Redox Regulation of the DNA Binding Activity in Transcription Factor PEBP2

Transcription factor PEBP2/CBF consists of a DNA binding subunit, α, and a regulatory subunit, β. The α subunit has an evolutionarily conserved 128-amino acid region termed “Runt domain” that is responsible for both DNA binding and heterodimerization with the β subunit. The Runt domain in all mammalian submembers of the α 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 PEBP2αA 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 β 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 β subunit as key modulators. Transcription factor PEBP2/CBF consists of a DNA binding subunit, α, and a regulatory subunit, β. The α subunit has an evolutionarily conserved 128-amino acid region termed “Runt domain” that is responsible for both DNA binding and heterodimerization with the β subunit. The Runt domain in all mammalian submembers of the α 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 PEBP2αA 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 β 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 β subunit as key modulators. Polyoma virus enhancer-binding protein 2 (PEBP2) 1The abbreviations used are:PEBPpolyoma virus enhancer-binding proteinTrxthioredoxinTrxRthioredoxin reductaseEMSAelectrophoretic mobility shift assayDTTdithiothreitol (1Ogawa E. Maruyama M. Kagoshima H. Inuzuka M. Lu J. Satake M. Shigesada K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6859-6863Google Scholar), also called core binding factor (2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.J. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Google Scholar), is a heterodimeric transcription factor composed of two different subunits, α and β. The α subunit binds directly to a specific DNA sequence, RACCRCA, while the β subunit does not by itself contact with DNA but facilitates the DNA binding activity of the α subunit through allosteric interactions (1Ogawa E. Maruyama M. Kagoshima H. Inuzuka M. Lu J. Satake M. Shigesada K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6859-6863Google Scholar, 2Wang S. Wang Q. Crute B.E. Melnikova I.N. Keller S.J. Speck N.A. Mol. Cell. Biol. 1993; 13: 3324-3339Google Scholar). The α subunit shares a 128-amino acid region of high homology with theDrosophila segmentation gene runt (3Kania M.A. Bonner A.S. Duffy J.B. Gergen J.P. Genes Dev. 1990; 4: 1701-1713Google Scholar) and the human AML1 gene (4Miyoshi H. Shimizu K. Kozu T. Maseki N. Kaneko Y. Ohki M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10431-10434Google Scholar). The conserved region, termed “Runt domain,” is responsible for both DNA binding and heterodimerization with the β subunit (5Kagoshima H. Shigesada K. Satake M. Ito Y. Miyoshi H. Ohki M. Pepling M. Gergen J.P. Trends Genet. 1993; 9: 338-341Google Scholar, 6Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Google Scholar). After the identification of the first member of the α subunit, PEBP2αA (1Ogawa E. Maruyama M. Kagoshima H. Inuzuka M. Lu J. Satake M. Shigesada K. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6859-6863Google Scholar), two additional members of the Runt family have subsequently been isolated in mice and humans: PEBP2αB (mouse homolog of AML1) (7Bae S.C. Yamaguchi-Iwai Y. Ogawa E. Maruyama M. Inuzuka M. Kagoshima H. Shigesada K. Satake M. Ito Y. Oncogene. 1993; 8: 809-814Google Scholar), and PEBP2αC (8Bae S.C. Takahashi E., W. Zhang Y. Ogawa E. Shigesada K. Namba Y. Satake M. Ito Y. Gene ( Amst. ). 1995; 159: 245-248Google Scholar) or AML2 (9Levanon D. Negreanu V. Bernstein Y. Bar-Am I. Avivi L. Groner Y. Genomics. 1994; 23: 425-432Google Scholar). polyoma virus enhancer-binding protein thioredoxin thioredoxin reductase electrophoretic mobility shift assay dithiothreitol 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. 10Speck N.A. Stacy T. Crit. Rev. Eukaryot. Gene Exp. 1995; 5: 337-364Google Scholar). 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. 10Speck N.A. Stacy T. Crit. Rev. Eukaryot. Gene Exp. 1995; 5: 337-364Google Scholar and 11Nucifora G. Rowley J.D. Blood. 1995; 86: 1-10Google Scholar). Moreover, an inversion in human chromosome 16 that gives rise to a fusion product, PEBP2β-SMMHC (smooth muscle myosin heavy chain), was also found to cause a M4Eo subtype acute myeloid leukemia (12Liu P. Tarle S.A. Hajra A. Claxton D.F. Marlton P. Freedman M. Siciliano M.J. Collins F.S. Science. 1993; 261: 1041-1044Google Scholar). Recent gene disruption studies in mice of AML1 (13Okuda T. Deursen J.V. Hiebert S.W. Grosveld G. Downing J.R. Cell. 1996; 84: 321-330Google Scholar) as well as PEBP2/CBFβ (14Wang Q. Stacy T. Miller J.D. Lewis A.F. Gu T.-L. Huang X. Bushweller J.H. Bories J.-C. Alt F.W. Ryan G. Liu P.P. Wynshaw-Boris A. Binder M. Marin-Padilla M. Sharpe A.H. Speck N.A. Cell. 1996; 87: 697-708Google Scholar) have confirmed that these genes are essential for definitive hematopoiesis. Our previous functional characterization of PEBP2 (6Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Google Scholar) 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 (15Yodoi J. Uchiyama T. Immunol. Today. 1992; 13: 405-411Google Scholar), the importance of the thiol-mediated redox regulation has been well recognized in various biological responses (16Holmgren A. J. Biol. Chem. 1989; 284: 13963-13966Google Scholar, 17Schulze-Osthoff K. Los M. Baeuerle P.A. Biochem. Pharmacol. 1995; 50: 735-741Google Scholar). Particularly notable are accumulating examples of redox-responsive transcription factors, such as the AP-1 family (18Abate C. Patel L. Rausher III, F.J. Curran T. Science. 1990; 249: 1157-1161Google Scholar,19Xanthoudakis S. Miao G. Wang F. Pan Y.-C.E. Curran T. EMBO J. 1992; 11: 3323-3335Google Scholar), the Rel family (20Toledano M.B. Leonard W.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4328-4332Google Scholar, 21Matthews J.R. Wakasugi N. Virelizier J.-L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Google Scholar, 22Hayashi T. Ueno Y. Okamoto T. J. Biol. Chem. 1993; 268: 11380-11388Google Scholar), Myb (23Myrset A.H. Bostad A. Jamin N. Lirsac P. Toma F. Gabrielsen O.S. EMBO J. 1993; 12: 4625-4633Google Scholar), Ets-1 (24Wasylyk C. Wasylyk B. Nucleic Acids Res. 1993; 221: 523-529Google Scholar), and p53 (25Hainaut P. Milner J. Cancer Res. 1993; 53: 4469-4473Google Scholar). 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 α subunit contain two cysteine residues that are perfectly conserved among them (8Bae S.C. Takahashi E., W. Zhang Y. Ogawa E. Shigesada K. Namba Y. Satake M. Ito Y. Gene ( Amst. ). 1995; 159: 245-248Google Scholar). Our recent random mutagenesis study with the Runt domain of PEBP2αA (26Akamatsu Y. Tsukumo S. Kagoshima H. Tsurushita N. Shigesada K. Gene ( Amst. ). 1997; 185: 111-117Google Scholar) 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. Plasmid pQE-RD (6Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Google Scholar) encoding the Runt domain of PEBP2αA 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 (27Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene ( Amst. ). 1989; 77: 51-59Google Scholar). The substitutions of the targeted residues were confirmed by sequencing. His-tagged derivatives of the Runt domain and the β subunit were expressed inEscherichia coli and purified on a nickel-nitrilotriacetic acid resin (QIAGEN) as described (6Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Google Scholar). Trx was purchased from Ajinomoto. TrxR was purified as described (28Kitaoka Y. Sorachi K. Nakamura H. Masutani H. Mitsui A. Kobayashi F. Mori T. Yodoi J. Immunol. Lett. 1994; 41: 155-161Google Scholar). Ref-1 was purified as described (29Xanthoudakis S. Miao G. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 23-27Google Scholar). A DNA probe containing the wild type PEBP2 binding site was prepared as described (6Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Google Scholar). The DNA binding reaction was routinely carried out for 10 min at 25 °C in 10 μl of the EMSA buffer containing 20 mmHEPES-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 of32P-labeled probe, 5 ng of purified His-tagged Runt domain, and the β 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. The two conserved cysteine residues in the Runt domain of PEBP2αA 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 β subunit (Fig. 1B,left panel; see also Fig. 1C for quantitative comparison). In confirmation of our previous observation (26Akamatsu Y. Tsukumo S. Kagoshima H. Tsurushita N. Shigesada K. Gene ( Amst. ). 1997; 185: 111-117Google Scholar), 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 β 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 β subunit. This indicates that substitution of either cysteine residue to serine or aspartate is tolerable for the heterodimerization between the Runt domain and the β subunit as well as their allosteric regulatory interaction. 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 β 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 β 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 CS was only even at 100 mm. the β subunit to both Cys-115 and Cys-124 tolerable to increased of diamide by one of or such β was observed with the as In Fig. are also changes in the of DNA binding by the β subunit with the diamide for Runt domain first that showed which is with the diamide and to represent the regulatory of the β subunit in the of the Runt domain for DNA as by (6Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Google Scholar). contrast, CC and SC much than a of or more at 1 mm diamide. This over that observed with is to the of the β subunit on Cys-124 as the of CS was showed a rise with increasing concentrations of diamide, that of at 100 mm. Cys-115 is also subject to the of the β subunit. We further the of cellular could for activation of the Runt domain. were as and Ref-1. is to transcription factor under the with TrxR and (15Yodoi J. Uchiyama T. Immunol. Today. 1992; 13: 405-411Google Scholar, A. J. Biol. Chem. 1989; 284: 13963-13966Google Scholar, Y. Sorachi K. Nakamura H. Masutani H. Mitsui A. Kobayashi F. Mori T. Yodoi J. Immunol. Lett. 1994; 41: 155-161Google Scholar). Ref-1 S. Miao G. Wang F. Pan Y.-C.E. Curran T. EMBO J. 1992; 11: 3323-3335Google Scholar) has been as a factor that the DNA binding activity of transcription factor Ref-1, in be readily reduced by Trx S. Miao G. Wang F. Pan Y.-C.E. Curran T. EMBO J. 1992; 11: 3323-3335Google Scholar). in Fig. was virtually in the Runt domain in the absence of the β subunit. When the β subunit was present, showed activation at low concentrations and but became at 0.1 mm or In contrast, Trx showed activation at (Fig. Ref-1 was even more than Trx with concentrations Fig. Interestingly, the β subunit to the activation of the Runt domain by these proteins, Ref-1 (Fig. and regulatory of this be in Trx and Ref-1 were found to synergistically with When CC was with either of them with a of to the diamide, or only DNA binding was In they the DNA binding as effectively as 100 mm 2 and The of Trx was also by its with TrxR and In the of this the could be with only a in DNA binding which was by the of Ref-1 The study has that both of the two cysteine residues the Runt domain are responsible for redox regulation in their own which are in with other redox-responsive transcription Cys-124 the redox-responsive cysteine in and (18Abate C. Patel L. Rausher III, F.J. Curran T. Science. 1990; 249: 1157-1161Google Scholar) in its high to amino acids see also Ref. M.J. P. 20: Scholar) and DNA binding by its substitution to In and of cysteine to of is to on DNA binding. A to Cys-124 in the Runt domain. Conversely, the redox of Cys-115 is to the absence of any amino acid in its (Fig. the reduced DNA binding activity caused by its substitution to serine as well as aspartate that the be have been for J.R. Wakasugi N. Virelizier J.-L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Google Scholar), Myb (23Myrset A.H. Bostad A. Jamin N. Lirsac P. Toma F. Gabrielsen O.S. EMBO J. 1993; 12: 4625-4633Google Scholar), and Ets-1 (24Wasylyk C. Wasylyk B. Nucleic Acids Res. 1993; 221: 523-529Google Scholar). the x-ray of has of the of cysteine with its target DNA G. G. S. 1995; Scholar). A the two cysteines in the Runt domain form a as for p53 (25Hainaut P. Milner J. Cancer Res. 1993; 53: 4469-4473Google Scholar). However, their redox susceptibilities as observed the that the two cysteines redox reactions from a mechanism more regulation of DNA binding in to cellular redox than In of our previous observation (6Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Google Scholar), the results of this study also the potential of the β subunit as a of redox of the Runt domain, in to its in the DNA binding of the Runt domain. The β subunit to both cysteines from by diamide. the β subunit not the of the Runt domain by much Trx and Ref-1. then was the β subunit to the of We that the of these cysteine residues could be by β in the them in of their with amino acids or their in or than a The finding that Trx and Ref-1 the Runt domain in a with other and by the β subunit the of where and these protein with other the the α subunit is a the β subunit by itself is in the and to the through with the α subunit J. Maruyama M. Satake M. S.C. Ogawa E. Kagoshima H. Shigesada K. Ito Y. Mol. Cell. Biol. 1995; Scholar). (6Kagoshima H. Akamatsu Y. Ito Y. Shigesada K. J. Biol. Chem. 1996; 271: 33074-33082Google Scholar, J. Maruyama M. Satake M. S.C. Ogawa E. Kagoshima H. Shigesada K. Ito Y. Mol. Cell. Biol. 1995; Scholar) has suggested that the of the β subunit with the α subunit is both in and in a between the Runt domain and its and that a specific mechanism to this Ref-1 is a protein S. Miao G. Wang F. Pan Y.-C.E. Curran T. EMBO J. 1992; 11: 3323-3335Google Scholar), Trx is in the but is the exposure of to various and such as and K. M. S. A. Mori K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar). The of Trx also be by various oxidative Y. K. Masutani H. K. Okamoto T. M. Yodoi J. Immunol. Lett. 1995; Scholar). In the of these is to that the DNA binding activity of PEBP2 be regulated by of the β subunit and Trx in to oxidative this work was in in vivo the functional importance of cysteine residues in the Runt domain protein was by the M. T. K. N. Ogawa S. K. Y. H. J. Biol. Chem. 1996; 271: Scholar) that the and of were by substitution of serine for to Cys-115 in To further the and of redox regulation of PEBP2 to their should be to such functional studies using a of mutants as in with various conditions the cellular redox is the of of cysteine in the Runt domain This protein family has been in a of from from the C. Kagoshima and T. to mammals, and to contain at three in (8Bae S.C. Takahashi E., W. Zhang Y. Ogawa E. Shigesada K. Namba Y. Satake M. Ito Y. Gene ( Amst. ). 1995; 159: 245-248Google Scholar, D. Negreanu V. Bernstein Y. Bar-Am I. Avivi L. Groner Y. Genomics. 1994; 23: 425-432Google Scholar, 10Speck N.A. Stacy T. Crit. Rev. Eukaryot. Gene Exp. 1995; 5: 337-364Google Scholar) and two (3Kania M.A. Bonner A.S. Duffy J.B. Gergen J.P. Genes Dev. 1990; 4: 1701-1713Google Scholar) and A. K. U. Genes Dev. 1996; in all the members in including Dev. Biol. 1996; Scholar), P. M. A. F. A. F. Exp. Res. 1996; Scholar), and (8Bae S.C. Takahashi E., W. Zhang Y. Ogawa E. Shigesada K. Namba Y. Satake M. Ito Y. Gene ( Amst. ). 1995; 159: 245-248Google Scholar, D. Negreanu V. Bernstein Y. Bar-Am I. Avivi L. Groner Y. Genomics. 1994; 23: 425-432Google Scholar, 10Speck N.A. Stacy T. Crit. Rev. Eukaryot. Gene Exp. 1995; 5: 337-364Google Scholar) the two conserved cysteine residues at and On the other hand, those found in C. which to have one or both of these cysteines to serine or also is an from cysteine to serine at in an with the of that were a of ways in cysteine to DNA binding by the Runt domain and its among them at of in the studies of the Runt domain proteins are to the potential of its redox regulation in We for the of C. homolog.