©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The DNA Binding Activity and the Dimerization Ability of the Thyroid Transcription Factor I Are Redox Regulated (*)

Maria I. Arnone , Mariastella Zannini , Roberto Di Lauro (§)

From the (1) Stazione Zoologica A. Dohrn, Villa Comunale, 81021 Napoli, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


MATERIALS AND METHODS

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(dIdC) 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(dIdC) and then with 2 volumes of same buffer without poly(dIdC); proteins were eluted with a 0.2-1.5 M linear KCl gradient in buffer F. The active fractions were pooled, poly(dIdC) was added to a final concentration of 2.5 µg/ml, and the affinity step was repeated.

Purified, recombinant, TTF-1 HD was prepared as described previously (21).

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.

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.


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.

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.

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] = PK[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) 10M and K = (3 ± 2) 10M, 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) .

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 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) .

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.


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.




FOOTNOTES

*
This work was supported by grants from the Progetto finalizzato Applicazioni Cliniche della Ricerca Oncologia of Consiglio Nazionale delle Ricerche, the Associazione Italiana per la Ricerca sul Cancro, and the Commission of the European Communities (BIO2 CT 930454). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 39-81-5833278; Fax: 39-81-5833285.

The abbreviations used are: DTT, dithiothreitol; HD, homeodomain; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

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.


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