
determined by measurement of A
for proteins of
known molecular mass sedimented in the same conditions as
above.
The sedimentation profiles of
TTF-1 mutants CSSS and SSSC (Fig. 3A) were also studied.
Each mutant contains only one cysteine residue, Cys-87 or Cys-363,
respectively. The presence of only one cysteine should allow the
formation of covalent dimers, but not of higher order covalent
multimers. As expected, upon oxidation, the mutant TTF-1 SCCC forms
dimers in solution, as shown in Fig. 5C. Interestingly,
noncovalent, higher molecular weight aggregates are also absent (data
not shown). Similar results were obtained with the mutant TTF-1 CSSS,
thus indicating that the formation of specific disulfide bridges is
crucial for the formation of higher order multimers.
Both Intra- and Intermolecular Disulfide Bridges Decrease
the DNA Binding Affinity of TTF-1
Since SDS-PAGE and rate-zonal
centrifugation analyses demonstrated the existence of multimeric forms
of TTF-1, we asked if any of these forms were able to bind DNA. In this
vein, we performed saturation binding experiments of highly purified
TTF-1 under both reducing and oxidizing conditions, using increasing
amounts of target DNA and in the absence of nonspecific DNA. Under
these conditions, we were able to observe the formation of a slower
migrating complex in the presence of diamide, probably due to a dimeric
form of TTF-1 (Fig. 6A, banda). The
faster migrating complex, as well as a fraction of the fully reduced
complex, were also obtained (Fig. 6Abandsc and b, respectively). The identification of
the slower complex as a dimeric form was indeed confirmed by analyzing
the DNA binding activity of TTF-1 CSSS and SSSC mutants. As shown in
Fig. 6B, these mutants are able to form a slower
migrating complex which, as demonstrated by rate-zonal centrifugation
analysis (see Fig. 5C), is due to a dimeric form of
TTF-1. This complex actually co-migrates with the slower species
(Fig. 6A, banda) observed with the
wild-type protein in the presence of diamide (data not shown). Moreover
these mutants, as expected, do not give rise to the faster migrating
complex (Fig. 6Abandb).
Figure 6:
Oxidation decreases the DNA binding
affinity of TTF-1. Saturation experiments. Purified TTF-1 at a fixed
concentration of 2 nM was incubated for 45 min in binding
buffer without poly(dIdC) containing increasing concentrations of
labeled oligo(C) (Fig. 1). Where indicated, DTT or diamide were added
to 5 mM and incubated for 10 min at room temperature before
the addition of the probe. Bound (a-c) and free
(F) DNA were visualized by autoradiography (panel A).
Data obtained from the titration in A are plotted in C where square and diamond symbols are referred to
as the points obtained in the presence of DTT and diamide,
respectively. The following equilibrium equation was fitted to the data
by nonlinear least-squares in order to calculate K ([Bound] =
P
K
[Free]/(1 +
K
[Free])) where P is the total
concentration of TTF-1, K is the association constant, and
[Bound] and [Free] are the concentrations of bound
and free oligo(C) probe, respectively. In the case of the data obtained
in the presence of diamide, [Bound] is referred to as the sum
of the concentrations of complexes a, b, and
c, thus accounting for an average binding. The values obtained
were K = (4 ± 2)
10
M
and K = (3 ±
2)
10
M
, for the
equilibrium studied in the presence of DTT and diamide, respectively.
Panel B shows the gel shift analysis for the expressed protein
mutants TTF-1 SSSC and TTF-1 CSSS (see Fig. 3 for nomenclature). The
slower migrating complex observed in the presence of diamide (lanes2 and 4) is assumed to derive from a covalent
dimeric form of these proteins (see for comparison Fig. 5, panel
C).
As far as
the DNA binding affinity is concerned, the saturation experiment shown
in Fig. 6clearly indicates that the overall binding affinity of
TTF-1 under oxidizing conditions is decreased at least 10-fold with
respect to that determined under reducing conditions. Taken together,
these data lead to the conclusion that both intra- and intermolecular
cross-linked forms of oxidized TTF-1 can still bind to DNA but,
overall, they exhibit a lower binding affinity. Furthermore, the
saturation curves exhibited in Fig. 5C show that, even
at high concentration of DNA, the oxidized protein shows a clear
decrease in total DNA binding activity, indicating that oxidation
induces the formation of inactive TTF-1 species. We presume that these
inactive forms are the high molecular weight TTF-1 aggregates detected
by SDS-PAGE (Fig. 4) and rate-zonal centrifugation analysis
(Fig. 5). In support of this view, no protein-DNA complexes
migrating slower than the dimer-containing form
(Fig. 5A, banda) are observed in band
shift experiments.
DISCUSSION
We show in this paper that oxidation of the
homeodomain-containing protein TTF-1 causes the formation of both
inter- and intramolecular disulfide bonds, resulting in a considerable
decrease of TTF-1 DNA binding activity. The redox modifications involve
only two of the four cysteine residues present in the primary structure
of TTF-1, namely Cys-87 and Cys-363. Interestingly, neither of these
cysteine residues is located within the TTF-1 homeodomain, which has
been shown to be by itself sufficient to elicit the specific TTF-1 DNA
binding activity (27). Moreover, neither the alkylation of the
sulfhydryl groups nor the simultaneous replacement of Cys-87 and
Cys-363 with serine residues have any adverse effects on the DNA
binding activity of TTF-1. Taken together, these observations indicate
that Cys-87 and Cys-363 are not directly involved in DNA recognition;
nonetheless, their oxidation to cysteine greatly decreases TTF-1 DNA
binding affinity. We suggest that the decreased DNA binding affinity
induced by oxidation relies on the formation of disulfide bridges,
which are responsible for the generation of TTF-1 oligomers inactive in
DNA binding. Oxidation seems to stabilize pre-formed dimers, as
demonstrated by the ability of detergents to inhibit diamide-induced
oligomerization. In fact, TTF-1 oligomers have been detected, under
both reducing and oxidizing conditions, by rate-zonal sedimentation
and, only after oxidation, by denaturing PAGE. This represents a novel
type of oxidation-sensitive DNA binding activity for
homeodomain-containing proteins. A large number of transcription
factors have been found to be inactivated in vitro by
oxidation of cysteine residues and, in many cases, to be inactivated by
alkylating reagents. In the majority of the cases described, the
targets of the redox regulation are cysteine residues that are directly
involved in recognition of the DNA or else closely associated with the
DNA
(4, 6, 9) . This does not seem to be the case
for TTF-1, which is not inactivated but rather it is protected by
alkylation toward oxidative damage. Interestingly, also, upstream
stimulatory factor (USF), a transcription factor that does not contain
a homeodomain, shows a similar behavior
(7) .
2
homeoproteins
(34, 35, 36) , and members of the
Arabidopsis HD-Zip class of proteins
(37) .
Representative of the second group are the POU proteins
(38, 39) and the paired-class homeodomains
(40) . Another
interesting case of homeoprotein that exhibits cooperative dimerization
on DNA is that of human HoxB5 protein, where a small protein domain,
adjacent to the homeodomain, is required for oxidation-induced
cooperative effect
(5) . Moreover, specific protein-protein
interactions among several members of the Hox gene family have recently
been found using affinity chromatography techniques
(41) .
Figure 7:
Alignment of TTF-1 binding sequences in
different promoters. The sequences shown derive from thyroglobulin
(Tg, 14), thyroperoxidase (TPO, 15), and surfactant
protein B (SPB, 48). The numbers indicates the
position of the sequences in the promoters as distance from the
transcriptional start site. TTF-1 binding sites, as demonstrated by
DNase I footprinting or methylation interference experiments (Refs.
14-16 and M. I. Arnone, unpublished results) have been
boxed. Arrows indicate the orientation of the TTF-1
binding sites on the DNA sequence. The boxed regions have been
aligned to the consensus sequence for TTF-1(42), and the bases matching
with it have been underlined.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.